Gert Müller-Berghaus, Lothar Thomas
Primary hemostasis: Following a vascular injury, the hemostasis is rapidly activated to initiate the formation of a platelet plug followed by a stable fibrin clot. During primary hemostasis platelets adhere to the extracellular matrix components at the side of injury, and to one onather, plugging vascular defects and often stopping bleeding.
Secondary hemostasis: The secondary hemostasis involves a series of enzyme-mediated activation of clotting factors occurring in distinct but overlapping steps of initiation, amplification, and propagation These stages of homeostasis take place on two main types of cells, tissue factor bearing cells and platelets, both of which control the coagulation process and regulate the regulation amount, and timing of thrombin generation. The ultimate goal is production of sufficient thrombin to convert fibrinogen to fibrin, and emesh the initial platelet plug in a stable fibrin meshwork.
- The system of blood vessels including the endothelial cells and subendothelial matrix of the vessel wall, vasoconstriction and the resulting reduction of blood flow
- Blood cells, especially thrombocytes (platelets)
- The plasma coagulation system with interacting platelets, coagulation factors and coagulation inhibitors
- The fibrinolytic system with its activators and inhibitors.
At rest the interaction of these components is a continuous perfectly organized series of events which includes a low level of continuing consumption and formation of all components. The interactions are activated by (e.g., vascular injury) in different ways:
- Normal regulation of hemostasis: activated platelets and plasma coagulation factors, come in contact with the subendothelial matrix causing platelet adhesion to the vessel wall. Platelet activation, the initial step of arrest of bleeding, is mediated by the von Willebrand factor. This leads to the formation of platelet aggregates, also referred to as platelet clot which seal the vascular leak (primary hemostasis). At the same time, the activation of the plasma coagulation system by factor VIIa takes place in combination with the tissue factor (TF) released from TF-containing cells as the cofactor.
- Deregulation of hemostasis: for example, in pathological conditions greatly activated hemostasis leads to the formation of cell aggregates and fibrin-rich clots in the blood vessels causing a reduction in blood flow and resulting in thrombosis and ischemia of tissues.
Hemostasis is subject to regulation similar to many other functional systems within the organism. Humoral and cellular systems, activators and inhibitors as well as positive and negative feedback mechanisms participate in this regulatory process. Defects in participating reactants or deregulation by activation or inhibition of the regulatory systems may result in hemostatic disequilibrium, thus followed by either hemorrhage (bleeding) or thrombosis (blood clot formation).
Hemostatic regulation takes place on the surfaces of TF-containing cells (e.g., on platelets or endothelial cells) or in injuries subendothelially, but not within the fluid phase. Physiologically, hemostatic reactants are activated to a small extent and thrombin is generated causing the conversion of minimal quantities of fibrinogen into fibrin. Fibrin, in turn, is subject to degradation by fibrinolysis. Fibrin synthesis is so minimal that under physiological conditions no blood clot formation is detectable by screening assays.
Interaction of the mechanisms
The vascular endothelium, platelets, plasma coagulation system and fibrinolytic system are not to be viewed as separate systems. The complexity of the reactions can be best exemplified by the hemostatic processes following vascular injury :
- If a blood vessel is injured to such an extent that subendothelial structures are exposed, vasoconstriction will take place initially. Parallel to this, platelet adhesion to the subendothelial structures will occur.
- Platelet activation leads to the perpetuation of the reaction and platelet aggregates form
- Subendothelial structures also initiate the activation of the plasma coagulation system. Contact factors as well as the tissue factor, which is a component of the subendothelial extracellular matrix, are part of this activation process.
- Platelets, activated by subendothelial collagen, express membrane glycoproteins (GPs) such as the GP Ib/IX/V receptor that reacts with the von Willebrand factor (VWF), thus binding and activating the platelets.
- The expression of specific platelet membrane phospholipids represents a surface for complex formation between the reactants. These complexes trigger the initial step of coagulation factor activation.
- During the initiation phase of coagulation, small amounts of thrombin are produced on tissue TF-bearing cells.
- This initial thrombin generation is critical for fully activating adjacent platelets into a highly procoagulant state.
- During the amplification phase, thrombin participates in positive feedback loops and activates FXI, FV, and FVIII. These events induce a marked generation of thrombin and acceleration of the plasma coagulation system.
- If a platelet fibrin clot develops at an intact vessel wall site, it will soon be lysed since endothelial cells release tissue plasminogen activator at an increased rate, thus inducing fibrinolysis activation.
- The platelet fibrin clot formed initially remains intact at the site of the vessel wall injury since the damaged endothelial cells are unable of producing or releasing fibrinolysis activators in adequate quantities. The inevitably associated lack in local fibrinolytic activity around the site of a vascular injury causes the platelet fibrin clot to stay in place.
Endothelial cells produce substances with pro aggregatory and anti aggregatory effects, adhesive proteins, and substances with impact on the vascular tone.
Modulation of the vascular tension
Since the muscular layer of arteries and arterioles is thicker than that of veins and venules, the vasomotor reaction of vasoactive substances is greater on the arterial side than on the venous side. Endothelial cells synthesize, among others /, /:
- Nitric oxide (NO), also referred to as endothelium-derived relaxing factor (EDRF). Besides its inhibitory effect on platelet aggregation, EDRF is a potent vasodilator. The release of EDRF from the endothelial cells is stimulated by bradykinin, histamine, acetylcholine, thrombin, vasopressin and ADP.
- Prostacyclin (PGI2), likewise inhibits platelet aggregation and causes vasodilation
- High-molecular-weight kininogen from which bradykinin with its vasodilatory effects is produced
- Endothelin-1, thromboxane A2 and angiotensin converting enzyme (ACE). The first two have a direct vasoconstrictor effect. ACE has an indirect vasoconstrictor effect since it transforms angiotensin I into the vasoconstrictor angiotensin II.
Adhesive proteins are not only responsible for the adhesion of endothelial cells to the substratum but also contribute to the attachment of platelets to subendothelial structures following a vascular wall injury. Endothelial cells influence the functions of platelets by synthesizing the following activators and inhibitors of platelet aggregation :
- Platelet-activating factor (PAF) released from endothelial cells following their stimulation. PAF also induces the adhesion and aggregation of platelets and stimulates the secretion of EDRF.
- Von Willebrand factor (VWF) which is mostly synthesized by endothelial cells and megakaryocytes. It mediates the adhesion of platelets to subendothelial connective tissue.
- Prostacyclin and EDRF. Both substances are the most potent inhibitors of platelet aggregation and have a vasodilatory effect.
Endothelial cells exert an antiaggregatory effect by releasing enzymes such as, for example, nucleotidases. ATP originating from platelets or endothelial cells is catabolized into ADP by these nucleotidases with subsequent further breakdown into AMP and adenosine. Adenosine thus being formed can be taken up and eliminated by endothelial cells. ADP is a potent substance triggering platelet aggregation whereas adenosine is an inhibitor of platelet activation by raising the concentration of cAMP. In addition, adenosine also causes vasodilation. Through these processes, endothelial cells contribute to the local regulation of platelet aggregation and platelet disaggregation .
Not only do endothelial cells act on platelets but, in reverse, platelets also influence the function of endothelial cells in many ways. Accordingly, serotonin and noradrenaline, after their release from stimulated platelets, may cause an isometric contraction of endothelial cells. Platelets also secrete substances such as vasopressin, ATP/ADP, PAF, and serotonin, which influence the metabolic capacity of endothelial cells, (e.g., the production of prostacyclin and EDRF) ().
Endothelial cells synthesize not only substances with a pro coagulant effect but also those with an anticoagulant effect. The release of these substances is regulated via a negative feedback mechanism.
Pro coagulant activity
Endothelial cells may exert their pro coagulant activities in different ways:
- The endothelial cells, similar to platelets, represent a surface to which coagulation factors bind, where enzyme-substrate complexes are formed and thus where complex formation of reaction partners occurs. Accordingly, prothrombinase, which is composed of F Xa, F Va and Ca2+, combines with prothrombin on the surface of endothelial cells .
- A specific receptor of the endothelial cell membrane for F IXa. The receptor-F IXa complex activates F X in the presence of F VIII .
- The F XII activator formed by the endothelial cell. It converts F XII into F XIIa which in turn may activate pre kallikrein and F XI, thus initiating the activation of the intrinsic pathway of the plasma coagulation system .
- The formation of tissue factor (TF) and its expression on the subendothelial extracellular matrix, thus enhancing the plasmatic coagulation system .
- The formation of vitronectin, an adhesive protein neutralizing heparin and heparin-like substances, thus reducing the inhibition of both F Xa and thrombin by antithrombin .
Via feedback mechanisms, endothelial cells are involved in the modulation of anticoagulant activities. The endothelial cells produce thrombomodulin, heparan sulfate, tissue factor pathway inhibitor and dermatan sulfate which carry different activities.
The membrane receptor thrombomodulin is the most important inhibitor of the coagulation system, binds thrombin with high affinity and exerts the following effects:
- Thrombin is disabled from cleaving fibrinogen and also from activating F V, F VIII and F XIII
- Thrombin-mediated aggregation of platelets will not occur if thrombin is bound to thrombomodulin
- The binding of thrombin to thrombomodulin initiates a negative feedback mechanism which leads to the inhibition of F Va and F VIIIa. After binding to thrombomodulin, thrombin changes its specificity and is capable of activating protein C which together with protein S proteolytically breaks down F Va and F VIIIa. This negative feedback mechanism is an effective regulatory system suited to locally modulate the plasmatic coagulation.
- Thrombin bound to thrombomodulin can be inhibited by antithrombin (AT).
Heparan sulfate on the surface of endothelial cells binds thrombin as well as antithrombin and, in a similar manner to heparin, accelerates the inactivation of thrombin. The binding of antithrombin to the endothelial cell membrane, as facilitated by heparan sulfate, may be inhibited by platelet factor 4 (PF4) which is released by platelets, because PF4 competes with heparan sulfate for binding to antithrombin.
The tissue factor pathway inhibitor (TFPI) is synthesized by endothelial cells and continuously released into the plasma. Together with F Xa and F VIIa/TF, it forms a quaternary complex and inhibits the extrinsic pathway of activation. Heparin injections lead to significant rises in plasma TFPI .
Dermatan sulfate expressed on the surface of endothelial cells accelerates thrombin inhibition by heparin cofactor II.
The TF, a transmembrane cell receptor with a MW of 47 kDa, is the activator of the extrinsic coagulation pathway. TF is activated by phosphatidylserine exposed in vascular injury and binds the activated F VII (F VIIa). The TF/FVIIa complex activates coagulation by proteolysis of F X and F IX. Relevant amounts of TF are only expressed by activated (and not by non-activated) platelets, endothelial cells and monocytes/macrophages.
- In hemostasis the platelets adhere to the injured blood vessel wall, form platelet aggregates and thus stop the bleeding
- In pro coagulation the platelets participate in the formation of a platelet/fibrin clot to seal the damaged vessel wall
- In the inhibition of fibrinolysis. Activated platelets release the plasminogen activator inhibitor type 1 and thus participate in the inhibition of fibrinolysis around the area of the fibrin clot.
GP Ib/IX/V is the receptor for the VWF. A reduced amount of functional receptors (e.g., in uremia) leads to reduced adhesion of platelets to the sub endothelium.
GP IIb/IIIa (αIIbβ3-integrin), is the receptor for fibrinogen. A modified conformation of the receptor (e.g., in uremia) results in failure of the platelets to aggregate.
GP VI and α2β1-integrin, are the receptors for collagen.
Platelets play an important role in hemostasis and are the first to respond to vessel injury. Activation of platelets results in multiple subpopulations:
- Platelets with expression of the active αIIbβ3-integrin and a lack of phosphatidylserine (PS) exposure. These aggregating, or spread, platelets mediate clot retraction.
- PS exposing platelets with characteristic baloon shape.They contain increased cytosolic Ca2+ and enhanced ability to bind coagulation factors and promote thrombin generation. A subset of PS exposing platelets are coated with several procoagulant α-granule proteins on their surface, such as fibrinogen, FV and von Willebrand factor.
The susceptibility of a thrombus to fibrinolysis is influenced by the platelet count and fibrin structure. Platelets anchor to fibrinogen via the αIIbβ3-integrin; this binding interaction destabilizes the forming thrombus and initiates the process of clot retraction.
The hemostatic function of the platelets is to stop bleeding following tissue injury and terminate exposure of the sub endothelium. The line between these physiological functions and abnormal thrombosis is narrow. Thus, platelets contribute to the formation of atherothrombosis, a leading cause of mortality.
The degree to which the platelets contribute to fatal events by clot formation depends on the location of the clots in the circulation:
- Venous thrombosis is mostly based on the activation of plasma components inducing a pro coagulant status
- In arterial thrombosis, platelets play an important part in the vascular occlusion process in the presence of atheromatous plaques.
The continuum of platelet functions in hemostasis is differentiated into the initiation, extension and consolidation phases.
Following vascular injury, circulating platelets and F VIII bound to von Willebrand factor (VWF) multimers are marginated. The initial contact of the platelets and their binding to the sub endothelium of the blood vessel are mediated by collagen fibrils which bind and activate VWF molecules. Interaction between the platelet receptor GP Ib/IX/V and the VWF bound to subendothelial collagen fibrils leads to the adhesion and activation of the platelets. In the presence of high local VWF concentration, the VWF also binds to the platelet receptor αIIbβ3-integrin, which is the physiological binding site for fibrinogen. The platelets aggregate to form a clot at the site of injury. Supported by vasoconstriction, the clot formation impedes blood loss, a procedure also referred to as primary hemostasis.
The GP Ib/IX/V complex, the complex αIIbβ3-integrin and the TF are important high-affinity platelet receptors that initiate and extend plasmatic coagulation. GP VI is a specific low-affinity receptor that has potent signalling capacity.
The central event in the platelet aggregation is the binding of fibrinogen to GP IIb/IIIa. This represents the mechanism by which platelet-to-platelet interaction is mediated.
In contrast to platelet adhesion in the initiation phase, platelet aggregation only takes place if prior platelet activation and cell membrane alteration have occurred, (e.g., organization of the complex αIIbβ3-integrin has taken place). Because of its special molecular structure as a double molecule, fibrinogen is predestined for exerting a bridging effect between complexes αIIbβ3-integrin of two platelets.
The formation of a platelet aggregate at the site of vascular injury mediated by collagen and the exposed VWF is followed by clot formation with further platelets recruited from the bloodstream. The synthesis of small amounts of thrombin at the site of injury initiates further platelet activation and triggers a highly pro coagulatory state of the platelets. The highly active platelets, also referred to as coat-plts (collagen and thrombin activated platelets), are loaded with coagulation factors and have a high granule content. They are capable of attaching to each other, a process referred to as platelet aggregation. This process is activated by the coat-plts releasing soluble agonists such as ADP, thromboxane A2 ,and adrenalin or binding agonists such as thrombin. Thus, platelet adhesion is enhanced.
The clot consolidation phase is the last phase in the coagulation cascade. In this phase, signals are generated by integrins after binding fibrinogen. These signals trigger events promoting the growth and consolidation of the clot, such as the reorganization of the cytoskeleton, formation and stabilization of large platelet aggregates, development of a pro coagulatory surface and clot retraction. Thus, the inter platelet space is consolidated and the local concentration of platelet activators is increased.
The pro coagulatory function of the platelets is the ability to provide the primary surface for thrombin formation . This allows the generation of fibrin and enables effective hemostasis. Thrombin also activates the platelets.
Small amounts of thrombin are immediately produced at sites of vascular injury with exposed subendothelial structures (). During the coagulation initiation phase, F VII binds to platelet TF and is rapidly activated to F VIIa. The F VIIa/TF complex catalyzes the formation of F IIa, the activation of F X to F Xa and of F IX to F IXa. The activity of F Xa is restricted to TF containing cells because once dissociated from the platelet surface fluid-phase F Xa is immediately inhibited by the TF pathway inhibitor and by antithrombin. This does not apply to F IXa.
The two factors have different functions:
- F Xa remains bound to platelet TF and interacts with F Va forming the pro thrombokinase complex (F Xa/F Va) that generates small amounts of prothrombin on platelets
- F IXa does not remain bound to TF, but passes on to the surface of other activated platelets, where it binds to a specific platelet receptor and interacts with F VIIIa forming the tenase complex (F IXa/F VIIIa) that activates F X on the platelet surface.
Small amounts of thrombin also induce the activation of platelet-bound F XI to F XIa and enhance thrombin formation by activation of F IX to F IXa. Factor IXa passes on to other platelets and contributes to the formation of tenase complexes on the platelet surface.
Large amounts of thrombin required for effective hemostasis are generated by association of F Xa, which is produced by platelet-bound tenase complex, (F VIIa, F IXa, Ca2+, phospholipids) and F Va to form the pro thrombokinase complex (F IIA, F VIIa; F IXa, F Xa).
Thrombin formation is not limited to platelets but also occurs on other cells, for example on activated cells of the vessel wall.
Thrombin is a potent platelet activator capable of inducing a whole range of platelet functions such as modified structure, TxA2 synthesis, Ca2+ mobilization, protein phosphorylation and platelet aggregation.
The conversion of activated platelets into a pro coagulatory state is associated with specific biochemical and morphological changes. These changes are comparable with those of apoptotic cells and comprise the activation of caspase, proteolysis of the cytoskeleton, exposure of phosphatidylserine (PS) on the membrane surface, contraction of the plasma membrane, membrane blebbing and micro vesiculation. It is assumed that exposure of PS that is trans located from the platelets onto the cell membrane leads to functional conversion to a pro coagulatory surface.
- Ca2+-dependent, caspase-independent pathways induced by agonists of the platelet function
- Bak/Bax-caspase-mediated pathways independent of platelet activation. Bak and Bax are essential mediators of the intrinsic pathway of cellular apoptosis. Caspases are cysteinyl aspartate specific proteases and important apoptotic enzymes. Caspases are subdivided into initiator caspases (caspase 8, 9) that contribute to triggering apoptosis and effector caspases (caspase 3, 7, 6) that cleave cellular proteins.
A thrombus builds up gradually and can at any time stop growing and become consolidated. EDRF and prostacyclin play an important role in the negative regulation of the platelets. Platelet activation can also be inhibited by the adhesion molecule PECAM-1 (CD31), a potent inhibitor of thrombus formation.
PECAM inhibits the effect of:
- GP VI and GP Ib/IX/V complex, proteins involved in thrombin-mediated platelet activation
- Integrin αIIbβ3 (GP IIb/IIIa complex) that mediates platelet inside-out signalling.
Following platelet activation, the contents of the storage granules are secreted into the environment within 10–120 sec. Platelets contain the following morphologically different storage granules:
- ∂-granules secreting ADP, ATP and serotonin
- α-granules secreting VWF, thromboglobulin, platelet factor 4, fibrinogen, high-molecular-weight kininogen, F V, thrombospondin and cellular growth factors such as PDGF, EGF, TGF-β
- Lysosomes excreting acidic hydrolases.
Platelets and endothelial cells synthesize eicosanoids in response to various stimuli. Stimuli for eicosanoid synthesis are the adhesion of platelets to collagen and the effect of agonists such as thrombin bound platelets and/or endothelial cells. Eicosanoids are oxygenated derivatives of arachidonic acid which contain 20 C-atoms. Eicosanoid synthesis is initiated by the release of arachidonic acid. Under the influence of cyclooxygenase, prostaglandin-endoperoxides are synthesized from arachidonic acid. These prostaglandin- endoperoxides are in subsequent metabolic reactions transformed into prostaglandins, thromboxanes or leukotrienes. Thromboxane and prostacyclin play important counterpart roles in the interaction between endothelial cells and platelets. Attempts have been undertaken to pharmacologically benefit from the differences between these two arachidonic acid derivatives. Aspirin alkylates a reactive serine within the cyclooxygenase, thus irreversibly inactivating this enzyme. Since platelets do not have a nucleus, they are unable to synthesize new cyclooxygenase; therefore, following treatment with aspirin, there is no synthesis of thromboxane and PGI2. Endothelial cells, however, which preferably synthesize prostacyclin, are capable of producing cyclooxygenase again within a few hours after the intake of aspirin. Low-dose aspirin administration, therefore, makes sense because it blocks the synthesis of thromboxane within the platelets and at the same time allows the biosynthesis of prostacyclin within the endothelial cells to restart relatively fast.
- The cascade model that contains two pathways (extrinsic and intrinsic pathway) acting independently of each other, involving a series of proteolytic reactions that converge to a common point of thrombin generation and fibrin deposition that result from FXa activation of prothrombin ().
- The alternative or cell based model. The model is based on the non-redundant function of thrombin generated on the surface of TF bearing cells and on platelets. For the formation of fibrin clots, it is essential that these thrombin generating reactions are localized to cells at the site of injury which is not accounted for in the cascade model or factor based assays. The cell based model is physiologically relevant as it recognizes the cells are pivotal to regulating the temporal and spatial activity of the coagulation proteins. This model highlights that assessment of thrombin generation rather than individual clotting factors, would provide a more complete picture of the blood clotting process in vivo.
Plasma coagulation factors are glycoproteins characterized by different levels and half-lives:
- The activated forms of factors II (prothrombin), VII, IX, X, XI and XII are serine proteases. They are present in blood as proenzymes and are transformed into their active form during the sequence of interactions in the formation of fibrin
- The activated factors V and VIII are not enzymes, they do, however, play an important role in the activation of the blood coagulation cascade
- The factors of the prothrombin complex (II, VII, IX and X) are synthesized by hepatocytes dependent on vitamin K. In the presence of vitamin K, postribosomal carboxylation of glutamic acid (glu) to γ-carboxyglutamic acid residues (gla) takes place. In the absence of vitamin K or in the presence of coumarin derivatives, the transformation into carboxylated proteins does not occur .
- The von Willebrand factor (VWF), also a plasma factor with a pro coagulant effect, plays a critical role in platelet adhesion; in the circulating blood, it is present as a complex with F VIII. The VWF is synthesized by endothelial cells and megakaryocytes while F VIII is produced by the sinusoidal cells of the liver .
- F XIII is considered to be one of the pro coagulant plasma factors. It is a transglutaminase which is primarily synthesized in the liver. It is responsible for cross linking of polymerized fibrin due to the development of covalent binding. Approximately 50% of the entire F XIII level in the blood is present within the cytosol of platelets .
Blood coagulation is continually ongoing on a very low level . Major activation take place when there is an injury or another stimulus which influences hemostasis. In this case the coagulation factors are measurably activated in the form of a cascade one after the other until the soluble plasma protein fibrinogen is transformed into a visible fibrinous clot. The plasma coagulation system is activated by the extrinsic pathway. The intrinsic pathway enhances the activated coagulation. The subdivision of the coagulation cascade into an intrinsic and an extrinsic activation system does not exist in vivo.
As a rule, the pro coagulatory activity of the tissue factor (TF) on the cell membrane is idle. It is activated by phospholipids in the event of vascular injury or cellular apoptosis. Phosphatidylserine of the inner cell membrane is externalized and exposed to enhance binding of TF and F VIIa as an initial coagulation complex triggering the coagulation cascade . This complex activates F X to F Xa together with F IX by proteolysis. During this process, small amounts of prothrombin are converted into thrombin. Together with F Va, F VIIIa and F IXa, an enhancing loop is formed causing significant amplification of thrombin formation ().
Significant potentiation of the initial stimulus which occurs in the event of a vessel wall injury or in case of the activation of cellular systems is caused by:
- The sequential activation of pro enzymes (inactive clotting factors) and involvement of enhancing loops
- The formation of coagulation factor complexes on endothelial cell and platelet surfaces, thus resulting in local concentrations of the individual factors which greatly exceed those observed in the plasma. The effect of F Xa is potentiated 300,000-fold by complex formation with prothrombin, F Va, phospholipids, and Ca2+.
In the event of vascular injury, the surface-sensitive coagulation factors F XII and F XI are activated by subendothelial structures, a process also referred to as contact activation. Contact activation is considered to be an enhancing loop. However, it is important to keep in mind that, according to clinical observation, only F XI deficiency but not F XII deficiency or a deficiency of pre kallikrein or high-molecular-weight kininogen results in hemorrhagic diathesis. In the intrinsic pathway, factors XI, IX, and X are activated in sequence.
Plasma coagulation is regulated by the following factors and mechanisms:
- Plasma inhibitors: serpins circulating in the blood as well as other inhibitors are responsible for the inactivation of activated coagulation factors
- Negative feedback mechanisms: parallel to the synthesis of coagulation factors with a proteolytic effect during the activation of the coagulation cascade, proteases are activated which proteolytically cleave co factors, thus down regulating the activation of the coagulation system via a negative feedback mechanism. The mechanism involving protein C is an example of this process
- Localization of the activation of the plasma coagulation system on cell surfaces: the activation of the coagulation system preferentially takes place on the surface of platelets and endothelial cells, whereby the activation of the blood coagulation cascade remains a local event
- Inhibition by coagulation end products: the circulating degradation products of fibrin and fibrinogen inhibit the polymerization of newly formed fibrin as well as the aggregation of platelets and lead to the increased release of plasminogen activators from the vascular wall
- Removal by the reticuloendothelial system: activated coagulation factors locally present in elevated concentrations are "washed out” by the blood stream and are subject to rapid removal from the circulation through the mononuclear phagocytic system in the liver and spleen.
Various inhibitors of the plasma coagulation system circulate in the plasma or are present within the platelets (). These proteinase inhibitors limit the activation of the blood coagulation system by inhibiting key factors of the enhancing loops by complex formation. They do not completely block the blood coagulation cascade. Instead, they limit the systemic activation to areas where blood coagulation i.e., bleeding cessation, is required. All inhibitors of the blood coagulation cascade are serine proteinase inhibitors (serpins).
- Antithrombin (AT)is the most important inhibitor of the plasma coagulation system. AT inhibits relatively slowly serine proteases, especially, however, F IXa, F Xa as well as thrombin (). In the presence of heparin, the rate of inhibition is significantly increased. This is the reason why AT is also referred to as heparin cofactor I. When thrombin is bound to fibrin, it can be inhibited neither by AT nor by the AT-heparin complex .
- Heparin cofactor II (HC-II). Heparin accelerates the inhibition of serine proteases by this inhibitor. In contrast to AT, HC-II does not inhibit F Xa. Dermatan sulfate is capable of increasing the inhibitory effect of HC-II but not that of AT.
- Tissue factor pathway inhibitor (TFPI), which down regulates the activation of the extrinsic pathway of the coagulation system (). TFPI is synthesized by endothelial cells and directly inhibits F Xa and forms a quarternary complex with F VIIa/TF. The inhibitory effect of TFPI can be increased 5-fold by an injection of heparin since heparin promotes the release of TFPI from the endothelial cells. Approximately 10% of the TFPI circulating in the blood is stored within the platelets and is released from them following stimulation with thrombin.
- Further inhibitors of the plasma coagulation system include α1-proteinase inhibitor (α1PI), C1-esterase inhibitor (C1-Inh) and α2-macroglobulin (α2M). α1PI and C1-Inh are involved in the inhibition of the intrinsic activation of the plasma coagulation system. α2M is considered to be a secondary inhibitor or a back-up inhibitor which is capable of inhibiting kallikrein, thrombin and plasmin.
Two important negative feedback mechanisms of the plasma coagulation system are:
- The protein C system which includes protein C, protein S, and thrombomodulin (). Protein C and protein S are synthesized in the liver dependent on vitamin K. Protein C, together with thrombin, binds to the endothelial cell membrane receptor thrombomodulin, thus becoming activated protein C (APC). APC is an enzyme which, in conjunction with protein S, cleaves F Va and F VIIIa, thereby again down regulating the “upregulated” activation of the plasma coagulation system, since protein C can only exert its activity after being activated exclusively by thrombin. The protein C system is the most important negative feedback mechanism of the plasma coagulation system.
- Thrombin, which has two different modes of action: as part of a positive feedback mechanism, it inactivates factors V, VIII, and XI; via the protein C mechanism, it inactivates F Va and F VIIIa.
Both platelets and endothelial cells represent an ideal surface for the activation of the blood coagulation system for the following reasons:
- The cells feature special receptors for the coagulation factors and express anionic phospholipids which function as binding sites for F IXa and F Xa
- Activated platelets expose receptors for F Va and F VIIIa
- Activated platelets release platelet factor 4 which neutralizes heparin, thus accounting for this additional prothrombotic effect exerted by platelets
- Non-activated, endothelial cells tend to reveal anticoagulant properties rather than pro coagulant activity. Non-activated endothelial cells express the membrane receptor thrombomodulin which inhibits thrombin and simultaneously activates protein C. Furthermore, AT which has bound to the heparan sulfate of the endothelial cell membrane may inhibit thrombin. The balance between the components with pro- and anticoagulant effects shifts, however, whenever endothelial cell function is impaired by various agonists, thus reflecting activation of the endothelial cell. Tissue thromboplastin, the strongest activator of the plasma coagulation system, may be expressed on cell surfaces following activation of the cells. In addition, on its surface, the activated endothelial cell provides phospholipids and binding sites to F IXa and F Xa, thus allowing the formation of activated coagulation factor complexes similar to the process observed in platelets.
Hemorrhagic diathesis and thrombophilia are the most significant forms of deregulation of the plasma coagulation system.
Hemmorhagic disorders may result from a decrease in the activity of fibrinogen, prothrombin, factors VII, IX, X, XI and XIII as well as factors V and VIII. Deficiencies in F XII, prekallikrein, and high-molecular-weight kininogen, however, do not cause hemorrhagic diathesis.
A thrombotic risk is associated with both an increase in F VIIa activity and elevated concentrations of fibrinogen and F VIII. The expression of tissue factor in many disease states has been recognized as the cause of thrombophilia. The negative feedback mechanism via the protein C system is of utmost importance for the modulation of hemostasis (). Patients with deficiencies of protein C, protein S or thrombomodulin are afflicted by thrombophilia. APC resistance refers to the inability of activated protein C (APC) to cleave its substrates F Va and F VIIIa. This is caused either by a mutation of the substrates , or APC by circumstances under which APC cannot reach the substrate due to inhibition by antibodies (e.g., as seen in the presence of lupus anticoagulant) .
Fibrinogen is the plasma protein with the highest plasma concentration in the coagulation system and plays a key role in the hemostatic system. Thrombin-catalyzed cleavage of fibrinopeptides A and B converts fibrinogen into fibrin, which spontaneously polymerizes and forms double stranded protofibrils that assemble into branched fibrin fibers, forming the fibrin clot. Low fibrinogen concentrations are associated with bleeding and high ones are associated with thrombosis . The conversion of fibrinogen to fibrin is shown in .
In two reactions which take place parallel to each other, thrombin initially cleaves off fibrinopeptide A from the fibrinogen molecule. A fibrin monomer remains which, due to a change in configuration, is able to polymerize with other fibrin monomers and, in the same way, also with fibrinogen. The fibrinopeptides are only cleaved off if desAA-fibrin has polymerized. The cleavage of fibrinopeptide B is not essential for the development of a fibrin clot.
Several fibrin monomers form protofibrils by polymerization which attach to each other by lateral association.
In the presence of high concentrations of fibrinogen or fibrin degradation products, the fibrin polymerization is delayed or even inhibited, thus accounting for the fact that fibrin does not polymerize into a fibrin clot. Fibrin which remains in solution within the plasma is referred to as soluble fibrin (). Soluble fibrin is composed of fibrin oligomers, which do not polymerize into a fibrin clot since fibrinogen and/or fibrin degradation products block the fibrin polymerization sites . In the case of limited proteolysis, soluble fibrin in the plasma represents an intermediary product in the formation of a fibrin clot .
Fibrin cross linking
A fibrin clot but also soluble fibrin may be crosslinked due to the action of F XIIIa. The cross linking process represents a transpeptidation (i.e., a covalent binding, between two γ-chains or two α-chains of adjacent fibrinogen or fibrin molecules). The covalent binding is an ε-(γ-glutamyl)-lysyl binding.
Other functions of fibrinogen
Fibrinogen is not only a component of fibrin-rich clots but is also involved in various other biological reactions as an adhesive molecule. Fibrinogen interacts with platelets, endothelial cells, macrophages and fibroblasts. During the process of platelet aggregation, platelets are linked to each other primarily by fibrinogen which binds to the glycoproteins of any two adjacent platelets. Endothelial cells also have a fibrinogen receptor which binds these cells on their contra luminal side to the extracellular matrix. In addition, fibrinogen has an impact on the agglutination of red blood cells and is involved in the process whereby bacteria and malignant cells adhere to cell surfaces and to the extracellular matrix.
If fibrin is formed in the intravascular or extravascular space as part of bleeding arrest or as part of intravascular coagulation or inflammatory reaction, it is degraded by proteolysis after having fulfilled its physiological function. Under these physiological conditions, this is referred to as fibrinolysis whereas the dissolution of a thrombus under pathological conditions is called thrombolysis. Fibrin can also be eliminated by cellular degradation and phagocytosis.
Fibrin is split by plasmin into fibrin degradation products (FDP). If plasmin is present in high concentrations, it may also transform fibrinogen into FDP. Different FDPs are formed depending on whether plasmin splits fibrin crosslinked or not crosslinked by F XIIIa, fibrinogen or soluble fibrin ().
FDP have anticoagulant properties due to the fact that they inhibit:
- Polymerization of fibrin by blocking the polymerization sites on the fibrin molecule, thus acting as a solvent mediator for soluble fibrin
- Platelet aggregation since they interfere with the binding of fibrinogen to platelets.
Fibrinolysis leads to the breakdown of:
- Fibrinogen into two D-fragments each plus one E-fragment since the symmetrical fibrinogen molecule is composed of two terminal D-domains and one central E-domain. These fragments are also referred to as D-dimers.
- Crosslinked fibrin into fragments of varying composition by plasmin. In the circulation only FDPs but no D-dimers are detectable; these degradation products are composed of crosslinked Y-fragments and D-domains, thus making Y-D the smallest crosslinked unit detectable in the plasma ().
Fibrinolysis is an essential physiological mechanism. The fibrinolytic system shares similarities with the coagulation system and like in coagulation requires activation steps for pro enzymes and cofactor functions. The central enzyme is plasminogen, a precursor of the serine protease plasmin.
There are two major pathways which trigger the activation of fibrinolysis:
- The clot formation, a fibrin dependent step
- The release of plasminogen activator inhibitor 1 (PAI-1) from activated platelets.
Fibrinogen has two important functions in hemostasis:
- It degrades fibrin-rich clots after they have fulfilled their physiological function
- It limits clot formation.
Consequently, this implies that fibrinolysis is involved in the wound healing process and recanalization of blood vessels obstructed by thrombus.
The intrinsic activation pathway of fibrinolysis involves the factors of the so-called contact activation, F XIIa, pre kallikrein and high-molecular-weight kininogen (HK). The components of the fibrinolytic system are listed in .
Plasminogen is synthesized by hepatocytes and in its native form has glutamic acid at its N-terminal end, thus also being referred to as Glu-plasminogen. After the activation of Glu-plasminogen by plasmin, Lys-plasminogen with an N-terminal lysine is produced initially . The cleavage of Glu-plasminogen into Lys-plasminogen results in a change in configuration, rendering Lys-plasminogen more susceptible to activation by plasminogen activators. The degradation of fibrin by plasmin enhances the binding of Glu-plasminogen to fibrin, thereby accelerating the thrombolytic process (positive feedback mechanism). By this process, thrombolysis becomes localized to the site of fibrin synthesis. Plasminogen binds to fibrin, α2-antiplasmin, histidine-rich glycoprotein, thrombospondin, tetranectin, and the extracellular matrix via a region referred to as cringle structure.
Endothelial cells are the primary site of synthesis for t-PA. In addition, t-PA is synthesized by mesothelial cells, megakaryocytes and monocytes. In plasma, t-PA is present as a complex with plasminogen activator inhibitor type 1 (PAI-1); therefore, the concentration of free t-PA in plasma is only 20%. The half-life of t-PA is 4 min., which is similar to that of PAI-1.
Fibrin binds t-PA with high affinity. If fibrin is crosslinked by F XIIIa, the ability of t-PA to bind to fibrin is impaired. The explanation for this is that the binding site for t-PA on fibrin is not accessible under these circumstances or the binding site for t-PA is sterically blocked by the cross linking of α2-antiplasmin to fibrin.
Urokinase cleaves plasminogen at the same site as t-PA. Urokinase, also referred to as urinary-type plasminogen activator (u-PA), is synthesized in the epithelial cells of the renal tubules and eliminated by urinary excretion. Urokinase is also synthesized by monocytes. Endothelial cells are capable of producing the precursor of urokinase known as pro-urokinase. If endothelial cells are stimulated, u-PA is secreted preferentially on the contra luminal side while t-PA secretion occurs on the luminal side.
Urokinase is produced from pro-urokinase which is also referred to as single-chain u-PA (scu-PA). Minimal concentrations of plasmin are sufficient to transform the single-chain molecule into a two-chain molecule or so-called two-chain u-PA (tcu-PA), of which only the latter is in fact urokinase. The half-life of u-PA is 5–10 min.
Under physiological conditions, continuous activation of fibrinolysis takes place since fibrinolytic degradation products are detectable also under resting conditions. An intrinsic and an extrinsic activation pathway are differentiated ().
Intrinsic activation pathway
Extrinsic activation pathway
t-PA is continuously secreted at a basal rate in the extrinsic activation pathway. In the event of hypoxia or following neurohumoral stimulation, t-PA is released at an increased rate. Even strenuous physical exercise or venous compression will lead to the release of t-PA from endothelial cells. Glu-plasminogen, the native plasminogen, is transformed auto catalytically or by trace amounts of plasmin into Lys-plasminogen which has an increased affinity for fibrin and which is rapidly transformed further by t-PA in the presence of fibrin.
Urokinase is a second, important activator of the extrinsic fibrinolytic system. Because of the fact that only scu-PA is detectable in the plasma, it is assumed that t-PA and pro-urokinase complement each other in the activation of fibrinolysis. Although urokinase does not bind to fibrin, the activation of pro-urokinase to urokinase occurs much faster in the presence of fibrin. During basal fibrinolysis, minimal amounts of plasmin are possibly produced via t-PA; plasmin transforms pro-urokinase into urokinase. The activation of the contact system leads to the biosynthesis of kallikrein which is a very potent activator of pro-urokinase. The catalytic activity of urokinase is significantly increased when urokinase binds to the u-PA receptor which is detectable on many cells including, for example, endothelial cells and monocytes. Urokinase is involved in the fibrinolysis of fibrin clots since monocytes penetrate fibrin-rich clots and contribute to thrombolysis.
The regulation of fibrinolysis reveals three characteristic features:
- There is continuous activation of the fibrinolytic system, thus resulting in basal fibrinolysis
- Beyond basal fibrinolysis, the fibrinolytic system can only be slowly activated
- If the inhibitors of fibrinolysis are neutralized, activation of the fibrinolytic pathway begins very rapidly and strongly.
Similarly to other biological systems, the fibrinolytic system is subject to regulation and under physiological conditions is in a dynamic steady state. The continuous activation of fibrinolysis is associated with the formation of plasmin.
Fibrinolysis regulation is also suggested by the observations that:
- Patients with congenital α2-antiplasmin deficiency clinically present with a predisposition to hemorrhagic diathesis
- A disorder of fibrinolysis such as seen, for example, in dysfibrinogenemia-type thrombophilia results in elevated rates of thrombotic episodes.
Systematic examinations allow the conclusion that plasma coagulation and fibrinolysis do not take place systemically and that both are activated locally.
Several mechanisms and systems are involved in the regulation of fibrinolysis:
- The biosynthesis of t-PA is regulated
- Inhibitors circulating in the plasma induce an inhibition of fibrinolysis, thus contributing to its regulation
- Systemic activation is prevented by the localization of fibrinolysis
- Fibrinolysis is modulated by links existing between the fibrinolytic system and activation of the plasma coagulation system as well as the aggregation of platelets.
Fibrinolytic activators locally present in elevated concentrations are little effective because they are “washed out” by the blood stream and are subsequently removed from the circulation by the mononuclear phagocytic system in the liver and spleen.
Regulation of t-PA synthesis
The biosynthesis of t-PA is subject to a circadian rhythm and steroid hormones are involved in the regulation of t-PA synthesis. Stimuli and substances influencing the synthesis and activity of t-PA are:
- Intravenously administered acetylcholine which leads to a rise in fibrinolytic activity
- Epinephrine which releases t-PA from endothelial cells
- Bradykinin, whose production results from the cleavage of high-molecular-weight kininogen during the activation of the contact phase and which represents a potent vasodilator, also releases t-PA from endothelial cells.
In contrast to these stimulants, cAMP causes down regulation of the t-PA synthesis within the endothelial cells.
Modulation of the activation of fibrinolysis
Under physiological conditions, t-PA is a weak activator of plasminogen, as long as both components are not bound to fibrin. Even a 20–100-fold increase in the concentration of t-PA (e.g., following strenuous physical activity) does not lead to a systemic activation of fibrinolysis. The formation of free plasmin with subsequent fibrinolysis will not occur unless there is an excessive, 1,000–5,000-fold rise in the t-PA concentration, as is seen, for example, under therapeutic conditions. Since the body aims to prevent intravascular fibrin formation, t-PA and plasminogen are quickly bound in the event of fibrin production in order to generate plasmin for the degradation of fibrin. The resulting fibrin degradation products limit the further production of fibrin by inhibiting the polymerization of fibrin as well as the aggregation of platelets ().
PAI-1 causes additional modulation of fibrinolysis activation. PAI-1 prevents the activation of plasminogen in the circulation due to its molecular excess in contrast to the activators of plasminogen. The concentration of PAI-1 and thus the inhibition of local fibrinolysis in close proximity to a platelet fibrin clot can be increased since approximately 80% of the PAI-1 contained in the blood is stored within platelets which release it when they are activated. Thus, the degradation of the blood clot which is necessary for the wound closure can be prevented.
Regulation of fibrinolysis by inhibitors
The regulation of fibrinolysis by inhibitors takes place at several levels. Only about 50% of the plasminogen circulating in the blood are available for transformation into plasmin; the remaining portion is bound to histidine-rich glycoprotein and can only be released slowly for activation.
If activation of fibrinolysis occurs within the fluid phase, plasmin is quickly inhibited by α2-antiplasmin (α2-AP) since the binding of plasmin and t-PA to fibrinogen is minimal. If, however, fibrin is present, the affinities of the components of the fibrinolytic system to each other change. Plasminogen and t-PA bind to fibrin, allowing plasmin to be produced in minimal quantities. During the next step, plasmin activates pro-urokinase to urokinase and initiates further activation of fibrinolysis.
α2-AP may be bound to fibrin by F XIIIa thus allowing it to be concentrated. α2-AP bound to fibrin is an effective inhibitor of plasmin and contributes to the maintenance of the fibrin clot.
Localization of fibrinolysis
Plasmin which is bound to fibrin can only be partially inhibited by α2-AP thus maintaining some fibrinolytic activity at the site of the fibrin clot. On the other hand, free plasmin can still be inhibited if α2-AP is bound covalently to fibrin by F XIIIa.
Free plasmin released during the degradation of the fibrin-rich clot can effectively be inhibited in the circulation by α2-AP, thus allowing fibrinolysis to remain focussed on the local area of the fibrin clot. Further localization of fibrinolysis is mediated by the platelets which release PAI-1 during the coagulation process.
The different regulation systems guarantee the localization of the fibrinolytic process to the fibrin clot and stabilization of the clot by inhibiting fibrinolysis. Due to this mechanism, fibrinolysis is initiated locally and only with a delay, allowing on the one hand fibrin to fulfill its function as a vascular occluding agent and as a component in inflammatory reactions and on the other hand to be slowly broken down again.
Release of t-PA and activation of the blood coagulation cascade
Thrombin increases the release of t-PA from endothelial cells. The concentration of t-PA in the circulation can be raised even more significantly by an injection of F Xa. The potentiation of plasmin formation after the binding of t-PA to fibrin leads to plasmin being formed at the site of its substrate (i.e., fibrinolysis is achieved very specifically and locally). Plasmin breaks down fibrin into fibrin degradation products (FDPs). If plasmin is present in a high concentration, it can also convert fibrinogen into FDP.
In classic thrombolytic therapy with urokinase or streptokinase, FDPs are preferentially produced.
Clearance by the reticulo-endothelial system
Activated components of the fibrinolytic system as well as enzyme-inhibitor complexes are removed from the circulation by the reticuloendothelial system with various half-lives. If the circulation or the hepatosplenic function are impaired, fibrinolytic activity remains elevated, as is typically observed in severe liver disease.
If the balance between activators and inhibitors is disturbed within the fibrinolytic system, either thrombophilia or a predisposition to bleeding may ensue.
In severe liver disease, the inhibitors α2AP and histidine-rich glycoprotein are decreased. The inhibitor PAI-1 evidently is not sufficient to adequately compensate for the pro fibrinolytic effect of t-PA. In orthotopic liver transplantation, massive hyper fibrinolysis occurs during the anhepatic phase. The lack of fibrinolysis inhibitors after removal of the liver is most likely the explanation for these findings. Systemic activation of fibrinolysis is also observed in conjunction with surgery involving organs rich in pro fibrinolytic components, such as the lung and the liver.
In the absence of fibrin, t-PA is capable of only slowly activating plasminogen. In the presence of fibrin, the catalytic activity increases about 1,000-fold, involving the formation of a ternary complex between t-PA, plasminogen and fibrin. If the binding sites for plasminogen or t-PA are absent or defective due to a mutation (e.g., as in dysfibrinogenemia-type thrombophilia) the fibrinolytic system cannot effectively be activated. These patients have an increased risk of thrombosis.
The posttraumatic or postoperative reduction in the fibrinolytic activity is probably mediated by the increased synthesis of PAI-1 which occurs as a result of increased cytokine release following tissue injury. The reduction of fibrinolytic activity may possibly represent a protection against tissue dissolution of a fresh hemostatic vascular occlusion. At the same time, it also explains, however, the increased occurrence of postoperative venous thrombosis.
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6. Brass LF. The biochemistry of platelet activation. In: Hoffman R, Benz jr EJ, Shattil SJ, Furie B, Cohen HJ (eds). Hematology. Basic principles and practice. New York: Churchill Livingstone, 1991: 1176–97.
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22. Pötzsch B, Kawamura H, Preissner KT, Schmidt M, Seelig C, Müller-Berghaus G. Thrombophilia in patients with lupus anticoagulant correlates with impaired anticoagulant activity of activated protein C but not with decreased thrombomodulin activity. J Lab Clin Med 1995; 125: 56–65.
28. Declerck PJ, de Mol M, Alessi MC, Baudner S, Pâques EP, Preissner KT, et al. Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. Identification as a multimeric form of S protein (vitronectin). J Biol Chem 1988; 263: 15454–61.
Hans D. Bruhn, Lothar Thomas
- The blood-clotting system leading to coagulation disorders
- The fibrinolytic pathway causing hyper fibrinolysis-induced hemorrhagic disorders
- The platelet count (thrombocytopenia) and the function of platelets (thrombocytopathy): in both cases, the hemostatic system may be dysfunctional causing bleeding predisposition
- The blood vessel wall causing vascular disorders and bleeding predisposition.
Diagnostic investigations of hemorrhagic disorders must detect the various possible causes of a bleeding predisposition. From a differential diagnostic point of view, the following clinical observations must be emphasized:
- Local or widespread hemorrhages (hematomas, suffusions) occur in conjunction with coagulation disorders
- Mucocutaneous petechial hemorrhages (purpura) are associated with thrombocytopenia and thrombocytopathy
- Hemarthroses occur mostly in conjunction with the two coagulation disorders hemophilia A and hemophilia B and are rather rare in thrombocytopenia.
In many cases, the clinical impression provides important information regarding the cause of a bleeding tendency and diagnostic investigations allow differential diagnostic conclusions and final diagnosis.
It is important in connection with a clinically manifested predisposition to bleeding to selectively take the patient’s history. Detailed questions in the anamnesis of bleeding should provide information as to whether predisposition to bleeding is familial, as in most hemophilias, or whether it is only the patient who is afflicted. It is also important to know whether the patient recently developed a bleeding tendency, for example under the influence of drugs, and had not presented with any such symptoms in previous years .
Disorders in the synthesis of blood coagulation factors are based on congenital or acquired defects which cause hematomas, suffusions, hemarthroses, deep hematomas, hematuria and bleeding into body cavities.
Coagulation disorders are subdivided into:
- Type I: a coagulation factor is not synthesized or synthesized at a reduced rate because of complete or almost complete deletion of the gene. Functional blood coagulation tests and immunochemical assays indicate the same degree of reduction.
- Type II: a variant of the affected coagulation factor is produced with an absent or altered function caused by a minor alteration in the gene. Immunochemical assays indicate low level and possibly even normal level results in comparison to functional tests.
Prevalence: 1 in 8,000 individuals; hemophilia A, hemophilia B and the von Willebrand disease make up 94% of these cases.
- Absent or decreased F VIII protein
- Qualitative disorders where F VIII protein concentration is relative high in relation to the clotting activity.
Hemophilia A and B are inherited in an X-linked recessive pattern or occur spontaneously. The absence or a lack of activity of F VIII:C (hemophilia A) or F IX (hemophilia B) result in hemorrhagic disorders which are characterized by a prolongation of the activated partial thromboplastin time (aPTT) in conjunction with a normal prothrombin time (PT) and a normal bleeding time. The half-life time in plasma for F VIII is 10–12 hours and for F IX 16–18 hours .
The incidence of X-linked hemophilia A is 1 in 5000 males. The incidence of hemophilia B is 5-fold lower.
Hemophilia A can be caused by a multitude of genetic defects. Defects in the gene F8C have been identified. The most frequent mutations in F8C are intron 22 and intron 1 inversions, which occur in 50% and 30% of patients, respectively . The X-chromosomal mode of inheritance in hemophilia A is the reason why only men are clinically affected by the disease whereas women, as carriers, transmit the gene to their offspring. In the differential diagnosis, distinction must be made between hemophilia A and von Willebrand syndrome which in addition leads to a disorder in platelet adhesion and aggregation.
The analysis of individual factors (using plasma deficient in F VIII) allows the determination of the coagulation activity of F VIII (F VIII:C).
An overview of the desirable F VIII:C levels in the event of various types of bleeding is presented in . Specific treatment consists of substituting the missing coagulation factor. The F VIII target concentration and the duration of therapy must be adapted to the given situation. As a rule of thumb for dose calculation, 1 unit of F VIII concentrate/kg body weight increases the F VIII plasma level by 2%. The maximum F VIII concentration is reached immediately after injection
The decrease in F VIII activity is bi-exponential with a short initial half-life of 4–6 h and a second phase with a half-life of 14 h. Mean half-life is 12 h. The F VIII therapy dose should be administered at intervals of 8–12 h. In their consensus recommendations, the German Hemophilia Society recommends a mean initial dose of 20–40 units/kg body weight in hemarthrosis and muscular hemorrhage in patients with severe hemophilia A and 50–70 units/kg body weight in severe to life-threatening hemorrhage. For maintenance therapy, half of the initial dose is administered every 8–12 h until cessation of bleeding, depending on the type of bleeding disorder and the response to therapy.
According to some authors, the initial dose requirement is calculated based on the formula (Dose requirement = A × G × F), with A indicating the desired increase in units, G indicating the body weight in kilograms and F representing a factor of 0.6 in hemophilia A and 1.0 in hemophilia B. Monitoring of the plasma levels of F VIII or F IX or the aPTT are necessary during therapy.
Detection of female gene carriers: Daughters of hemophiliacs (hemophilia A) and mothers of more than one hemophilic son are obligatory hemophilia gene carriers. In order to detect these carriers, the ratio between functional and immunochemical factor determination is used. The ratio of F VIII:C/F VIII R:Ag may decrease to 0.5 in female gene carriers. The diagnosis can also be verified by molecular biological tests.
Hemophilia B is a disease with an X-chromosomal recessive mode of inheritance. The diagnosis is based on a prolonged aPTT and the subsequent individual factor analysis. In this test, plasma deficient in F IX is supplemented by patient plasma and the aPTT measured.
In the event of bleeding, F IX substitution is performed employing the same dose as in hemophilia A although the different half-lives result in dissimilar dose intervals. Prophylactic F IX treatment in hemophilia B consists of administering F IX at a dose of 18 units/kg body weight once a week. As a rule of thumb for dose calculation in hemophilia B, 1 unit of F IX concentrate/kg body weight increases the F IX concentration by 1%. The maximum F IX concentration is reached immediately after injection. Contrary to F VIII, F IX has a longer half-life (17 h); therefore, dose intervals are 12–24 h.
Specific inhibitors of coagulation factors are a rare phenomenon associated with a high risk of bleeding.
F VIII antibodies
F VIII antibodies occur as allo- and autoantibodies. In patients with severe hemophilia, F VIII alloantibodies lead to complete inhibition of the F VIII activity; there is a linear correlation between antibody concentration and the logarithm of residual F VIII activity. Even in the presence of high levels, autoantibodies do not completely block the F VIII activity.
In patients with hemophilia A, F VIII is inhibited by alloantibodies and very rarely by autoantibodies. The risk of inhibitor formation is 20–30%; this type of complication can already occur after an average of 10–15 days of therapy . In rare cases, inhibitors may develop in patients after long years of therapy. The development of an inhibitor compromises the ability to effectively manage hemorrhage because the plasma F VIII concentration does not increase following the infusion of F VIII concentrate.
The incidence of clinically relevant F VIII autoantibodies is 1 to 1 million. The risk of bleeding in affected patients is higher than 80%. F VIII autoantibodies are detected at an increased rate during pregnancy, in rheumatoid arthritis, malignancies, drug-induced, in systemic lupus erythematosus and other autoimmune diseases.
The treatment of patients with bi-specific antibody emicizumab results in reduction of the rate in bleedings in patients with F VIII hemophilia. The antibody binds with one tethering arm to the ligand F IXa and with the other to the ligand F Xa. During this step the F IXa mediated activation of F X and formation of the tenase complex (F VIIIa, F IXa, phospholipids and Ca2+) is initiated. In this way the blood clotting cascade works without F VIII .
F IX antibodies
F IX antibodies occur as allo- and autoantibodies. F IX- alloantibodies are detected in hemophilia B patients at an incidence of 1–4%. The inhibitors are oligo- or polyclonal IgG antibodies. Autoantibodies to F IX are rarer than autoantibodies to F VIII. Their occurrence is associated with pregnancy, surgery and autoimmune disease.
Laboratory findings in hemophilia
Isolated prolongation of the aPTT is the main finding. Differentiation from genuine F VIII or F IX deficiency is achieved mixing patient plasma with normal plasma. In order to determine the presence of F VIII and/or F IX inhibitors, the diluted and undiluted patient sample is mixed with normal plasma and then analyzed to find out whether F VIII and/or F IX are inactivated by the patient plasma. A patient sample producing a residual F VIII activity of 50% of the normal value is considered to contain 1 Bethesda unit of F VIII inhibitor/mL by definition. See ).
The VWS is caused by genetic alterations in the gene VWF that lead to a decrease or qualitative defect of the VWF protein. Predisposition to bleeding results from the loss of two key functions of the VWF protein:
- Binding and consolidation of F VIII
- Binding of platelets to the subendothelial matrix.
In general, all coagulation factors of the intrinsic and extrinsic pathways may be congenitally decreased, thus resulting in a predisposition to bleeding . These deficiencies include the following: F XI, pre kallikrein (Fletcher factor), high-molecular-weight kininogen (Fitzgerald trait), F VII, F II, F X, F V, fibrinogen, fibrinogen variants (dysfibrinogenamia), F XIII, α2-antiplasmin.
Hereditary F XI deficiency
This rare disease the prevalence is 1 : 1,000,000. F XI deficiency is autosomal recessively inherited. The bleeding tendency occurs under posttraumatic or postoperative conditions. The half-life of F XI of 60–80 h must be taken into account when administering fresh frozen plasma.
The prevalence of this rare disorder is 1–2 per 1,000,000. Carriers may have umbilical cord hemorrhage in the first days of life or secondary hemorrhage following venipuncture. Fibrinogen concentrates can be used for therapy to raise fibrinogen concentration to 500–1,000 mg/L.
Hereditary F XIII deficiency
This disorder has a prevalence of 1 : 5,000,000. Major clinical symptoms include wound healing deficits and cerebral hemorrhage. F XIII concentrates are used for therapy.
Homozygous deficiency states of F II, V, VII and X may cause hemorrhagic diathesis as in hemophilia. Prothrombin concentrates are used for therapy in such cases, and fresh frozen plasma is used in F V deficiency.
In many cases, significantly prolonged aPTT points to the correct diagnosis. Subsequent individual factor analysis then reveals F XII deficiency. Acquired F XII deficiency is often based on hepatic damage, including drug-induced damage.
Contrary to other factor deficiencies, congenital F XII deficiency does not imply predisposition to bleeding but rather to thrombosis. F XII deficiency was for the first time described in the patient Hageman. It induced pulmonary embolism in the patient’s older age. Increased activity of the fibrinolytic pathway has been discussed as the underlying cause of thrombophilic tendency in patients with F XII deficiency.
Clinically, there is no bleeding tendency despite significant changes in aPTT.
Plasma pre kallikrein
Plasma deficient in pre kallikrein is suited for analysis. An amidolytic assay is also available where a pre kallikrein activator converts pre kallikrein to kallikrein. Kallikrein cleaves chomogenic tripeptide substrate releasing p-nitroaniline which is measured at 405 nm .
High molecular weight kininogen (HMWK)
Plasma deficient in HMWK is difficult to prepare. A new easier and reproducible HMWK assay was developed using a chromogenic substrate which is incubated with an excess of F XI and F XIIa in the presence of kaolin in order to form activated F XIa. The formed F XIa is measured under the selected assay prerequisites using the chromogenic substrate glu-Pro-Arg-p-nitroanilide .
α2-antiplasmin deficiency is an autosomal recessive hereditary disease of the hemophilia type that may cause severe hemorrhagic diathesis.
Autosomal dominant hereditary defects of antithrombin, heparin cofactor II, protein C and protein S cause a predisposition to the development of thromboses due to a inhibitor deficit resulting in inadequate neutralization of coagulation activation. Homozygous protein C deficiency becomes manifest immediately after the delivery of an affected newborn in the form of purpura fulminans. Factor concentrates and fresh frozen plasma are employed for the treatment. Refer to:
For disorders of hemostasis following organic diseases, refer to:
If vitamin K is deficient (e.g., in maldigestion and malabsorption) or if vitamin K antagonists (coumarins) are present, the synthesis of the factors II, VII, IX, X, protein C and protein S is incomplete. As a result of this, no carboxyl group is incorporated in the glutamic acids at the N-terminal end of the factor peptide chains. The coagulation state in vitamin K deficiency is characterized by a decrease in factors II, VII, IX and X in conjunction with normal F V activity. The activities of protein C and protein S are also reduced.
Vitamin K deficiency is observed in conjunction with total parenteral nutrition, impaired absorption, for example, malabsorption, biliary atresia, biliary fistulas and antibiotics-induced changes in the intestinal flora. For treatment, vitamin K is perorally administered in a dose of 10–20 mg; in the case of absorption problem, it is administered parenterally. The intravenous administration of vitamin K may in rare cases cause severe allergic reactions. Therefore, an accurate medical history should be obtained concerning the possibility of such an allergy.
The onset of the effect of perorally or parenterally administered vitamin K takes 36 h or longer and is measurable as clinically relevant improved hemostasis. Hence, parenteral administration of a factor concentrate is the preferred approach in acute hemorrhagic events involving prolonged, vitamin K deficiency induced prothrombin time.
Coumarins block the vitamin K dependent carboxylation of the coagulation factors II, VII, IX and X as well as protein C and protein S. Depending on their half-life, F VII and protein C decline at first, followed by a decrease in factors X, IX and II. F V remains normal.
Antibiotics can have a similar effect as coumarins by destroying the vitamin K forming intestinal flora, for example β-lactam antibiotics and/or destroying vitamin K epoxide hydrolase (cephalosporins), thus interfering with the synthesis of vitamin K dependent coagulation factors. Inhibition of the hepatic vitamin K epoxide reductase leads to a coumarin-like inhibition of the synthesis of vitamin K dependent coagulation factors.
Coumarin therapy monitoring
Therapy is usually monitored by determining the prothrombin time (PT) and calculating the INR value. The therapeutic range of the PT is defined as 15–30% of normal. A range of 35–45% is recommended in specific indications (low-dose oral anticoagulation) in non-rheumatic atrial fibrillation and long-term venous thromboprophylaxis.
Risk of bleeding under coumarin therapy
The total risk is 2–3% per year and the risk of cerebral hemorrhage is 0.2–0.4%. In hemorrhagic diathesis induced by coumarins, orally administered vitamin K only leads to slow normalization of the coagulation value. Therefore, administration of fresh frozen plasma together with a prothrombin complex concentrate is recommended.
Heparin therapy is monitored by aPTT. In the treatment of thromboses, a 1.5–2.5-fold prolongation of the aPTT is considered to be the therapeutic range. Heparin can be neutralized by protamine chloride.
Excessive heparin doses resulting in a predisposition to bleeding are possible (e.g., in conjunction with the treatment of thromboses, extra corporeal circulation, procedures requiring cardiopulmonary bypass and hemodialysis). Elevated heparin concentrations prolonging the aPTT are also measured in urticaria pigmentosa. Predisposition to bleeding and thrombosis in heparin-induced thrombocytopenia. See ).
Immunologically mediated coagulation disorders are caused by antibodies (IgG or IgM) which display two different modes of action. They either inactivate a coagulation factor or a receptor at the platelet membrane as part of a time-dependent reaction (neutralizing inhibitors) or they interfere with one of the phases of coagulation (interfering inhibitors), thus causing the clinical picture of severe hemophilia (see ).
They are found in hemophilia A, rheumatoid arthritis, lupus erythematosus, ulcerative colitis, polymyositis, bronchial asthma, monoclonal gammopathies, mycosis fungoides, pemphigus, bullous dermatitis, penicillin-related hypersensitivity angiitis and lymphomas.
Lupus anticoagulants: they are immunoglobulins directed against membrane phospholipids (e.g. found after viral or drug-induced tissue damage). These interfering inhibitors do not cause bleeding but they commonly induce arterial or venous thromboses and in women may lead to spontaneous abortion.
Disseminated intravascular coagulation (DIC) is an acquired coagulation disorder due to the systemic intravascular activation of the coagulation system along with the formation of thrombi in the microcirculation and secondary hyper fibrinolysis. This processes causes the consumption of coagulation factors and platelets and, with hyper fibrinolysis, the onset of hemorrhagic diathesis. The formation of micro thrombi within life-sustaining organs and hemorrhagic diathesis lead to organ damage and impairment of vital functions due, for example, to respiratory insufficiency (acute respiratory distress syndrome/shock lung) and renal insufficiency.
It is a form of DIC with a chronic course presenting in small infants with the triad of giant hematoma, thrombocytopenic purpura and afibrinogenemia, as a result of the stasis.
It is characterized by an extremely dramatic course and in 90% of the cases can be traced back to the presence of meningococcal sepsis. Untreated, it results in death within a few hours. Therapeutically, heparin, thrombolytics or antibiotics and, in the case of extremely pronounced adrenal insufficiency, even corticosteroids have all been employed.
This disorder is a form of DIC with symmetrical necroses involving the extremities and the trunk; it occurs following infectious diseases and is characterized by the simultaneous presence of high fever, leukocytosis, circulatory decompensation and shock.
Hereditary thrombotic thrombocytopenic purpura is a rare autosomal recessive disorder caused by ADAMTS13 mutations that result in the absence or severe deficiency of the plasma metalloprotease ADAMTS13. The protein is required for cleavage of newly synthesized von Willebrand factor multimers. The patient’s age may help to distinguish between hereditary TTP and acquired TTP. Acquired TTP is much less common in young children than in adults. The presence of a functional ADAMTS13 or an increased anti-ADAMTS13 IgG antibody titer argues against the diagnosis of of hereditary TTP .
Thrombocytopathies are disorders of platelet function that may be associated with mild hemorrhagic diathesis. However, hemostasis can be severely impaired depending on the type of platelet dysfunction and the additive effect of other, concurrently present hemostatic disorders. Congenital and acquired thrombocytopathies are distinguished.
Vascular disorders triggering hemorrhagic diathesis must be differentiated into congenital and acquired forms. A predisposition to bleeding in vascular disorders can derive from circumscribed morphological alterations involving the blood vessel walls, on changes in vascular permeability or on changes in vessel fragility.
Diagnostically, vascular hemorrhagic disorder may be present in cases with:
- Prolonged bleeding time in conjunction with a normal coagulation profile (PT, aPTT)
- Normal platelet count and function
- Normal von Willebrand factor.
The following tests can be used for differentiating vascular hemorrhagic diathesis from platelet-induced hemorrhagic diathesis.
The subaqueous bleeding time according to Marx is a screening test. In this test, the fingertip is pricked with a sterile vaccination lancet and then immersed into a cup with sterilized water (37 °C), with the blood flowing into the surrounding water like a thread. The sudden end of the thread represents the end of the bleeding time.
Upper reference interval: up to 4 min.
Clinical significance: the bleeding time comprises the reaction of the vessel wall, the number and function of the platelets and the presence of the von Willebrand factor.
To perform the test, a blood pressure cuff is applied around the patient’s upper arm and inflated to 10 mm Hg above the patient’s diastolic blood pressure for 5 min. The test is positive if clearly visible petechia are detected in the cubital fossa after removing the cuff .
Clinical significance: vascular disorder can be discussed if the number and function of the platelets are normal and analyses do not point to von Willebrand syndrome.
- Tests for primary hemostasis, comprising activation, adhesion and aggregation of platelets
- Tests for the coagulation system, and importantly familial history (pointing to congenital disorders)
- Drug history because acquired tendency to bleeding is usually induced by drugs.
The first step is to perform basic diagnostic screening assays if there is no positive bleeding history. This applies, for example to preoperative preparation: if the bleeding history is positive, further diagnostic testing is required. This also applies to all occasions when the basic test results are within the reference interval.
The proenzyme plasminogen in human blood is activated to plasmin by therapeutically administered activators such as urokinase, streptokinase or tissue-type plasminogen activator for thrombolytic therapy of arterial or venous vascular occlusions .
Tissue plasminogen activator (t-PA)
t-PA preferentially activates plasminogen which is bound to fibrin, thus exerting mostly localized fibrinolytic activity at appropriate dose for therapeutic solubilization of a clot. At higher doses, t-PA also induces systemic fibrinolytic activity.
Urokinase and streptokinase
As therapeutic fibrinolysis activators, they cause a systemic activation of plasminogen which subsequently exerts its effect on the coagulation factors. Fibrinogen, F V and F VIII and to a lesser degree factors II, VII, IX, X and kininogen are cleaved.
Systemic administration of urokinase causes the conversion of plasminogen to plasmin. The administration of streptokinase causes the formation of streptokinase-plasminogen complexes. Plasmin is formed by modifying the conformation of plasminogen due to autocatalytic proteolytic activation. The resulting plasmin effect leads to a decrease in fibrinogen with a simultaneous increase in fibrinogen degradation products. Fibrinogen degradation products have an inhibitory effect on coagulation since they can interfere with the reaction between thrombin and fibrinogen. This effect of the fibrinogen degradation products leads to a prolongation of the thrombin time (TT). The aPTT may be prolonged in a similar way; while in general this also applies to the PT, but the prolongation is less pronounced .
The TT has the highest sensitivity regarding therapeutically induced fibrinolytic and/or thrombolytic activity. Hence, the TT determination should have priority in fibrinolytic/thrombolytic therapy monitoring over all other tests. However, the aPTT and PT also provide important information on the therapy-related changes in the hemostasis system.
Tests for monitoring fibrinolytic therapy
The following tests should be performed before and during thrombolytic therapy:
- Complete blood count including the platelet count, PT, aPTT, fibrinogen, blood group determination and urinalysis
- 4 h after the start of therapy and then, afterwards, twice each day, monitoring should be performed by the following assays: TT, aPTT, possibly fibrinogen and urinalysis
- Often, a heparin infusion is applied simultaneously with thrombolytic therapy, therefore possibly making it necessary for therapeutic reasons to assess the thrombolytic activity separately from the heparin effect. In order to do so, the reptilase (batroxobin) time can be determined. Both enzymes have a thrombin-like effect, however, they are not affected by heparin. For this reason, they are suited to assist in the differentiation between fibrin degradation products which have accumulated at a higher rate due to thrombolysis and the effect of thrombin inhibitors such as heparin and hirudin which do not prolong the batroxobin time. Besides thrombolytic therapy, low fibrinogen concentrations and dysfibrinogenemias may prolong the batroxobin time.
None of the available laboratory tests (i.e., neither the TT nor the aPTT) can predict the success or failure of thrombolytic therapy or indicate impending bleeding. However, a lack of TT prolongation suggests inadequate activation of the fibrinolytic system during thrombolytic therapy, while significant prolongation of the aPTT to more than 2 min. or of the PT to more than 60 sec indicates at least a predisposition of the hemostatic system to possible bleeding.
Under such circumstances, further tests should be performed, for example:
- Plasminogen concentration analysis; congenital or acquired plasminogen deficiency, possibly dysplasminogenemia
- Determination of the anti-streptokinase titer in the case of low-dose streptokinase therapy; during high-dose streptokinase therapy, the anti-streptokinase titer is usually easily masked.
Local causes for bleeding
Bleeding (e.g., resulting from atherosclerotic changes in cerebral blood vessels) can, due to the nature, not be detected by any laboratory test but may lead to the feared complication of cerebral hemorrhage in patients undergoing thrombolytic therapy, especially those > 60 years of age. Cerebral hemorrhage is primarily feared to occur within the scope of acute lysis of a recent myocardial infarction, especially in concurrent administration of a thrombolytic agent and an anticoagulant such as heparin.
In general, local causes for bleeding which may complicate thrombolytic therapy (e.g., gastric ulcer, renal stones, malignant tumor, diabetic retinopathy) are not detected by any laboratory test. In this regard, the information obtained during the medical history and clinical analysis prior to start of therapy is often more important than any of the laboratory tests.
Prior to the start of thrombolytic therapy, the following should be taken into account:
- Existing therapy with inhibitors of platelet function (e.g., aspirin, clopidogrel) since these medications may significantly increase the risk of bleeding during thrombolytic therapy
- Predisposition to bleeding known to the patient.
A possible bleeding tendency during thrombolytic therapy is indicated by:
- Markedly decreased fibrinogen levels (≤ 0.5 g/L)
- Marked prolongation of the PT
- Marked prolongation of the aPTT and TT.
Coumarin derivatives, as vitamin K antagonists, block the γ-carboxylation of the coagulation factors II, VII, IX and X during their synthesis in the hepatocytes. This results in the loss of the Ca2+-binding ability of these factors, thus causing the formation of functionally inactive factors (PIVKA; protein-induced by vitamin K absence).
Depending on the half-life of the vitamin K dependent coagulation factors, it ranges from 6 h in F VII to 70 h in F II, the coagulability of plasma decreases due to this anticoagulant effect. Adequate and stable anticoagulation is usually not reached until 3–4 days after initiation of therapy.
The coagulation inhibitor protein C, also a vitamin K dependent protein, decreases rapidly, like F VII, because of its short half-life (7 h) whereas prothrombin (70 h) and F X (50 h) decline more slowly. Accordingly, during the first few days of oral anticoagulant therapy, a state of blood hyper coagulability may develop, especially in the case of preexisting protein C deficiency, leading to the possible development of coumarin necrosis.
Therapy is usually monitored by determining the PT. The therapeutic range for the PT was defined as 15–30% of normal. The standardization of the PT and, thus, the comparability of the available PT is achieved by mathematical conversion into a so-called International Normalized Ratio (INR).
The PT is not suited to control the efficacy of low-dose oral anticoagulant therapy. The advantage of low-dose oral anticoagulant therapy lies in the fact that the bleeding tendency is lower than at higher dose. Indications, for which low-dose oral anticoagulant therapy is recommended, include:
- Secondary prophylaxis of deep venous thrombosis
- Peri operative thrombosis
- Peri operative thromboprophylaxis as part of surgical procedures
- Permanent central venous catheters
- Non-rheumatic atrial fibrillation, especially in elderly patients, possibly also certain types of heart valve implants.
Orally administered thrombin inhibitors
Contrary to vitamin-K antagonist therapy with orally administered coumarins, orally administered, direct thrombin antagonists have a much more specific site of action regarding the inhibition of thrombin-mediated clot formation and thromboembolism. For further information. See .
Unfractionated heparin is a mixture of mucopolysaccharides with different chain lengths; these mucopolysaccharides are mainly collected from the intestinal mucosa of pigs or the lungs of cattle. The molecular weight of unfractionated heparin ranges from 3 to 30 kDa while that of low molecular weight heparin ranges from 1.5 to 12 kDa. Unfractionated heparin and low molecular weight heparin differ with regard to half-life, bio availability and their effects exerted on platelets and lipolysis ().
Low molecular weight heparin has the following advantages:
- Higher t-PA release from vascular endothelium
- No increase in platelet aggregation
- No release of platelet factor 4
- Low probability of osteoporosis in long-term treatment
- Less frequent occurrence of heparin-induced thrombocytopenia.
Unfractionated heparin exerts its effect by significantly accelerating the inactivation of activated coagulation factors, especially of F Xa and thrombin; it does so by forming a complex with the naturally occurring coagulation inhibitor antithrombin (heparin-antithrombin complex). Significantly lower heparin doses can inhibit F Xa in comparison to those required for the inhibition of thrombin. This is the mechanism on which the effect of low-dose heparin is based in thromboprophylaxis.
Distinction is made between high-dose heparin therapy (30,000–50,000 IU/24 h) and low-dose heparin therapy or heparin prophylaxis using approximately 15,000 IU/24 h (up to 20,000 or 25,000 IU/24 h).
Monitoring of high-dose heparin therapy
The aPTT is used for monitoring high-dose heparin therapy. In the treatment of thromboses, a 1.5–2.5-fold prolongation of the aPTT is considered to be the therapeutic range. Alternatively, the heparin effect can be monitored by determining the thrombin time. Some laboratories measure two thrombin times parallel to each other i.e., one with a low and one with a high thrombin concentration, thus allowing the determination of lower and higher heparin activity levels.
Monitoring of low-dose heparin prophylaxis with fractionated heparin
Low-dose heparin therapy usually does not require monitoring as long as hemorrhagic diathesis or a bleeding tendency have been excluded in the patients prior to start of therapy. If monitoring is required (e.g., in patients with chronic renal insufficiency) an anti-F Xa test is used for determination. See .
The maternal coagulation factors do not cross the placental barrier. Independent fibrinogen synthesis starts at embryonic week 5–6 and coagulability of the blood starts at gestational week 11. Fetal plasma concentrations which are present from gestational week 19 are markedly lower than those of the full-term newborn. The concentration of the following plasma coagulation factors in full-term newborns is only approximately 50% of that in adults :
- Vitamin K-dependent factors II, VII, IX and X
- Contact factors XI, XII and pre kallikrein.
During the first 6 months of life, the concentration of vitamin K-dependent factors and contact factors increases to 80% and remains approximately at this level throughout childhood.
The levels of fibrinogen, factors V, VIII, XIII and von Willebrand factor (VWF) in newborns and children are not decreased and correspond to those in adults.
The VWF and its high-molecular-weight multimers are elevated during the first two months of life and then gradually decline to adulthood concentrations.
The neonatal form of fibrinogen in newborns differs from the form present in adulthood by higher sialylation. The fibrinogen concentration at birth is similar to that in adults, increases in the first weeks of life and then decreases again to adulthood levels.
- Mild prolongation of the prothombin time (PT) due to a decrease in vitamin K dependent factors
- Prolongation of the activated partial thromboplastin time (aPTT) due to the decreased concentration of contact factors.
During the first weeks of life, antithrombin, protein C, protein S and heparin cofactor II are decreased to levels corresponding to those of heterozygous deficiency in adults. The concentration of protein C during childhood and adolescence is lower than that in adults.
C1-esterase inhibitor and α2-macroglobulin have roughly adult concentrations at birth and increase to 150% and 200%, respectively, at the age of 6 months. α2-macroglobulin is a more potent thrombin inhibitor in neonates and children than in adults and compensates antithrombin deficiency .
- 80% in childhood
- 35% in full-term neonates
- 25% in pre term infants
In the neonate, plasminogen occurs in a fetal form with increased sialylation and in an adult form. The neonatal levels are 50% of adult ones . Plasminogen activator inhibitor is elevated and α2-antiplasmin concentration at birth corresponds to 80% of the adult value.
Reference intervals for coagulation factors are listed
The medical history is especially important for investigating a predisposition to bleeding in children because their hemostatic system has not yet been exposed to major challenges.
The type of bleeding is of differential diagnostic significance:
- Mucosal bleeding (gingiva, menorrhagia), petechiae and nosebleed point to thrombocytopenia or von Willebrand syndrome
- Spontaneous deep muscular hemorrhage and hemarthrosis, widespread suffusions and hematomas rather point to factor deficiency (e.g., hemophilia).
- An acute onset and persistence for several days suggests an acquired disorder. This applies, for example, vitamin K deficiency, liver disease and disseminated intravascular coagulation. Common causes of liver disease include viral hepatitis, hypoxic liver damage, biliary atresia and total parenteral nutrition. In many cases, renal diseases are associated with platelet dysfunction, and a malabsorption syndrome can cause bleeding due to vitamin K deficiency.
- Although acquired hemostatic disorders occur in great excess over congenital problems, the latter need to be considered because they frequently present initially during the first weeks or months of life. The most congenital hemorrhagic disorders to present with bleeding at birth or during early infancy are deficiencies of F VIII and F IX. In contrast, von Willebrand disease, which is the most common congenital hemorrhagic disorder to present during childhood and in adults, rarely presents with bleeding during the first weeks of life.
Complete blood count
The complete blood count is significant in determining thrombocytopenia and anemia. Anemia in combination with bleeding (e.g., frequent nosebleed) points to primary hemostatic disorders, whereas microcytic anemia points to long-term loss of blood. The combination of anemia and thrombocytopenia can indicate impaired megakaryopoiesis in leukemia, lymphoma or toxic bone marrow damage.
A blood smear can be used for estimating the platelet count and platelet quality in suspected thrombocytopenia or thrombocytopathy. Using a 100x objective, one platelet/field will give an estimated platelet count of 15 × 109/L. Platelet clumps, which are not taken into account by the analyzer, are visible under the microscope. Giant platelets are seen in the Bernard-Soulier syndrome, May-Hegglin syndrome and after major bleeding.
PT and aPTT
The clotting time in one of the two tests is prolonged if the activity of the relevant coagulation factor analyzed in the test is decreased to below 40%.
Plasma mixing test
If PT or aPTT are prolonged, a plasma mixing study should be performed using a mixture of patient plasma and the same amount of pool plasma of healthy individuals. Thus, any factor deficiency is compensated. Normalization of the clotting time indicated by aPTT or PT points to factor deficiency. If the clotting time does not normalize, the presence of an inhibitor to a coagulation factor must be assumed. In most children, coagulation factor inhibitors will not cause hemorrhagic diathesis.
Factor analysis is required depending on the information from family history or the results of the plasma mixing test and is performed using a modified PT or aPTT test. If the aPTT plasma mixing test points to factor deficiency, it is important:
- To analyze the sample for a deficiency in factors VIII, IX and XI because a deficiency in these factors may be associated with bleeding
- To determine the factors XII, pre kallikrein and high molecular weight kininogen. A deficiency in these factors can also lead to prolonged aPTT but is not associated with bleeding.
The functional activity of fibrinogen is determined by measuring clot formation. Clot formation is decreased in the rare disorder dysfibrinogenemia, but the immunologically determined fibrinogen concentration is normal.
Laboratory tests for primary hemostasis
The following tests are required to determine platelet dysfunction and the von Willebrand syndrome:
- Bleeding time: used as a screening test; however, the diagnostic reliability of this test is low
- Platelet aggregation test: the test should primarily be focused on determining von Willebrand syndrome. If the result is negative, further investigation of the platelet function is necessary.
Severe congenital hemostatic disorders can already manifest in the peri-/postnatal period. For example, umbilical cord bleeding, suffusions, gastrointestinal and more rarely intracerebral bleeding are typical symptoms of a deficiency in factors II, V, VII, X and XIII and afibrinogenemia . Hemophilia A and B will be diagnosed no later than after injury in toddlers. Inborn disturbances of platelet function e.g., Bernard-Soulier syndrome (VWF receptor defect) and Morbus Glanzmann-Naegeli (deficiency of GPIIb/IIIa) can be conspicuous with petechial bleeding after birth.
Acquired coagulatory bleeding, mostly results from organic disease of the child in the setting of hepatic disease, hematopoiesis, lymphatic system disorder, severe infection, or after medication with valproic acid or asparaginase.
Prolonged PT and/or aPTT occasionally measured in asymptomatic children can be caused by:
- Presence of an inhibitor not associated with bleeding
- Deficiency in contact factors (e.g., high molecular weight kininogen, pre kallikrein or F XII): the aPTT is prolonged
- Vitamin K deficiency. The National Institute for Health and Excellence recommends a dose of 1 mg of konakion postnatally administered by intramuscular injection. According to a study , 11 in 3.15 million newborns in the United Kingdom had vitamin K deficiency bleeding (VKDB). Six babies with early bleeding received no VK prophylaxis, two babies with late VKDB received incomplete oral prophylaxis and three babies with late VKDB had liver disease
Neonatal major bleeding occurs in about 5–15% of pre term neonates admitted to a neonatal intensive care unit. A dynamic prediction of bleeding risk model was developed that included the variables thrombocyte count, gestational age, postnatal age, intrauterine growth retardation, necrotizing enterocolitis, sepsis and mechanical ventilation. The median cross-validated c-index was 0.74. A c-Index of 1.0 indicates perfect discrimination between newborns with and without major bleeding .
The incidence of symptomatic thromboembolism in children is lower than in adults . It is 5.1 per 100,000 births, but 5% of sick newborns develop a thrombosis . The congenital risk factors in children are the same as in adults. Most of the pre term neonates have a thrombocyte count below 50 × 109/L
1. Male C, Johnston M, Sparling C, Brooker LA, Andrew M, Massicotte P. The influence of developmental hemostasis on the laboratory diagnosis and management of hemostatic disorders during infancy and childhood. Clin Lab Med 1999; 19: 39–69.
6. Busfield A, Samuel R, McNinch A, Tripp JH. Vitamin K deficiency after NICE guidance and withdrawal of konakion neonatal: British pediatric surveillance unit study, 2006–2008. Arch Dis Child 2013; 98: 41–7.
During normal pregnancy hormonal and physiological modifications cause changes in thrombocyte count, coagulation and fibrinolytic activity leading to a pro coagulant state . The advantage is a decreased probability of bleeding during delivery, but the drawback is an increased risk of venous thromboembolism .
Thrombocytopenia is observed in 6–15% of pregnant women at the end of pregnancy and is defined as a platelet count below 150 × 109/L. In mild thrombocytopenia, the number lies within a range of down to 75 × 109/L. Criteria for gestational thrombocytopenia are :
- Asymptomatic thrombocytopenia > 75 × 109/L
- No history of thrombocytopenia in the past except during a previous pregnancy
- Thrombocytopenia occurred during the last trimester
- No fetal or neonatal thrombocythemia
- Spontaneous resolution postpartum.
- Preeclampsia and HELLP syndrome (approximately 20% of the cases)
- Idiopathic thrombocytopenic purpura (ITP) (5% of the cases). Approximately 5–40% of the neonates from mothers with ITP have a platelet count below 150 × 109/L.
Serial measurements of coagulation activity are determined in serious pregnancy complications e.g., postpartum hemorrhage, disseminated intravascular coagulation, amniotic fluid embolism, preeclampsia.
Indicators of fibrinolysis activation: tissue plasminogen activator (t-PA) and urinary plasminogen activator (u-PA) are increased. D-dimer concentration increases continuously and finally markedly in the early postpartal period .
In preeclampsia, increased amounts of F VIII are consumed and the levels of markers indicating increased thrombin activity (thrombin-antithrombin complex and prothrombin fragments F1+2) are increased .
Factor VIII and von Willebrand factor are determined if a hereditary bleeding or a carrier status is suspected.
Factor VIII:C: the concentration increases about the factor 2,3 e.g., from 138% in the gestational weeks 5–9 to 320% in the gestational weeks 37–40 . Normalization 6 weeks post partum. Reported symptomatic cases of hemophilia A in pregnant women are not based on homozygously mutated alleles, but on extensive lyonization. Acquired inhibitors against F VIII occur at a prevalence of 1 in 1 million and cause severe bleeding. In most cases, the affected patients are older than 50 years (i.e., pregnant women are affected even more rarely). The plasma mixing test is used for diagnosing.
Von Wllebrand factor antigen (vWF:Ag): the level increases about the factor 2.6 e.g., from 98% in the gestational weeks 5–9 to 257% in the gestational weeks 37–40 . Normalization 6 weeks post partum. The clinical presentation of the von Willebrand syndrome varies greatly in pregnant women. Most cases are asymptomatic, except in extreme situations such as surgery. Occurring bleeding is of the platelet type i.e., mucosal bleeding, susceptibility to bruises, increased bleeding postpartum. See also ).
Very rare coagulation factor deficiencies: hereditary F XIII deficiency (prevalence 1 in 2 million pregnancies) and fibrinogen deficiency (afibrinogenemia, hypo fibrinogenemia, dysfibrinogenemia) are associated with the inability to carry pregnancies to term .
VTE is a leading cause of morbidity and mortality in pregnancy and puerperium. The risk of VTE events in pregnancy is approximately 5-fold higher than in non-pregnant women. The incidence of pregnancy associated VTE ranges from 1 in 1,000 to 1 in 2,000 deliveries. In absolute numbers per 1,000 pregnancies, the prevalence is :
- 0.57 prepartum (venous thrombosis 0.50, pulmonary embolism 0.07)
- 0.29 postpartum (venous thrombosis 0.21, pulmonary embolism 0.08).
Maternal deep venous thrombosis is more common in the left leg. Pulmonary embolism occurs in about 16% of pregnant women with untreated deep venous thrombosis and remains the most frequent cause of maternal death.
- Age above 35 years
- Cesarean section
- Obesity (above 80 kg)
- Multiparity (more than 4 pregnancies)
- Deep vein thrombosis in medical history.
In women with a prior idiopathic VTE who carry an additional hereditary risk factor or who have a positive family history of thrombosis, a high risk of VTE in pregnancy can be expected (> 10%); approximately 70% of the women with VTE in pregnancy have such history. Previous thromboembolism, in particular, represents an increased risk of recurrent VTE during pregnancy.
Congenital and acquired risk factors are listed in . However, the majority of pregnant women heterozygous for a risk factor (e.g., factor V Leiden) do not suffer from a thromboembolic event. The occurrence of such an event is enhanced by the presence of several genetic or acquired risk factors /, /.
Successful pregnancy outcome is dependent on the development and maintenance of adequate placental circulation. The inability of the vascular system, abnormalities of placental vasculature and of the hemostatic system to ensure adequate hemostasis may result in a number of gestational pathologies, including first and second trimesters miscarriages, intrauterine growth retardations, intrauterine fetal death, pre-eclampsia and detachment of the placenta.
Recurrent miscarriage is caused by chromosomal aberration (7%), approximately 15% in hormonal defects (progesterone, estrogen), diabetes mellitus, thyroid disorders, unknown reasons (about 6%) and blood coagulation or platelet defects (55–62%) .
- Increase in coagulation (elevation in fibrinogen, F VIII:C and von Willebrand factor)
- Decrease in anticoagulation (reduction in antithrombin and protein S)
- Inhibition of fibrinolysis (increase in plasminogen activator inhibitors PAI-1 and PAI-2).
In total the hemostatic equilibrium is shifted toward hyper coagulability, presumably to prevent hemorrhagic complications during childbirth. Therefore, the pregnant women has a higher potential of pro coagulant factors and an increased fibrinogen concentration . Activation of the blood coagulation system increases continuously with gestational age, while fibrinolytic activity is decreased.
During labor, the blood flow is initially stopped by myometrial contraction and vessels are then blocked with a thrombus by the coagulation system. There is a significant consumption of platelets, coagulation factors and fibrinogen during delivery.
Fibrinolytic activity increases again after the child has been born and the placenta expelled and all hemostatic processes that had changed during pregnancy return to normal 4–6 weeks postpartum.
2. Jacobsen AF, Skjeldestad FE, Sandset PM. Incidence and risk patterns of venous thromboembolism in pregnancy and puerperium- a register-based case control study. Am J Obstet Gynecol 2008; 198: 233 e1–7.
4. Kristoffersen AH, Petersen PH, Bjorge L, Roraas T, Sandberg S. Within-subject biological variation of activated partial thromboplastin time, prothrombin time, fibrinogen, factor VII and von Willebrand factor in pregnant women. Clin Chem Lab Med 2018; 56: 1297–1308.
11. Cerneca F, Ricci G, Simeone R, et al. Coagulation and fibrinolysis changes in normal pregnancy, increased levels of procoagulants and reduced levels of inhibitors during pregnancy induce a hypercoagulable state, combined with a reactive fibrinolysis. Eur J Obstet Gynecol Reprod Biol 1997; 73: 31–6.
14. Robertson L, Wu O, Langhorne P, et al. For the Thrombosis Risk and Economic Assessment of Thrombophilia Screening (TREATS) study. Thrombophilia in pregnancy: a systemic review. Br J Haematol 2005; 132: 171–96.
Coagulopathy is a condition in which the blood’s ability to clot is impaired. Coagulopathies resulting from injured sites leading to major bleeding are often triggered by massive transfusion after major surgery, severe trauma, gastrointestinal or obstetric hemorrhage. Four major risk factors for coagulopathy and their relative risk (RR) are identified:
- pH below 7.1 (RR 12.3)
- Core temperature below 34 °C (RR 8.7)
- Injury severity score higher than 25 (RR 7.7)
- Systolic blood pressure below 70 mmHg (RR 5.8).
The coagulopathy of trauma is a syndrome of non-surgical bleeding from mucosal lesions, serosal surfaces, and wound and vascular access sites, the tissue oozing that continues after identifiable vascular bleeding has been controlled. It occurs in the presence of profoundly decreased platelet count and coagulation factors .
- Congenital and acquired causes of bleeding
- Bleeding due to anticoagulants or inhibitors of platelet aggregation
- Bleeding due to hypothermia, acidosis or massive transfusion used to treat hypovolemia
- Disseminated intravascular coagulation (DIC) due to initial tissue injury or treatment thereof. An early and a late type of DIC are distinguished. The early type is associated with initial tissue injury and the late one results from organ failure.
The outcome of the interaction between cellular components and coagulation factors of the hemostatic pathways in massive tissue injury is decisive as to whether the body responds with adequate hemostasis, bleeding or thrombosis. Massive bleeding is the second most common cause of death following severe trauma. Patients die in the course of a bloody vicious cycle of bleeding, emergency therapy, hemodilution, coagulation disorder and continuous bleeding.
Coagulation disorder in severe trauma is mainly triggered by the following risk factors:
- pH value ≤ 7.2. At such pH values, the activity of the F VIIa/TF complex, the F Xa/Va complex and F VIIa decreases by up to 90% and interaction with phospholipids is impaired
- Hypothermia ≤ 34 °C. The activity of the coagulation factors decreases and especially the interaction between the von Willebrand factor and the platelet glycoprotein Ib/IX complex is reduced
- Systolic blood pressure < 70 mmHg
- Massive transfusion. When the blood components, erythrocytes, thrombocytes, and fresh frozen plasma, are mixed in an equal unit ratio in massively transfused trauma patients, the resulting hematocrit, thrombocyte count, and coagulation factor activities will all be below common transfusion triggers. Thus, dilutional reductions in clotting activity are inevitable in massively transfused patients and coagulopathy is very likely .
The best approach of determining the coagulation state prior to planned surgery and identifying patients with increased risk of peri operative bleeding is by anamnesis and medical examination . For laboratory tests, see .
In trauma or a peri operative setting, bleeding may occur due to dilution resulting from a decrease in coagulation factors or due to consumption resulting from the release of tissue factor from ischemic tissue induced by shock. Hyper fibrinolysis, presumably resulting from protein C activation, may also make a major contribution to bleeding.
Coagulopathy of trauma is a syndrome of non-surgical bleeding from mucosal lesions, serosal surfaces, and wound and vascular access sites associated with serious injury, hypothermia, acidosis, hemodilution and occasionally with classic disseminated intravascular coagulation /, /. Bleeding in massively injured patients increases with additional coagulopathy. Acute coagulopathy of trauma correlates independently with an 8-fold increase in mortality within 24 h and a quadrupled total mortality . Tissue injury results in hypo perfusion and hyper fibrinolysis which, in turn, correlate with the severity of the trauma. Approximately 30% of the severely traumatized patients suffer from coagulation disorders on admission to the emergency department. Coagulation disorders in severe trauma may have multiple causes and can result from a combination of factor depletion and dilutional coagulopathy, hypothermia, acidosis and hyperfibrinolysis . Main effects on the hemostatic system such as hypothermia ≤ 34 °C, pH ≤ 7.2, ionized calcium ≤ 0.9 mmol/L and anemia ≤ 100 g/L are not identified by laboratory coagulation tests.
The systemic damage of vascular endothelium caused by the bleeding shock and systemic inflammation is the main pathologic event of acute trauma . Vessel wall injury activates the blood coagulation cascade through the release of tissue factor. Since the fibrinolytic system is also activated at the same time (above all, plasminogen is activated by the tissue plasminogen activator) in order to maintain the hemostatic equilibrium, coagulation factors and platelets will rapidly decrease in severely injured patients.
The term dilutional coagulopathy summarizes coagulation disorders developing during surgery in the absence of a disease that might impair the coagulation pathways. This coagulopathy is caused by massive transfusion used to treat hypovolemia. The extent, severity and changes in loss of blood and hemodilution are based on hypovolemia. The loss of ≤ 750 mL of blood (up to 15% of the blood volume) does not cause major changes. A loss of blood of 15–30% results in the stimulation of the sympathetic nervous system in conjunction with tachycardia and mild hypotension, and hemorrhagic shock in conjunction with coagulation disorders occurs at a loss of > 40% of the blood volume.
DIC is defined by the International Society on Thrombosis and Hemostasis (ISTH) as “an acquired syndrome characterized by the intravascular activation of coagulation with loss of localization arising from different causes”. This condition typically originates in the microvasculature and can cause damage of such severity that it leads to organ dysfunction. DIC usually presents as hemorrhage, with only 5–10% of cases presenting with micro thrombi. Sepsis is the most common cause of DIC. The up regulation of tissue factor activates coagulation, leading to the widespread deposition of fibrin and to microvascular thromboses and may contribute to organ dysfunction, such as development of renal insufficiency and ARDS, hypotension, and circulatory failure. The consumption of coagulation factors and platelets produces a bleeding tendency, with thrombocytopenia, prolonged PT and aPTT, hypo fibrinogenemia and elevated levels of fibrin degradation products, such as D-dimers. The coagulation localizing capacity is lost due to the loss of antithrombin.
- Activation of the coagulation system and hyper coagulability
- Secondary response of the fibrinolytic system resulting in temporarily elevated coagulation and fibrinolysis biomarkers
- Impaired fibrinolysis caused by elevated levels of plasminogen activator inhibitor.
This has the following consequences:
- Thrombotic complications due to excessive fibrin formation
- Consumption of coagulation factors (consumptive coagulopathy) and platelets. Bleeding primarily occurs at wounds and vascular accesses, but profuse hemorrhage is also possible.
The ISTH/SSC score system facilitates assessment of the diagnostic laboratory test results (). A D-dimer level above the reference intervals is assigned the point value 1 and a level 5-fold higher than the upper reference interval is assigned the point value 2 .
It is important for therapy to maintain a platelet count above 5 × 109/L and to prevent prolongation of the PT and aPTT to more than 1.5-fold the normal level and a decrease in the fibrinogen concentration to below 1.5 g/L by the administration of fresh frozen plasma.
- from subclinical activation in conjunction with an increase in biomarkers of activated coagulation pathways
- to fulminant DIC culminating in intravascular fibrin formation and deposition in the microvasculature, multiple organ dysfunction syndrome (MODS), hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP) and vasculitis.
- Systemic inflammatory response syndrome (SIRS) from trauma, acute pancreatitis, snake bite. Coagulation activation is indirectly influenced by pro inflammatory cytokines such as TNF-α, IL-6 and by the complement system.
- Sepsis mediated by components of microorganisms (lipopolysaccharides, endotoxin) or by bacterial exotoxins such as the staphylococcal α-hemolysin.
The clinical picture can be dominated by hemorrhage, thrombosis or a combination of the two. None of the syndromes (DIC, MODS, HUS, TTP, vasculitis) results from a specific cause or specific microbial pathogen. Manifestation of the syndromes depends on the patient ’s condition, the location and severity of trauma and criteria such as virulence, amount and site of entry of the pathogens in sepsis and antibiotic treatment.
The pro coagulatory activity induced by sepsis is generally more severe than in severe trauma because of the systemic nature of prothrombotic diathesis. Thrombin is not only generated by vascular endothelium but also by activated macrophages. Consequently, tissue factor is available in almost unlimited quantities and the general activation of the coagulation system leads to a high consumption of coagulation factors.
- Systemic inflammation during sepsis leads to the generation of pro inflammatory cytokines TNF-α, ΙL-1 and IL-6 that orchestrate coagulation activation as well as the down-regulation of fibrinolysis. This results in an imbalance between intravascular fibrin formation and its removal.
- Reduced anticoagulant capacity leads to excessive fibrin formation and consumption of clotting factors and anticoagulants, causing microvascular thrombosis in conjunction with multiple ischemic organ damage (MODS) and skin necrosis.
The exogenous pathway is activated by the tissue factor (TF). TF is synthesized by activated monocytes/macrophages and endothelial cells. After binding to exposed TF, F VII is activated (F VIIa). The TF/F VIIa complex then activates F X (F Xa), by which prothrombin is converted to thrombin (see also ).
- Inactivation of antithrombin (AT) by elastase released from granulocytes as well as rapid clearance of thrombin-antithrombin (TAT) complexes from circulation
- Decreased thrombomodulin synthesis due to pro inflammatory cytokines, leading to inadequate activation of protein C
- Relative deficiency of tissue factor pathway inhibitor (TFPI) which inhibits the activity of the FVIIa/TF complex.
The decrease in the physiological anticoagulant effect leads to a thrombin burst and, as a result, enhances fibrin formation.
Generation of thrombin in sepsis also initiates fibrinolysis, primarily through the release of tissue-type plasminogen activator (t-PA) and the urokinase-like plasminogen activator (u-PA). Endothelial cells are the principle source of t-PA. The initially increased fibrinolytic activity in sepsis is markedly decreased by the following processes :
- Increasing PAI-1 levels. PAI-1 forms stable complexes with t-PA and u-PA thus reducing fibrinolysis
- Increased synthesis of α2-antiplasmin which inactivates plasminogen by forming plasminogen-α2-antiplasmin complexes.
3. Segal JB, Dzik WH. Paucity of studies to support the abnormal coagulation test results predecting bleeding in the setting of invasive procedures: an evidence based review. Transfusion 2005; 45: 1413–25.
4. Blome M, Isgro F, Kiessling AH, Skuras J, Haubelt H, Hellstern P, et al. Relationship between factor XIII activity, fibrinogen, haemostasis screening tests and postoperative bleeding in cardiopulmonary bypass surgery. Thromb Haemost 2005; 89: 1101–7.
6. Korte W, Gabi K, Rohner M, Gähler A, Szadkowski C, Schnider DW, et al. Preoperative fibrin monomer measurement allows risk stratification for high intraoperative blood loss in elective surgery. Thromb Haemost 2005; 94: 211–5.
- Most factors of the coagulation system are synthesized by liver parenchymal cells
- The inhibitors of the coagulation system which establish an coagulation equilibrium by counter regulation are synthesized by parenchymal cells. Inhibitors include antithrombin, protein C, protein S, plasminogen activator inhibitor (PAI-1) and α2-anti plasmin
- The activators of fibrinolysis (e.g., tissue-plasminogen activator; t-PA), are removed from circulation. The half-life of t-PA is prolonged in liver injury, resulting in increased fibrinolysis in conjunction with bleeding tendency. The clearance not only of activated coagulation and fibrinolysis factors but also of activation complexes and end products of the fibrinogen/fibrin conversion is accomplished by the reticuloendothelial system of the liver.
The von Willebrand factor (VWF) and t-PA are produced by endothelial cells and urinary plasminogen activator (u-PA) by kidney cells.
The vitamin K dependent coagulation factors II, VII, IX and X as well as the inhibitors protein C and protein S require post translational modification in order to acquire their physiological function. All of these proteins have a number of glutamic acid residues in their amino terminal region that must be converted to γ-glutamic acids by a carboxylase that requires vitamin K. Only in their carboxylated form can these proteins bind to phospholipid surfaces via Ca2+ bridges, thus contributing to the formation of activation complexes such as the prothrombin complex. The unavailability of the binding site for Ca2+ due to vitamin K deficiency leads to reduced coagulation capacity.
Liver diseases may generally be associated with the following hemostatic problems:
- Decreased biosynthesis of coagulation factors
- Increased consumption of coagulation factors
- Synthesis of abnormal coagulation factors
- Abnormal clearance of activated components of the coagulation system.
Vitamin K deficiency may also lead to significant coagulation problems. The pattern of hemostatic proteins observed in various liver diseases and in conjunction with vitamin K deficiency are presented in .
6. Jalan R, Saliba F, Pavesi M, Amoros A, Mroreau R, Gines P, et al. Development and validation of a prognostic score to predict mortality in patients with acute-on- chronic liver failure. J Hepatol 2014; 61: 1038–47.
Patients with decreased renal function may display not only disorders of the plasma coagulation system and hemorrhage but also thromboembolic complications.
Thrombocyte dysfunction and increased bleeding time are due to a defect in primary hemostasis. Prolonged may be the coagulation time of the PT (extrinsic pathway) and the aPTT (intrinsic pathway) of the coagulation system because of a decline in F II, F VII and F X. The factors V and VIII are normal, fibrinogen is most often increased. Hemostatic disorders in renal disease are shown .
Thromboembolic complications are the major threat to the nephrotic patient. The incidence is about 35%. Patients with membranous glomerulopathy and severe proteinuria are at highest risk . Common events include deep vein thrombosis, pulmonary embolism, renal vein thrombosis and also thrombosis involving peripheral arteries and the coronary arteries.
Patients with chronic renal failure often suffer from ecchymoses, purpura, epistaxis and bleeding from tapping sites due to impaired primary hemostasis because of platelet dysfunction. Heavy bleeding may occur following trauma and blood loss during surgery . Bleeding time is prolonged, caused by dysfunctional von Willebrand factor, reduced thromboxane synthesis, the presence of uremic toxins and modified platelet granules. The probability of hemostatic disorder is correlated with the extent of urea increase . Bleeding can be induced by taking platelet function-inhibiting drugs (steroidal antiphlogistics, antibiotics) .
On the other hand, uremic patients also have an increased incidence of thromboembolic events such as cardiovascular disease, ischemic stroke and thromboses of arteriovenous fistulae.
Deep vein thrombosis occurs with higher frequency in renal transplant recipients as compared to age-matched patients after other major surgery . The prevalence of renal vein thrombosis is 0.5–4%, especially during the first months following transplantation. At later stages, when graft function has stabilized thromboembolism may be associated with the use of immunosuppressive therapy and is thought to be caused by cyclosporine. In severe acute vascular rejection thrombosis is widespread and gives rise to a large number of small infarcts in the renal cortex. The thrombosis may involve the main vessels, with eventual development of total graft necrosis.
3. Mezzano D, Tagle R, Panes O, et al. Hemostatic disorder of uremia: the platelet defect, main determinant of the prolonged bleeding time, is correlated with indices of activation of coagulation and fibrinolysis. Thromb Haemost 1996; 76: 312–21.
- The formation of new blood vessels (tumor angiogenesis)
- The activation of the coagulation system (coagulopathy).
Tumor activated blood vessels exhibit compromised ability to contain plasma proteins (are leaky), poorly sustain blood flow (are prone to stasis) and provide inadequate anti thrombotic luminal surfaces (promote intravascular thrombosis) .
The tissue factor (TF), frequently over expressed in malignancies, plays a major role in tumor progression, metastasis and angiogenesis through signalling via its intracellular domain. TF may involve three different compartments: cancer cells, their adjacent stroma (blood vessels, fibroblastic and inflammatory cells) and the circulating blood.
- Venous thromboembolism (VTE), inclusive deep vein thrombosis
- Pulmonary embolism
- Syndromes comparable to low disseminated intravascular coagulation.
Up to 90% of patients with metastatic tumors are affected by one of these coagulopathies in the course of their disease. Besides the spontaneously occurring hemostatic disorders during the course of malignancy enhancement of the para neoplastic effects must be expected during tumor therapy (surgery, chemotherapy or irradiation), possibly resulting in clinical manifestations. Whereas solid cancers lead predominantly to VTE, myeloproliferative diseases are associated with thrombotic and hemorrhagic complications at about the same rate and in some cases even feature both at the same time .
Tumor tissues per se activate blood coagulation, and mechanisms that are involved in the malignant transformation may also be involved in the regulation of tumor cell pro coagulant factors. This applies, for example, to the JAK2V617F mutation expression in patients with myeloproliferative disorders and for PML-RARα hybrid gene expression in patients with acute pro myelocytic leukemia. In both cases, the gene expression is associated with a pro coagulatory phenotype .
- Tissue factor (TF), a transmembrane glycoprotein that is the primary activator of normal blood coagulation (see ). Tf forms a complex with F VIIa, activates F X and starts the extrinsic pathway (). The TF concentration in tumor patients can be up to 1,000-fold as high as in healthy controls.
- Cancer pro coagulant, a cysteine protease which can activate F X
- TF-bearing micro particles, small membrane vesicles released from tumor cells, monocytes, platelets and endothelial cells after activation of apoptosis play an important role in tumor progression, metastasis and angiogenesis
- Fibrinolysis proteins, for example urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA), as well as plasminogen activator inhibitor 1 and 2 (PAI-1 and PAI-2)
- Cytokines and vascular endothelial growth factors (VEGF). Cytokines stimulate the synthesis of the fibrinolysis inhibitor PAI-1 and down regulate the expression of thrombomodulin (T M). T M has a potent anticoagulant function. It forms a complex with thrombin to activate the anticoagulant protein C that causes the inhibition of F Va and F VIIa.
- Expression of adhesion molecules on the tumor cell membrane allows interaction with host cells (endothel cells, platelets) and promotes localized clotting activation to the vessel wall and to start thrombus formation.
Approximately 20% of all new cases of VTE are associated with cancer whereas 26% of incident cases have idiopathic VTE /, /. The relative risk of developing VTE is 7-fold higher in patients with active cancer. The incidence of VTE is higher in the first few months (30–60 days) after cancer diagnosis and thereafter the incidence decreases with time. The VTE incidence rate is higher in metastatic cancer and a faster growing cancer, as evidenced by early recurrence and death. The metastasis stage at the time of diagnosis is an independent risk factor for the development of VTE within the first year after tumor diagnosis. Mucinous adenocarcinomas (lung, colon, ovarian cancer) are associated with a higher VTE incidence than other solid tumors. Patients with hematological malignancies (leukemia, lymphoma, myeloma) have relative high rates of VTE.
The incidence of cancer associated VTE is strongly increased in the presence of chronic co morbid conditions such as renal insufficiency, liver disease, hypertension, cardiac disease or psychiatric diseases.
Conventional cytotoxic chemotherapy (thalidomide, lenalidomide) are associated with increased risk of VTE, especially when combined with high dose dexamethasone for the treatment of multiple myeloma. Anti-angiogenic therapy is associated with a higher risk of both arterial and venous thrombosis.
Nonspecific activation of the coagulation cascade can be caused by stasis, immobilization of the patient, necrosis of tumor tissue, in combination with inflammation or infection, due to irradiation or surgery and by foreign objects (e.g., venous catheters and ports).
Tumor-related hemorrhagic diathesis is less common than VTE and primarily occurs in myeloproliferative diseases and metastatic advanced solid tumors as well as under chemotherapy and irradiation therapy. The causes are disseminated intravascular coagulation and thrombocytopenia.
Hemorrhage may proceed the overt clinical diagnosis of leukemia by several months . The most common pre diagnostic manifestations are petechiae, purpura and ecchymoses and are present is 40–70% at the time of leukemia diagnosis. Hemorrhage is most common in acute promyelocytic leukemia. Hemorrhage is less commonly a problem in the chronic myeloid or lymphoid leukemias.
Hemorrhage may be a significant problem in solid tumors as well . Intravascular coagulation in solid tumors may manifest as low-grade disseminated intravascular coagulation (DIC) or as acute fulminant DIC with massive hemorrhage and thrombosis. The acute fulminant DIC in conjunction with thrombocytopenia occurs with bleeding usually being from three unrelated sites simultaneously. The most commonly cancers are pancreatic cancer and mucinous adenocarcinoma of the bronchus, prostate, ovaries and gastrointestinal tract.
The low-grade DIC is manifested clinically by mild to moderate bleeding, usually of the integument or mucous membranes. In many cases, these events are associated with thrombosis triggered by chemotherapy .
Suppression of the bone marrow
Megakaryopoiesis is ultimately suppressed in almost all types of leukemia, advanced lymphoma, multiple myeloma and solid tumors with bone marrow metastases.
Hodgkin’s lymphoma and other lymphoproliferative diseases in conjunction with hypersplenism may cause thrombocytopenia by platelet sequestration in the spleen.
A small portion of tumor patients, especially those suffering from lymphoproliferative disease, may develop antibodies to platelets. Alloimmunization to donor HLA determinants is seen in up to 50% of patients after platelet transfusion.
The thrombocyte count increases again within 1–3 weeks after termination of therapy. However, several substances (e.g., mitomycin and nitrosoureas) have a prolonging effect on the suppression of megakaryopoiesis.
Laboratory tests that are associated with an increased risk of hemostasis disorders in tumor patients may identify high-risk cancer patients. A number of hemostatic tests are suggested for detecting the activation of the coagulation cascade, including D-dimers, thrombin-antithrombin complex, prothrombin fragments, thrombin generation test, PAI-1 and thrombocyte count .
Risk factors for VTE comprise:
- Pre chemotherapy platelet count ≥ 350 × 109/L. For example, 21.9% of tumor outpatients had these levels and presented a 3-fold higher rate of VTE as compared to patients with counts below 200 × 109/L who had a rate of 1.25%
- A leukocyte count ≥ 10 × 109/L
- Elevated CRP and increased D-dimer concentrations (significant indicators)
- A risk model for chemotherapy-associated VTE is depicted in. The different pre chemotherapy patient characteristics were assigned a risk score. A score ≥ 3 points to a VTE risk under chemotherapy.
Preanalytics covers the procedures for the collection, transport, and processing of specimens for plasma-based coagulation. Many pre analytical variables may affect test results of hemostasis assays and may influence important diagnostic and therapeutic decisions. This is especially important in institutions where coagulation analysis is centralized and collection of the blood specimens is decentralized. Such circumstances necessitate the standardization of the collection, transport and storage of the blood samples prior to centrifugation and analysis .
- The traditional methods measure the clotting system in general and are based on detection of a blood clot in a test tube
- The second type determines the level of the individual plasmatic coagulation protein, inhibitory protein or fibrinolytic protein either by immunologic (antigenic) methodology or by analysis of enzyme-specific synthetic substrate release (amidolytic assay).
To obtain venous blood from an antecubital vein, 19- to 21 gauge needles or butterfly needles are ideal. Smaller needles are used in adults with compromised veins or in children and newborns . Using needles with a smaller diameter (gauge of 25 and smaller), whether used with an evacuated tube system or syringe may activate platelets or induce hemolysis. Needles larger than 16-gauge may cause turbulence-induced hemolysis. Winged blood collection sets used in combination with smaller-gauge needles, because of their longer path between vein and anticoagulant, may cause activation of platelets and coagulation.
Blood collection tube
Polypropylene or siliconized glass.
Blood specimens for plasma-based coagulation assays should be collected by venipuncture using a system that collects the specimen directly into a glass or plastic evacuated tube containing the appropriate additive. The blood collection system typically includes a straight needle attached to an adapter used to hold and pierce the collection container . The drawing of a discard tube when performing a PT, INR, or aPTT is abandoned in the recommendations of the Clinical and Laboratory Standards Institute (CLSI) . This is also true for other parameters tested : antithrombin, protein C, and factors II, V, VIII, IX and X.
It is important to avoid contamination with infusion solutions when blood specimens for coagulation assays are drawn from a vascular access device using a blood collection system or a syringe. The infusion should be stopped approximately 5 min. before compression. If blood is obtained from a normal saline lock (a capped-off intravenous port), two dead space volumes of the catheder and extension set should be discarded .
In intensive care patients, blood specimens may be collected from catheters placed in the radial artery and in the subclavian vein. Patients with sepsis present no difference between arterial and venous blood regarding PT (INR), aPTT, fibrinogen and erythrocyte, leukocyte and thrombocyte counts. However, decreased AT activity and increased D-dimer concentrations have been determined in venous plasma. This is thought to be due to the formation of capillary micro thrombi .
Paravenous needle placement or excessively rapid blood collection causes release or aspiration of tissue thromboplastin with subsequent activation of coagulation, also as a result of bubble or foam formation.
Venous compression too long
Compression for too long a period of time causes local activation of fibrinolysis.
Citrate is the standard anticoagulant. In order to inactivate the Ca2+ ions, a trisodium citrate solution (0.109 mol/L or 3.2%) is mixed with the blood at a ratio of 1 : 10 (1 + 9) immediately after the sample is collected. Two citrate molecules bind 3 Ca2+ in the complex C12H10Ca3O14 × 4 H2O.
Incorrect citrate-plasma ratio
A decreased filling of the collection device decreases the blood to anticoagulant ratio below 9 : 1, clotting time in seconds tend to increase . At least a 90% fill volume is recommended. The effect of fill volume on clotting results will also be dependent on the PT and APTT reagent. INR prolongation was reported with less than 90% fill volume .
In common practice 0.10 mL of the citrate can be removed for the majority of samples with HCT values between 0.55 and 0.65 (5 mL collection device) . The citrate solution contained in the blood collection device also plays a role. For example, when the PT was determined using reagents of different manufacturers, differences in the clotting time of up to 8.8% and in the INR of up to 10% were found . The differences between the tubes was explained mostly by the effect of Mg2+ contamination in the citrate solution.
- Below 200 × 109/L for determining the PT/INR, aPTT, antithrombin, protein S, protein C and von Willebrand factor. A relative centrifugal force (RCF) of (1200–1500) × g for 10 min. is recommended.
- Below 10 × 109/L (platelet poor plasma) for determining anti F Xa and lupus anticoagulant. A RCF of 1500 × g for at least 15 min. is required.
After centrifugation, the analysis is performed directly from the collection tube or the sample is transferred to a secondary vessel for storage.
PT: specimens for PT assay can be stored centrifuged or uncentrifuged in an unopened tube for up to 24 hours.
APTT: can be stored centrifuged or uncentrifuged in an unopened tube for up to 4 hours. Specimens containing unfractionated heparin should be kept at room temperature and centrifuged within one hour of collection.
Thrombin time, fibrinogen, AT and D-dimers, protein C, protein S, anti FXa, FV: can be stored centrifuged or uncentrifuged at room temperature and should be tested within 4 hours.
Long-term storage: for longer storage of plasma samples, rapid freezing by immersion of sample tubes in liquid nitrogen at –70 °C is recommended. Plasma samples transferred directly to the storage compartments and stored at -20 °C will yield different results. Depending on the type of coagulation test, frozen and thawed samples generate different results compared to fresh samples . The PT in samples stored at –20 °C decreased by 15% after 2 months and 25% after 4 months; however, in snap-frozen samples stored at –70 °C it only decreased by 15% after 4 months. The aPTT is generally prolonged by up to 10% within 3 months in not snap-frozen samples and by up to 5% in snap-frozen samples stored at –70 °C. Frozen and thawed samples generate slightly higher fibrinogen levels compared to fresh samples. PT and aPTT should be measured in fresh samples, since freezing has an inconstant and unpredictable effect on the results .
Citrated whole blood
PT, aPTT, thrombin time, fibrinogen, antithrobin D-dimer : the mean percentage change after 8 and 24-h storage at ambient temperature was below 10%. Considering the changes in individual samples all parameters can be reliably tested after 8-h storage, since less than 15% of the samples demonstrated individual changes above 10%. Clinical relevant changes over 10% were detected after 24-h storage for aPTT (41% of samples). Antithrombin and fibrinogen demonstrated individual changes of above 15% in about 10% of samples .
Platelet function: for determining the platelet function in platelet aggregation studies, the citrated blood should be analyzed within 4 h after blood withdrawal because of the decline in sensitivity to platelet agonists such as ADP. Platelet testing by light transmission aggregometry and measurement of thrombin adhesion can be conducted after storage of blood for 24 h in heparin (150 U/mL, 1 : 9 v/v) with stimulation by ADP, arachidonic acid, TRAP-6, U46619 and adrenaline without any loss of activity, but not with collagen or ristocetin stimulation .
The Clinical and Laboratory Standards Institute in its guidelines for PT and aPTT testing, states that samples with visible hemolysis (≥ 0.3 g Hb/L or ≥ 0.1% hemolyzed red blood cells) should not be used. It is speculated that :
- Exposure of anionic membrane phospholipids during erythrocytolysis could provide a phospholipid-rich surface to accelerate coagulation reactions and, hence, shorten the coagulation time
- Exposure of membrane phospholipids could compete with thromboplastin for F VIIa availability and increase the coagulation time.
In a study in paired patient specimens with an Hb below 0.3 g/L and up to 7 g/L, with mechanical clot detection assays hemolysis only prolonged the PT from 15.8 ± 8.4 to 16.3 ± 8.7 seconds and the aPTT from 31.6 ± 18 to 32.5 ± 19 seconds. In a different study in samples containing a final lysate concentration of 0.5%, increase in PT was measured and a decrease in aPTT and fibrinogen in samples containing a final lysate concentration of 0.9%.
Hyperbilirubinemia, hyperlipidemia and hemolysis do not have a major effect on coagulation tests using clot detection assays, but interfere with optical detection methods. Aiming to reduce interferences by optical properties of plasma samples, some analyzers enable light transmission to be measured not only at 405 nm, but also at 570 nm. Changing the wavelength should lead to more valid test results, because light transmission at 570 nm is less influenced by absorbance of triglycerides, bilirubin and hemoglobin . The optical spectra of bilirubin, hemoglobin and triglycerides are shown in .
In a study , the results of spectrophotometer measurements were compared with clot detection measurement. The results obtained with a clot detection analyzer is unaffected by the optical properties of the sample and was defined as the reference method for measuring PT, aPTT and fibrinogen.
shows that there are pronounced differences for triglyceride concentrations above 200 mg/dL (2.3 mmol/L), bilirubin above 5 mg/dL (85 μmol/L) and hemoglobin above 0.30 g/L. Changing the spectrophotometer wave length from 405 to 570 nm was associated with a marked increase in valid results.
The quantitative determination of coagulation factors and inhibitors is performed by functional testing and concentration measurements. Functional tests measure the activity of one or several coagulation factors and have the advantage of reflecting the functional behavior of the relevant coagulation factor. Since the enzymatic activity is measured, changes in test conditions (ionic strength, pH, temperature, surface) will have an effect on the result. The following functional tests are distinguished:
- Classical coagulation tests (clot detection assays)
- Tests using synthetic peptide substrates and spectrophotometrical detection (amidolytic assay).
The clotting assays measure the time in which the mixture of reagents and sample starts to clot after Ca2+ are added. For the determination of PT the coagulation process is started by incubation of a plasma sample with thromboplastin and Ca2+. The time to formation of a fibrin clot is measured (clotting time). The clotting time correlates with the decrease in activity of the relevant coagulation factor(s). The measurement is mechanical, for example, the time when the coagulation process is started until the standstill of the ball rotating in the coagulometer due to the formed clot is recorded (). The results in percentage of normal are derived from a standard curve from measurement of PT in standard human plasma.
Turbidimetric testing: during the coagulation process in the reaction mixture agglutinates are formed and the preparation becomes increasingly turbid. The increase in agglutination is an indicator of clot formation and reduces the amount of light passing through the cuvette or increases the amount of scattered light. Measurement results are calculated from light transmission or scattered light.
Chromogenic (amidolytic) assays
The peptide substrates used in these assays are composed of a short peptide chain (2 to 4 amino acids) labeled with a chromophore (e.g., para-nitroaniline) behind the cleavage side. The peptide sequence can be selected to ensure high binding specificity for the relevant coagulation factor. The chromophore labeled peptide chain shows an absorbance maximum at shorter wavelengths than the free chromophore.
Assessment of hemostasis involves plasma based assays that measure plasma clotting time after addition of Ca2+ Plasma clotting time contains a series of reactions including contact reaction, thromboplastin formation, prothrombin conversion, and thrombin-fibrinogen interaction ().
The plasma clotting tests are first line tests for diagnosis, intervention, management of hemorrhage and thromboembolic events, and include the following tests:
- PT for evaluation of the extrinsic activation of plasma clotting (see )
- aPTT for evaluation of the intrinsic activation of plasma clotting (see )
- Thrombin time (TT), a routine screening test to specifically test for disorders of the thrombin-fibrinogen reaction (see )
- Fibrinogen for quantitatively measuring fibrinogen because of the lack of sensitivity of the TT to changes in fibrinogen levels
- Tests using endogenous thrombin potential for determining the amount of thrombin that can be activated in patients with suspected hyper- or hypo coagulability (see ).
Global hemostasis assays like thrombelastography (TEG) measure the viscoelastic changes occurring during clot formation. In peri operative transfusion management TEG is better suited than the functional tests to estimate the complex interactions among pro coagulant proteins, their natural inhibitors and blood cells .
- Peri operative blood product use by providing specific information about coagulation status and the nature of coagulation dysfunction
- Trauma-associated coagulopathy
- Identification of hyper fibrinolysis
- The combination of hyper coagulability and hyper fibrinolysis
- Advanced disseminated intravascular coagulation
- Thrombolytic drug therapy.
TEG supplements conventional tests (PT, aPTT, thrombocyte count) and provides information on the coagulation status and the type of coagulation disorder, especially during the peri operative phase.
Principle: the TEG consists of a heated sample cup that oscillates at ± 4° 45’ every 5 seconds, into which a pin is suspended by a torsion wire. During coagulation, the forming clot results in a physical connection between each cup and pin, transferring the torque of the cup to the pin. The rate of clot formation and its elastic strength affect the magnitude of motion of the pin and its range of oscillation. Computer software produces both quantitative parameters and a graphic representation of the phases of clot formation. Refer to:
Citrated venous whole blood (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 3 mL
Example of a system:
- Coagulation time (R): 100–240 sec.
- Clot formation time (K): 30–110 sec.
- Maximum clot firmness (MA): 53–72 mm
- Clot lysis at 30 min. and 60 min. after MA: below 15%.
The TEG result is substantially influenced by the thrombocyte count, fibrinogen concentration and presence of hyper fibrinolysis. The latter is an important problem in trauma-associated coagulopathy which may require antifibrinolytic therapy in addition to substitution of blood and blood products. However, hyper fibrinolysis is not detected by conventional tests in the laboratory .
Other biological influence factors affecting the TEG include F XIII activity and fibrin polymerization impaired, for example, by D-dimers and thrombin inhibitors.
1. Clinical and Laboratory Standards Institute. Collection, transport, and processing of blood specimens for testing plasma-based coagulation assays and molecular hemostasis assays; approved guideline – fifth edition. Wayne, Pennsylvania 2008.
7. Van den Besselaar AMPH, Hoekstra MCL, Witteveen E, Didden JH, van der Meer FJM. Influence of blood collection systems on the prothrombin time and international sensitivity index determined with human rabbit thromboplastin reagents. Am J Clin Pathol 2007; 127: 724–9.
8. Christensen TD, Jensen C, Larsen TB, Maegaard M, Christiansen K, Sorensen B. International normalized ratio (INR), coagulation factor activities and calibrated automated thrombin generation – influence of 24 h storage at ambient temperature. Int Jnl Lab Hem 2010; 32: 206–14.
10. Kemkes-Matthes B, Fischer R, Peetz D. Influence of 8 and 24-h storage of whole blood at ambient temperature on prothrombin time, activated partial thromboplastin time, fibrinogen, thrombin time, antithrombin and D-dimer. Coagulation and fibrinolysis 2011; 22: 215–20.
14. Junker R, Käse M, Schulte H, Bäumer R, Langer C, Nowak-Göttl U. Interferences in coagulation tests – evaluation of the 570 nm method on the Dade Behring BCS analyser. Clin Chem Lab Med 2005; 43: 244–52.
The PT is the best screening test for the reactions of the extrinsic activation way. It measures the extrinsic activation of factor X by the tissue thromboplastin -factor VII complex and the resulting common pathway reaction. The test is sensitive for deficiencies of F VII, F X, F V, F II and for fibrinogen (see ). The PT specified as International Normalized Ratio (INR) is used to assess the effect of oral anticoagulant therapy.
- Acquired or congenital deficiency of F VII, F X, F V, F II and fibrinogen
- Monitoring of oral anticoagulant therapy
- Diagnosing of vitamin K deficiency (neonates)
- Monitoring of substitution therapy with plasma (fresh frozen plasma, prothrombin complex)
- Assessment of the liver function
- Criterion of score systems.
The PT measures the clotting time of plasma in the presence of an optimal concentration of tissue factor extract.
In citrated plasma the transformation of prothrombin into thrombin is started after the addition of tissue thromboplastin and Ca2+. Fibrinogen is converted into fibrin by the generated thrombin and the fibrin formation time (clotting time) is measured (see ). Tissue thromboplastin is produced from tissue (rabbit brain, human placenta) and contains tissue factor and phospholipids as active components. The PT is expressed in seconds or as Quick value in % of normal . The percentage values are determined by producing a serial dilution of a plasma pool of healthy individuals with distilled water. A Quick value of 50% of normal means that the sample has the same clotting time as one volume part of plasma diluted with one volume part of distilled water. The lower the coagulation capacity of the sample, the longer the clotting time (PT) and the lower the Quick value in %.
A distinction is made between PT according to the Quick method and according to the Owren method . The Quick method determines fibrinogen and factors II, V, VII and X. It has the advantage of diagnosing coagulation factor deficiency because it measures F V. PT according to Owren only measures factors II, VII and X. In oral anticoagulant therapy, PT according to Owren has the advantage that the F V, which is not affected by vitamin K antagonists, is not taken into account in the determination.
Principle: Analogously to the coagulation test, the reaction in the chromogenic assay is started by adding a thromboplastin reagent which, besides Ca2+ contains a thrombin-specific chromogenic substrate (). Due to the higher affinity of the chromogenic substrate for thrombin in comparison with fibrinogen, the chromogenic substrate is cleaved first as soon as traces of thrombin are produced, thus releasing a chromophor. When a certain, predefined threshold value of the optical density is reached during the spectrophotometric monitoring of the chromophor (usually p-NA), this difference is specified as the “clotting time”. Only after the chromogenic substrate has been almost completely consumed, will fibrinogen be cleaved as well. Since the products thus produced will also cause additional optical density, the fibrinogen concentration (derived fibrinogen) can be calculated based on the total absorption after complete consumption of both substrates (chromogen and fibrinogen) minus the constant portion of the added chromogenic substrate.
This method is mainly used in outpatient care and patient self-monitoring.
A battery-powered laser photometric device is used for measuring . A drop of capillary blood is applied to the application field of a pre warmed test pad and drawn by diffusion to the reagent pad, where it is mixed with rabbit brain thromboplastin bound to iron oxide particles, thus triggering the coagulation cascade. The blood continues to diffuse into the reagent pad and coagulates. The content of iron oxide particles in the agglutinates is dependent on the activity of the clotting system. An electromagnet located below the reagent pad in the measuring device initiates the iron oxide particles to regular pulsation at a frequency of 2 Hz. The regular pulsation pattern is recorded by reflectance photometry. As a fibrin matrix is formed, the iron oxide particle movement is inhibited and eventually stopped. This causes a decrease in reflectance and recorded as start of coagulation. Calibration is performed by the manufacturer and stored in the device.
The use of International Normalized Ratio (INR) is recommended to achieve international harmonization of measured PT values and therapeutic PT ranges for monitoring anticoagulant therapy with vitamin K antagonists . The prothrombin time ratio is determined by the equation in . By using the ISI, the INR can be calculated from the prothrombin time ratio.
The International Sensitivity Index (ISI) is a value that describes the ratio of the manufacturers thromboplastin in relation to the WHO reference thromboplastin preparation (IRP 47/60) /, /. Contemporary thromboplastins have usually ISI values of about 1 . Less sensitive reagents have ISI values higher than 1.
Reporting of results
The clotting time of the PT tests is expressed in seconds or in % of normal. In patients undergoing oral anticoagulant therapy, the PT should be expressed as International Normalized Ratio (INR).
- Venous citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 1 mL
- Capillary blood, obtained by using a sodium citrate containing capillary: 0.025 to 0.1 mL
The PT is a measure of the integrity of the extrinsic pathway and the final step of fibrin formation (). The PT test does not take account of factors VIII, IX, XI, XII, XIII and high-molecular-weight kininogen (HMWK).
Prolonged PT is caused by:
- Oral anticoagulants (vitamin K antagonists)
- Factor deficiency
- Dysfibrinogenemia and fibrin(ogen) degradation products
- Antibodies to coagulation factors
- New oral anticoagulants.
Except for F IX, the PT test according to Quick records 3 of 4 vitamin K dependent factors (F II, F VII and F X). If the platelet count, the bleeding time and the thrombin time are normal, the prolongation of the PT suggests a reduction in the vitamin K dependent factors or in F V, regardless of the aPTT value.
The extrinsic pathway is checked by means of F VII while the other factors X, V, II and fibrinogen represent the common final steps of both extrinsic and intrinsic pathways of the coagulation system. Since all of these factors are produced in the liver, the PT is a good criterion for assessing the protein synthesis capacity of the liver. F V deficiency is not detected by the PT test according to Owren.
Vitamin K antagonists result in the functional alteration of the synthesis of the four coagulation factors II, VII, IX and X as well as of the inhibitors protein C and protein S. Since the activity of factors II, VII and X is measured by the PT, its value represents a good tool for monitoring thromboprophylaxis ().
Method of determination
The underlying cause for the lack of comparability between the PT values of different manufacturers can be:
- Thromboplastin preparations present various sensitivities toward factors II, V, X and VII
- PIVKAs (protein-induced vitamin K absence), which possibly enter into the PT determination, interfere with the activation of the normal coagulation factors. PIVKA involves the non-γ-carboxylated precursor proteins of the coagulation factors II, VII, IX and X, as a result of therapy with vitamin K antagonists.
New oral anticoagulants: rivaroxaban administered at normal dose prolongs the PT clotting time.
Inadequate pre warming of the reagents: prolonged PT clotting time.
Heparin: depending on the reagent used, heparin may (at concentrations of 0.8–2 U/mL plasma) prolong the PT.
Fibrinogen degradation products: lead, usually at concentrations > 50 mg/L, to a prolongation of the PT.
Comparability of the PT measurement results: expression in INR establishes comparability even when different thromboplastins have been used.
Drugs: penicillins cause a decrease in the PT; this is especially important in children.
3. Dati F, Barthels M, Conard J, Flückiger J, Girolami A, Hänseler E, et al. Multicenter evaluation of a chromogenic substrate method for photometric determination of prothrombin time. Thromb Haemostas 1987; 58: 856–65.
4. Burri S, Biasutti FD, Lämmle B, Wuillemin WA. Vergleich der Quick-/INR-Werte aus kapillärem Vollblut (CoaguChek Plus) und venösem Citratplasma bei Patienten mit und ohne orale Antikoagulation. Schweiz Med Wochenschr 1998; 128: 1723–9.
5. International Committee for Standardization in Haematology, International Committee on Thrombosis and Haemostasis. ICSH/ICTH recommendations for reporting prothrombin time in oral anticoagulant control. Thromb Haemostas 1985; 53: 155–6.
12. Baglin T, Hillarp A, Tripodi A, Elalamy I, Buller H, Ageno W. Measuring oral direct inhibitors of thrombin and factor Xa: a recommendation from the Subcommittee on Control of Anticoagulation of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. J Thrombos Haemostas 2013; 11: 756–60.
14. Gest Daalderop JHH, Mulder AB, Boonman-De Winter LJM, Hoekstra MMMCL van den Besselaar AMPH. Preanalytical variables and off-site blood collection. Influences on the results of PT/INR test and implications for oral anticoagulant therapy. Clin Chem 2005; 51: 561–8.
The aPTT is the best screening test for the reactions of the intrinsic activation. The test is sensitive for deficiencies of F IX, F VIII, F X, F V, F II and for fibrinogen (see ).Thus, it evaluates all coagulation factors except for F VII, albeit with a sensitivity different from that of the PT. The aPTT is not sensitive to minor coagulation factor deficiencies, and since the activities of many factors are measured together, overall prolongation may be masked by elevated levels of one or more individual factors. The aPTT is sensitive for the influence of heparin or similar anticoagulants on blood coagulation /, /.
- Congenital deficiency of F VIII (hemophilia A) , FIX (hemophilia B) or of von Willebrand factor which sometimes is the reason for F VIII deficiency
- Congenital deficiency of F IX (hemophilia B)
- Acquired deficiency of F VIII, F IX and F XI
- Monitoring of therapy with parenterally administrated anticoagulants (unfractionated heparin, hirudin, aprotinin)
- Screening test for anamnestic or clinically manifest bleeding tendency or predisposition to thrombosis
- Suspected presence of inhibitors (lupus anticoagulant, F VIII inhibitor) in the plasma mixing test.
In general, the aPTT is mainly used for preoperative screening and for heparin monitoring
Partial thromboplastins are lipid reagents that are used as substitutes for platelets in clotting tests. Activated partial thromboplastins are mixtures of contact activators (micronized silica, ellagic acid or kaolin) and partial thromboplastins and are used to perform the activated partial thromboplastin time (aPTT) .
The PTT is a is a modification of the plasma recalcification time, a test performed in platelet rich plasma. In the aPTT, a platelet substitute (contact activator) is added and platelet activity is not tested for .
The addition of a partial thromboplastin reagent to patient plasma gives maximum platelet activity (aPTT assay ) than that obtained with the simple addition of platelet poor plasma to partial thromboplastin (plasma recalcification time assay) .
Activated partial thromboplastin is added to re calcified citrated plasma, followed by incubation at 37 °C for a few minutes. This results in the contact activation of F XII and F XI. The measured clotting time (seconds until formation of a coagulation clot) is expressed in seconds. The clotting time depends on the activation of the intrinsic pathway factors .
Chromogenic (amidolytic) assay
Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 1 mL
- In cases of F XI, F IX and F VIII deficiency in the range of 30–40% of normal
- In cases of F II, F V and F X deficiency in the range below 30%.
Impaired fibrin polymerization (dysfibrinogenemia) and fibrin(ogen) degradation products (FDP) have only little influence on the coagulation time. The aPTT is only prolonged in high concentrations of FDP.
The detection limit of the aPTT depends on the reagents used. This applies also to the suitability for the therapeutic monitoring of heparin therapy and the lupus anticoagulant sensitivity.
Approximately 95% of congenital bleeding disorders are associated with prolonged aPTT. A factor deficiency may not cause prolonged aPTT until its activity has decreased to below 30–40%. Hence, mild factor deficiencies are often not identified .
If the platelet count, the bleeding time and the screening tests of the coagulation system such as the PT and the thrombin time are normal, a prolonged aPTT suggests the presence of hemophilia A or B, especially when additional corresponding data in the medical history support this diagnosis. A reduction in F VIII:C and F IX activity to below 40% of normal may be associated with a significantly increased perioperative bleeding tendency. Therefore, the aPTT is prolonged in such cases. F VIII:C is the low-molecular portion of F VIII that mediates coagulation activity.
The following tests are indicated for differentiation:
- Thrombin time and possibly reptilase time can identify a heparin effect and hypo- or dysfibrinogenemia
- Plasma mixing test can differentiate between factor deficiency and an antibody against a coagulation factor. Normalization of aPTT after addition of normal plasma to the patient plasma indicates factor deficiency. If the aPTT stays abnormal the presence of an antibody or lupus anticoagulant is possible.
- Since the aPTT is less influenced by fibrin(ogen) degradation products (FDP) than the thrombin time (TT), there is no need to examine the sample for the presence of FDP if the thrombin time test is normal.
Intravenous administration of unfractionated heparin enhances the inhibitory effect of AT on factors XII, XI, IX, X and II, thus resulting in a decreased activity of the intrinsic pathway of the coagulation system. In therapeutic anticoagulation with unfractionated heparin, plasma heparin concentrations of 0.2–0.5 IU/mL of plasma are achieved which corresponds to a mean 1.5–2.5-fold increase of the baseline aPTT value . Values below 1.5 indicate an inadequate level of anticoagulation and are associated with an increased risk of thromboembolism. Prolongations over 2.5 times above the baseline value may indicate the risk of bleeding.
- Commercial aPTT reagents represent individual test systems which differ in the source of their partial thromboplastins. Therefore, the general specification of 1.5–2.5-fold prolongation as the therapeutic range is incorrect and may result in inappropriate heparin doses.
- aPTT values and heparin sensitivity are lower in patients with acute-phase response than in normal controls, possibly due to an increase in fibrinogen and F VIII. Especially F VIII is thought to compete for heparin with heparin binding protein.
- Unless the aPTT assay is performed on heparinized plasma within 30 min. after blood collection, unduly prolonged aPTT values will be recorded. Delays of 1 h result in approximately 20% and 90 min. result in approximately 60% prolongation of the aPTT baseline value.
Since the low-molecular-weight heparins are primarily directed against F Xa, the aPTT is only slightly prolonged (low activity of low-molecular-weight heparins against thrombin).
Depending on the aPTT reagent used, recombinant hirudin prolongs the clotting time by 17–40% . Above a certain value the aPTT will no longer show a linerar dose response relationship to hirudin (plateau effect), thus overdosing will not be identified.
Shortened aPTT may be related to pre analytical problems such as improper procedure during blood collection (venous compression too long), improper handling of the sample (heavy shaking of the sample tube) or too long a storage time. Therefore, it is recommended to collect another sample and perform the analysis again. If the clotting time is still shortened, this may point to :
- Increased activity of FVIII, an acute phase protein
- Increased risk of a thromboembolic event
- Multiple biological influence factors such as pregnancy, hyperthyroidism, diabetes mellitus, malignant tumor, myocardial infarction.
The reference interval depends on the individual laboratory (aPTT reagent, method, analyzer). If possible, each laboratory should determine its own reference interval (2.5th and 97.5th percentile of the clotting times of at least 40 healthy individuals) for the aPTT . The lower reference interval value should also be specified since a shortened aPTT indicates hyper coagulability as well as premature activation of the blood sample due to improper blood collection procedure.
The properties of the reagents depend on the type and concentration of the partial thromboplastin and the surface activator . Phospholipids from placental tissue, brain extracts and plants are used as a substitute for platelet factor 3 while kaolin, ellag acid and silica are used as surface activators. The detection limit of a certain aPTT method therefore depends on the reagent used. This applies not only to the detection limit in regard to a factor deficiency but also to the suitability for the monitoring of a therapy using heparin or to the detection of lupus anticoagulant.
Performance of the aPTT determination
The incubation period specified for each aPTT reagent must be strictly observed. Too short an incubation period results in a non reproducible prolongation of the aPTT.
New oral anticoagulants: rivaroxaban prolongs the aPTT, as does dabigatran even more markedly.
5. Hellstern P, Oberfrank K, Köhler M, Heinkel K, Wenzel E. Die aktivierte partielle Thromboplastinzeit als Screeningtest für leichte Gerinnungsfaktorenmängel – Untersuchungen zur Sensitivität verschiedener Reagenzien. Lab Med 1989; 13: 83–6.
7. Strekerud FG, Abildgaard U. Activated partial thromboplastin time in heparinized plasma: influence of reagent, acute phase reaction, and interval between sampling and testing. Clin Appl Thrombosis/Hemostasis 1996; 2: 169–76.
12. Baglin T, Hillarp A, Tripodi A, Elalamy I, Buller H, Ageno W. Measuring oral direct inhibitors of thrombin and factor Xa: a recommendation from the Subcommittee on Control of Anticoagulation of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis. J Thrombos Haemostas 2013; 11: 756–60.
The endstage of both extrinsic and intrinsic activation of the plasmatic coagulation system is the conversion of fibrinogen to fibrin by the proteolytic action of thrombin. It is possible to isolate this reaction and estimate the quantity and reactivity of fibrinogen in plasma by adding a specific amount of exogenous thrombin and measuring the speed of clot formation. A standardized procedure of this kind is known as the thrombin time .
The TT is a screening test to specifically test for disorders of the thrombin-fibrinogen action. Among the factors of the plasma coagulation system, the TT only determines fibrinogen and not the fibrin-stabilizing F XIII.
- Testing for the presence of disseminated intravascular coagulation (DIC)
- Clarification of manifest bleeding tendencies
- Investigation of unclear pathological PT and aPTT findings (exclusion of impaired fibrin polymerization)
- Confirmed absence of heparin or hirudin in samples used for thrombophilia clarification
- Thrombolytic therapy. Evaluation of the total effect of decreased fibrinogen, fibrin(ogen) degradation products and (possibly) heparin
- Monitoring of dabigatran therapy.
The TT is the time elapsing between the addition of a standardized quantity of thrombin to patient plasma and the formation of fibrin. Thus, effects of plasma-inherent thrombin formation disorders on fibrin formation do no apply. The TT is a function of the fibrinogen concentration and the quality and presence of antithrombin .
Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 1 mL
- The aPTT clotting time is prolonged and the presence of thrombin inhibitors such as heparin and hirudin in the plasma is suspected
- Disorders in the fibrinogen-to-fibrin conversion are suspected. The TT is prolonged in hypo fibrinogenemia, dysfibrinogenemia, the presence of fibrin(ogen) degradation products (FDP) and monoclonal immunoglobulins.
If the TT is prolonged in the face of a normal platelet count, a normal bleeding time, a normal PT and a normal or slightly prolonged aPTT, this finding indicates the presence of heparin in the plasma or the presence of FDP (e.g., as seen in conjunction with disseminated intravascular coagulation).
The TT measures thrombin-induced fibrin formation and fibrin aggregation (i.e., the last step in the process of coagulation) but the test does not detect fibrin polymerization (i.e., the covalent cross linking between the fibrin chains by F XIII).
The TT is prolonged if:
- The function of the test thrombin added to the patient plasma is inhibited (e.g., as observed in conjunction with heparin therapy or in the presence of thrombin antibodies). Under recombinant hirudin therapy, the mean TT prolongation is 195% and the maximum prolongation is 282% .
- A defect in the aggregation of fibrin occurs due to the presence of FDP (e.g., as observed in thrombolytic therapy)
- Low fibrinogen concentrations (below 0.6 g/L) or dysfibrinogenemia (severe liver cell damage).
The desirable therapeutic range for heparin therapy and thrombolytic therapy using streptokinase or urokinase is 2–4-fold prolongation of the upper reference interval value.
In patients with severe infections (sepsis), hepatic disease and massive myocardial infarction, the TT is a better indicator of heparin therapy than the aPTT. The aPTT, due to prolongation independent from heparin, (e.g., as a result of impaired contact activation in conjunction with pre kallikrein deficiency, overestimates the heparin activity) .
Shortened TTs are of no clinical relevance and, at most, are indicators of increased fibrinogen concentrations.
Using the same reagents and reaction mixtures turbidimetric and nephelometric analyzers measure shorter TT values than the clotting assays.
High fibrinogen levels may, in the absence of heparin, prolong the TT. In the presence of heparin, however, they will shorten the TT and may suppress the detection of heparin, especially after freezing of the sample.
The TT depends on the concentration and the type of thrombin used. The final concentration in the test sample usually amounts to 1 IU/mL of plasma.
Under the influence of thrombin on fibrinogen, fibrin monomers are formed as the fibrinopeptides A and B are cleaved off. These fibrin monomers form soluble aggregates. The effects of thrombin-activated F XIII and of Ca2+ finally result in polymerization, thus yielding insoluble, crosslinked fibrin (see ).
Heparin (antithrombin effect) or FDP (inhibition of fibrin polymerization in concentrations > 50 mg/L) cause a, concentration-dependent, prolongation of the TT. The effect of heparin is identified by performing tests using thrombin-like enzymes (e.g., by determining the reptilase time).
The TT does not allow differentiation between defects in the thrombin-fibrinogen interaction and defects in the aggregation of fibrin monomers. This differentiation requires the parallel use of tests with thrombin-like enzymes.
5. Bounameaux H, Marbet GA, Lämmle B, Eichlisberger R, Duckert F. Comparison of thrombin time, activated partial thromboplastin time and plasma heparin concentration and analysis of the behavior of antithrombin III. AJCP 1980; 74: 68–73.
Batroxobin (reptilase) and thrombin coagulase are enzymes which are able to clot fibrinogen:
- Batroxobin cleaves fibrinopeptide A from fibrinogen
- Thrombin coagulase, a complex composed of prothrombin and coagulase from staphylococci, cleaves fibrinopeptide A and B.
Both thrombin-like enzymes are not influenced by heparin.
- Screening for impaired fibrin polymerization
- Differentiation between thrombin inhibition and impaired fibrin polymerization in the presence of prolonged thrombin time
- Thrombolytic therapy: evaluation of the total effect of decreased fibrinogen, fibrin(ogen) degradation products and (possibly) heparin
- Diagnosis of predisposition to bleeding based on clinical manifest symptoms or the medical history of the patient.
Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 1 mL
The batroxobin time and the thrombin coagulase clotting time are only influenced by disorders in the polymerization of fibrin monomers.
Causes of prolonged clotting time include:
- The inhibition by fibrin(ogen) degradation products (FDP) which form soluble complexes with fibrin monomers. As the inhibition of fibrin aggregration is concentration-dependent, the clotting times in tests using thrombin-like enzymes correlate with the concentration of the FDP. However, this only applies to concentrations above 50 mg/L.
- Low fibrinogen concentrations
Clotting times are not prolonged in the presence of:
- Thrombin inhibitors such as heparin and hirudin
- Immunoglobulins, penicillins, protamine chloride.
The inhibitory effect on polymerization of fibrin monomers only occurs at high immunoglobulin concentrations. Thrombin inhibitors that can be detected by a combination of tests using the thrombin time and thrombin-like enzymes are rare.
In combined thrombolytic and heparin therapy, assessment of the lytic activity with the batroxobin time and the thrombin coagulase time is more specific than with the heparin-dependent thrombin time because there is no interference by heparin-antithrombin complexes.
Tests using thrombin-like enzymes react more sensitive to decreases in the fibrinogen concentration than the thrombin time. Often, low or greatly elevated fibrinogen concentrations are observed in intensive-care patients without any significant rises in the fibrinogen degradation products. Under such circumstances, tests using thrombin-like enzymes produce slightly prolonged clotting times which may be falsely interpreted to reflect an increased state of fibrinolysis.
The generation of thrombin is a fundamental part of the coagulation system. The thrombin generation time test (TGT) measures the amount of active thrombin produced in plasma or whole blood after recalcification. The original TGT is technically difficult to perform and time consuming. Methods based on sensitive chromogenic or fluorogenic peptide substrates have been developed that measure the endogenous thrombin potential (ETP) . The ETP represents the total amount of thrombin that can be generated as a function of the balance between the pro coagulant drivers (thrombin formation) and anticoagulant drivers (thrombin consumption) operating in plasma. The pro coagulant drivers comprise the coagulation factors downstream from F XII, while the anticoagulant drivers are represented by antithrombin (AT) and protein C (PC) .
By measuring the ETP in a TGT, it is possible to determine the amount of activated thrombin formed after recalcification of the plasma or whole blood.
- Investigation of patients with a congenital deficiency of anticoagulant factors
- Investigation of patients with an acquired deficiency of coagulation in which both pro- and anticoagulant drivers are decreased (liver cirrhosis, neonatal period)
- Management of patients undergoing therapy with thromboprophylactic drugs
- Situations where the PT and aPTT are not conclusive.
The TGT measures the speed of the reactions of the intrinsic activation by recalcification of plasma when there is minimal thrombocyte activity and surface activation. In contrast to PT and aPTT which only measure the initiation phase the TGT measures the initiation and propagation phase .
In the propagation phase:
- The bulk of thrombin is generated via feedback loops on factors V, VIII and XI (see and )
- After that the thrombin formation declines by the anticoagulant pathways and all thrombin activity is inhibited by plasma protease inhibitors (termination phase).
Two fluorescence measurements of the patient plasma are required for each CAT assay. In the measurement tube tissue factor and synthetic phospholipid vesicles are added to the plasma to initiate coagulation and thrombin formation. In the calibration tube a known amount of substrate-converting activity (the thrombin calibrator) is added to plasma without activating coagulation. The thrombin calibrator consists of thrombin bound to α2-macroglobulin, thus thrombin is protected from inhibition by protease inhibitors of the plasma. A mixture of CaCl2 and fluorogenic substrate is subsequently added in both tubes and the developing fluorescence is recorded by a fluorometer. The fluorescence in the measurement tube is produced by thrombin generated in plasma following initiation of coagulation and forms the basis for the thrombin generation curve. In the calibration tube the fluorogenic substrate is converted at a constant rate by the added thrombin calibrator. A typical thrombin generation curve is shown in . A short lag phase (initiation) is followed by an intensive increase of thrombin (propagation) that subsequently disappears due to inhibition by mainly antithrombin (termination).
- T-lag; the lag time is defined as the time needed for thrombin concentration to reach 1/6 of the peak concentration and shows a good correlation with the plasma clotting time
- Peak time or T-max; the time elapsing until the peak of thrombin generation is reached
- Peak height or C-max; the maximum thrombin activity generated
- Endogenous thrombin potential (ETP), identical with or area under curve (AUC). The ETP represents the total enzymatic activity of thrombin during the time and is generally considered the most predictive parameter of bleeding/thrombosis risk.
Prolonged T-lag and decreased C-max or ETP indicate a hypo coagulable (pre hemorrhagic) state. Vice versa, short lag times and high ETP/peak heights point at a hyper coagulable (prothrombotic) state . A test method for whole blood has also been published .
Citrated plasma, citrated whole blood, depending on the method used.
Depending on the method used.
Thrombin is a multifunctional protease that, in addition to hemostasis, supports non hemostatic mechanisms, such as the regulation of vascular permeability, vascular tone, inflammation, and angiogenesis.
Among the functions of the coagulation system mediated by thrombin, the most important are the fibrinogen-to-fibrin conversion, the activation of factors V, VIII, XI, XIII and protein C, and the activation of platelets. Large amounts of thrombin are inactivated by antithrombin.
Thrombin enhances not only its own formation during the propagation phase of the coagulation system (see ), but is also self-inhibiting, in the following manner: thrombin and PC bind to thrombomodulin of the vascular endothelium, activating PC to APC. With involvement of PS, APC inactivates factors Va and VIIIa, thus lowering an upregulated coagulation (see .
Determination of the ETP is superior to PT and aPTT for indicating the overall hemostatic function . In these tests, plasma has been estimated to start to clot as soon as 5% of the total amount of thrombin has been generated, thus leaving the remaining 95% undetected. Furthermore, owing to the relatively short interval (10–30 sec.) from the initiation of coagulation to clot formation, naturally occurring anticoagulants operating in plasma (i.e., antithrombin and PC) cannot be fully activated, especially if one considers that they require heparin-like substances (antithrombin) and thrombomodulin for their activation. These substances are located on endothelial cells and not in the plasma. Therefore, the PT and aPTT tests are unlikely to account for the total amount of thrombin generated as a function of the pro coagulant drivers and for its inhibition as a function of the anticoagulant drivers. The PT and aPTT are well known to be suitable for detecting deficiencies of the pro coagulant factors, but they are much less suitable for detecting deficiencies of the anticoagulant factors .
In cases with hemorrhage, the ETP, bleeding tendency and factor deficiency are well correlated if platelet-poor plasma is used . In hemophiliacs with undetectable F VIII or F IX, ETP analyzed in platelet-rich plasma has been reported to have high potential for distinguishing between mild and severe bleeding tendencies .
In patients with the risk of venous thromboembolism (VTE) and congenital thrombophilic defects such as deficiencies in AT, PC, in carriers of factor V Leiden or prothrombin G20210A mutation and an imbalance of pro- versus anti-coagulation the TGT is a valuable indicator for hyper coagulability. Numerous epidemiological prospective and retrospective studies describe significant correlations between the ETP and the risk of VTE . This refers to both the first event and recurrent thrombosis. The determination of the ETP in acquired thrombophilia, for example in pregnancy and diabetes mellitus , is also of diagnostic and prognostic significance.
4. Duchemin J, Pan-Petesch B, Arnaud B, Blouch MT, Abgrall JF. Influence of coagulation factors and tissue factor concentration on the thrombin generation test in plasma. Thromb Haemost 2008; 99: 767–73.
5. Ninivaggi M, Apitz-Castro R, Dargaud Y, Bas de Laat, Hemker HC, Lindhout T. Whole-blood thrombin generation monitored with a calibrated automated thrombogram-based assay. Clin Chem 2012; 58: 1252–9.
6. Duckers C, Simoni P, Spiezia l, Radu C, Gavasso S, Rosing J, et al. Residual platelet factor V ensures thrombin generation in patients with severe congenital facor V deficiency and mild bleeding symptoms. Blood 2010; 115. 879–86.
7. Santagostino E, Mancuso ME, Tripodi A, Chantarangkul V, Clerici M, Garagiola I, et al. Severe hemophilia with mild bleeding phenotype: molecular characterization and global coagulation profile. J Thromb Haemost 2010: 8: 737–43.
8. Castoldi E, Simoni P, Tormene D, Thomassen MC, Spiezia L, Gavasso S, et al. Differential effects of high prothrombin levels on thrombin generation depending on the cause of hyperprothrombinemia. J Thromb Haemost 2007: 5: 971–9.
10. Tripodi A, Branchi A, Chantarangkul V, Clerizi M, Merati G, Artoni A, et al. Hypercoagulability in patients with type 2 diabetes mellitus detected by a thrombin generation assay. J Thromb Thrombolysis 2011; 31: 165–72.
11. van Hylckama Vlieg A, Christiansen SC, Luddington R, Cannegetier SC, Rosendaal FR, Baglin TP. Elevated endogenous thrombin potential is associated with increased risk of a first deep venous thrombosis but not with the risk of recurrence. Br J Haematol 2007; 138: 769–74.
13. Debaugnies F, Azerad MA, Noubouossie D, Rozen L, Hemker HC, Corazza D, et al. Evaluation of the procoagulant activity in the plasma of cancer patients using a thrombin generation assay. Thrombosis Res 2010; 126: 531–5.
15. Chantarangkul V, Clerici M, Bressi C, Giesen PLA, Tripodi A. Thrombin generation assessed as endogenous thrombin potential in patients with hyper- or hypocoagulability. Haematologica 2003; 88: 547–54.
Analyses of individual coagulation factors are performed in suspected hereditary defects, acquired decreases or isolated increases of a coagulation factor. The focus of the investigations is on factors VII, VIII, IX and on the von Willebrand factor (VWF). Screening tests such as the prothrombin time (PT) and activated partial thromboplastin time (aPTT) can provide hints, but the analysis of individual factors is usually required for further clarification.
Hereditary factor defects and acquired factor deficiencies are distinguished. Acquired deficiencies are due to defective synthesis or turnover and manifest in the presence of underlying disease. Except for F XII deficiency, they usually involve several coagulation factors.
Factors VIII, IX and VWF play a special role because they may cause hemorrhage and venous thrombosis.
- Suspected congenital or acquired deficiencies or defects in one or several coagulation factors in the presence of hemorrhage
- Investigation of abnormal results obtained by one or several screening tests: PT, aPTT, thrombin time
- Therapy monitoring in conjunction with the use of coagulation factor concentrates
- Suspected genetic defects of factors VIII, IX and VWF in thrombotic patients.
The activities of factors are determined mainly by one-stage assays such as the aPTT or PT and the use of factor deficient plasmas. In many cases, the antigen concentrations of individual factors are also determined by immunological methods.
Molecular biological analyses may be required to determine the presence and extent of genetic changes of the coagulation factor.
The activities of factors II (prothrombin), V, VII, VIII:C, IX, X, XI, XII, pre kallikrein (Fletcher factor) and HMWK (high molecular weight kininogen) are determined mainly by so-called one-stage assays. These assays are activity measurements which measure the fibrin formation time in one single reaction step; after all, they are variants of the aPTT (factors VIII:C, IX, XI, XII, pre kallikrein or HMWK) or the PT (factors II, V, VII, X). Factor deficient plasma to which diluted patient plasma is added is used for this purpose. The assays are adjusted in such a way that the factor to be examined is the exclusive determinant of the reaction speed . The residual activity of factors in deficiency plasma is below 1% . The measured activity is expressed as percentage of the activity in seconds by extrapolation to a reference curve.
Activity measurements can also be performed by specific chromogenic assays . These tests are mainly employed for FV III and F IX. In these tests, the speed with which an activated coagulation factor such as F Xa, splits a chromogenic substrate is measured.
- Direct genomic analysis; identification of mutations in the affected gene
- Indirect genomic analysis; characterization of markers, for example restriction fragment length polymorphisms (RFLPs), short tandem repeats or variable number tandem repeats (VNTRs), which are closely linked to the mutation located in the affected gene.
Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L, mixed with 9 parts of blood): 2 mL
Deficiency of clotting factors in hereditary and acquired coagulopathies are afibrinogenemia, prothrombin deficiency and deficiencies of FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII and congenital deficiency of Vitamin K dependent factors .
Among the congenital factor defects, dysproteinemias are distinguished from aproteinemias. The former are characterized by the exchange of one single amino acid due to a point mutation (e.g., as observed in certain forms of hemophilia A) whereas the genetic information in the latter is altered to such a degree that it can no longer be read at all or the abnormal mRNA is immediately broken down. Both forms can be inherited in a homozygous or heterozygous pattern.
Homozygous factor deficiencies are associated with extremely low residual activities, while heterozygotes display levels of 20–50% of normal. For the autosomally inherited and encoded coagulation factors this implies that the genetic information of both autosomes must be present in order for the activity to be normal.
Dysproteinemias can be differentiated from aproteinemias only by immunochemical methods (e.g., immunofixation electrophoresis). The clinically observed bleeding tendency correlates strictly with the remaining residual activity. Hereditary factor deficiencies are listed in .
Increased activities of coagulation factors and their genotypes are potentially associated with venous thromboembolism (VTE) and atherothrombosis. Factors VIII, IX and the von Willebrand factor (VWF) are important in this context.
The VWF is synthesized by endothelial cells and macrophages. It leads to the adhesion of platelets to vascular wall collagen released due to injury. It has been found that increased concentrations of VWF-F VIII complexes are associated with VTE and atherothrombosis. Individuals with A and B blood groups have an increased risk of VTE which is assumed to be linked to an 25–30% increased level of the VWF-F VIII complex .
A high plasma level of F VIII is a risk factor for VTE. In the Leiden thrombophilia study, patients with levels above 150 IU/dL had a 5-fold increased risk of VTE. The risk of recurrence within 30 months in patients with levels above the 90th percentile of control individuals (above 294 IU/dL) was 6.7-fold higher than in those with lower levels . In many cases, high levels of F VIII may be responsible for acquired resistance to APC if F V Leiden mutation is excluded as the underlying cause.
The F IXa plays a key role in maintenance of thrombin (and hence fibrin) formation within the intrinsic coagulation pathway. In a study , an A>G sequence variant encoding F IX (rs 6048, F9 Malmö) was identified that is associated with deep vein thrombosis.
Acquired reductions in coagulation factors are more common than hereditary-type ones and usually involve several coagulation factors. The causes of hemostatic disorders with associated reduction in coagulation factors are listed in .
Factor replacement therapy may be required in congenital hemostatic defects (e.g., in von Willebrand disease, hemophilia A, hemophilia B and in the more unusual cases of isolated deficiencies of factor II, VII, X, XI and XIII) /, /.
Acquired hemostatic defects usually occur in the form of acute bleeding complications during the peri- or postoperative period or in conjunction with advanced liver disease as well as disturbances in acid-base and electrolyte homeostasis such as the hemolytic-uremic syndrome.
Prior to major surgery, the activities of the coagulation factors should be at least 60%, whereas for less extensive surgical procedures in which the risk of bleeding can be limited by local hemostatic measures an activity of 35% will be appropriate.
If, in the case of a known factor deficiency, preoperative factor replacement is required, the levels should be determined prior to (baseline) and, for monitoring, after replacement therapy by means of a one-stage assay. Intraoperative monitoring of the levels is necessary in surgery lasting > 3 h and in all episodes of intraoperative bleeding. Further monitoring of the levels is required immediately, postoperatively, and once or twice daily until wound healing is complete.
A coagulation factor unit refers to the activity which is contained in 1 mL of pooled citrated plasma from healthy blood donors. WHO reference materials exist for factors II, VII, VIII:C, IX and X. The administration of 1 unit of a coagulation factor per 1 kg of body weight raises the activity by 1%. After surgical procedures, the activity level should be at least 60% for the first 8 days and 30% for the following 4–8 days.
Antithrombin must be determined prior to factor replacement (e.g., in surgical patients with liver parenchymal injury or in patients requiring massive intraoperative blood transfusions). The reason for this is that the replacement of pro coagulant substances may cause thromboses or disseminated intravascular coagulation in the presence of underlying antithrombin deficiency.
Factor replacement therapy can be accomplished by using:
- Fresh frozen plasma: after thawing, it contains all coagulation factors with a residual activity > 70% (≥ 0.7 U). In the case of more severe factor deficiencies, replacement is limited by the need to avoid volume overload; approximately 1 mL of plasma/kg of body weight is needed in order to raise the activity by 1%.
- PPSB: it contains the prothrombin complex (factors II, VII, X, IX as well as proteins C and S). Reduction in the prothrombin complex is the result of defective synthesis which is due to either liver parenchymal injury or vitamin K deficiency. Reduced prothrombin complex activity is also present in disseminated intravascular coagulation.
- Concentrates of factors VIII, IX, XIII and protein C.
Although the plasma to be investigated is diluted to such a degree that influences other than those of the factor to be investigated should be eliminated, high concentrations of heparin, autoantibodies, fibrin(ogen) degradation products or other inhibitory substances may still cause falsely low activities.
Falsely high activities may result from the blood collection or storage of activated samples. This applies, in particular, to the determination of F VIII and other factors of the intrinsic coagulation pathway.
Almost all coagulation factors are synthesized in the liver. Acquired factor deficiencies are the result of an underlying primary disease and are due either to defects in the synthesis (practically all liver diseases, especially toxic liver failure, shock liver) or increased turnover recognizable by shortened coagulation factor half-lives. The causes for an increased turnover (consumption) are primarily based on:
- Disseminated intravascular coagulation due to intravascular thrombin formation and hyper fibrinolysis (consumption coagulopathy)
- Increased fibrinolysis (plasminemia)
- Release of proteolytic enzymes (e.g., lysosomal enzymes such as the granulocyte elastase, liberated with the lysis of leukocytes)
- Abnormal losses such as observed in patients with nephrotic syndrome or exudative enteropathy as well as in ascites (exudation into the ascitic fluid) and heavy blood losses
- Adsorption to abnormal surfaces (e.g., to amyloid fibrils in amyloidosis, or to tumor cells as well as to areas of endothelial injury).
9. Repesse Y, Peyron I, Dimitrov JD, Dasgupta S, Moshai EF, Costa C, et al. Development of inhibitory antibodies to therapeutic factor VIII in severe hemophilia A is associated with microsatellite polymorphism in the HMOX1 promoter. Haematologica 2013; 98: 1650–5.
Fibrinogen is the plasma protein with the highest concentration in the coagulation system and plays a key role in the hemostatic system. Thrombin-catalyzed cleavage of fibrinopeptides A and B converts fibrinogen into fibrin, which spontaneously polymerizes and forms double stranded protofibrils that assemble into branched fibrin fibers, forming the fibrin clot . Low fibrinogen concentrations are associated with bleeding and high ones are associated with thrombosis.
- Preoperatively in patients with existing hemorrhage, a history of bleeding disorder or in clinical indication (elevated liver enzymes, HELLP syndrome)
- Intra- and peri operatively in severe blood loss and massive volume replacement
- Exclusion of disseminated intravascular coagulation in combination with antithrombin, D-dimers, TT and aPTT
- Detection of congenital or acquired deficiencies or defects in fibrinogen (hypo fibrinogenemia, dysfibrinogenemia or afibrinogenemia)
- Monitoring of thrombolytic therapy (e.g., with ancrod or defibrase)
- Detection of elevated fibrinogen concentration as a marker for atherothrombosis
- Abnormal results in screening tests (e.g., PT, aPTT and TT).
The fibrinogen concentration can be determined using various methods:
- Thrombin clotting rate assay according to Clauss
- Kinetic turbidimetry (often used on automated platforms)
- Immunochemical method (used for the evaluation of dysfibrinogenemia)
- Derived fibrinogen (results correlate well with the thrombin clotting rate assay with the exception of samples containing high concentrations of fibrin(ogen) degradation products).
Fibrinogen is determined by diluting the plasma sample (1 : 10) and adding an excess of thrombin (100 U/mL) to overcome the influence of inhibitors. The time required for clot formation is recorded (Clauss method). The clotting time of the plasma is inversely proportional to the fibrinogen concentration. The clotting time obtained is then compared with that of a normal plasma pool in which the fibrinogen concentration has been calibrated against an assayed reference material. Patient plasma is diluted 1 : 10 in 0.02 mol/L barbital puffer, warmed for 5 min. at 37 °C and 0.1 mL of thrombin (100 NIH U/mL) is added to start the coagulation /, /.
In the definitive reference method thrombin is added to citrated plasma and during prolonged incubation in the absence of Ca2+ converts all fibrinogen to a fibrin clot. Epsilon aminocarproic acid is added to inhibit plasmin degradation of the fibrin clot. Soluble proteins trapped within the clot are removed by gentle expression of serum from the clot and by subsequent washing of the clot. In the absence of Ca2+, the clot is not stabilized and remains soluble in concentrated urea solution. The fibrinogen concentration in g/L is measured either by light absorption at 282 nm or by determining the tyrosine content using Folin’s reagent /, /.
Batroxobin, a thrombin-like enzyme from the saliva of the snake Bothrops atrox, cleaves fibrinopeptide A from fibrinogen. The resulting fibrin monomers polymerize and lead to an increase in turbidity. This rise in turbidity is linear under the selected reaction conditions and can be measured at 340 or 405 nm. The increase in absorption per time unit is directly proportional to the fibrinogen concentration. The plasma from patients is used undiluted .
Using turbidimetric or nephelometric endpoint detection for PT determination, the total increase of turbidity is directly proportional to the clottable fibrinogen concentration. When in the PT measurement a certain, predefined threshold value of the optical density is reached this increase is specified as the “clotting time”. Only after the clotting time is reached, will fibrinogen be cleaved as well. Since the products thus produced will also cause additional optical density, the fibrinogen concentration (derived fibrinogen) can be calculated based on the difference of turbidity (endpoint turbidity minus clotting time turbidity) .
Fibrinogen or coagulation factor I:
- Is the substrate of thrombin, the last enzyme of the coagulation system
- Is the substrate of plasmin, the last enzyme of the fibrinolytic system
- Belongs to the group of acute phase proteins
- Is synthesized in the liver.
Hemostasis is the physiological process preventing loss of blood and involves the interaction of vessel wall, platelets and the pro- and anticoagulation systems.
Fibrinogen has two functions:
- In primary hemostasis, it plays a key role in platelet aggregation by linking activated platelets. On their membrane, the platelets express the glycoprotein receptor GPIIb/IIIa (integrin αIIbβ3). The receptor binds fibrinogen present in plasma and released by platelet granules (see )
- In secondary hemostasis as the activator of fibrinformation.
Not all of the plasma fibrinogen is functional (clottable) . This is because circulating fibrinogen shows considerable heterogeneity in structure and function, partly due to multiple genetic influences and partly to degradation by enzymes including thrombin, plasmin and neutrophil elastase. Therefore, assays such as the method according to Clauss which determine functional fibrinogen based on clot formation yield different results compared to immunological methods which determine the total fibrinogen, whether clottable or not. These assays are inappropriate for the assessment of bleeding risk. They are useful, however, to distinguish functional fibrinogen from dysfibrinogenemia. In low total fibrinogen (hypofibrinogenemia, afibrinogenemia) and dysfibrinogenemia clottable fibrinogen is low, however in dysfibrinogenemia the fibrinogen concentration is normal.
In general, the fibrinogen concentrations determined with the thrombin clotting rate assay is too low and those determined with derived methods are too high as compared to the reference method.
Clottable fibrinogen assays (Clauss method): although clottable fibrinogen assays use thrombin as the enzyme to transform the fibrinogen, no remarkable, undesirable sensitivity towards heparin is present because the sample is pre diluted (1 : 10) and a large thrombin excess is employed.
Derived fibrinogen: in the presence of fibrin(ogen) degradation products (FDP), this method produces higher levels than the clottable fibrinogen assays. FDP aggregate with fibrin fibrils and thus increase turbidity. Heparin may have an impact on the derived fibrinogen measurement depending on the heparin dependence of each of the thromboplastin reagents used. Usually, derived fibrinogen methods based on PT determination are reliable only if the PT values are ≥ 25% of normal. Compared to the method according to Clauss, the concentrations determined with this method are too high in patients with liver cirrhosis, renal insufficiency, disseminated intravascular coagulation and dysfibrinogenemia. For instance, in a comparative study involving patients with dysfibrinogenemia, the median fibrinogen was 0.40 g/L (0.30–2.07 g/L) determined by the Clauss assay and 2.41 g/L (0.97–4.87 g/L) determined by the derived method. Fibrinogen measured with the PT-derived method was about 5-fold higher and an existing dysfibrinogenemia would not have been detected.
Precipitation methods: like the method according to Ratnoff-Menzie, they mimic falsely elevated levels, as a result of the co precipitation of other proteins.
Immunochemical methods: in the presence of FDP, falsely elevated levels are measured because of cross antigenicity with fibrinogen.
Fibrinogen is synthesized in the liver and has a molecular weight of 340 kDa. It is a dimeric molecule, with each half containing three different 45 nm long polypeptide chains (Aα, Bβ, γ) which are linked by disulfide bridges. The fibrinogen chains are folded into distinct structural regions, comprising a central E region and two outer D regions linked by coiled-coil connections (). The E domain consists of the N-terminal ends of all six polypeptide chains, whereas the D domains primarily comprise the C-terminal ends of the Bβ and γ-chains . Thrombin cleaves the Aα and Bβ chains to release fibrinopeptides A and B. After release the resulting fibrin monomers undergo polymerization to form an insoluble fibrin clot.
In the blood, fibrinogen exists in various forms with slightly different molecular weights . For instance, a fibrinogen proportion includes a variably structured γ chain identified as heterodimer with an A-chain as γA/γ– chain . The γ– chain differs from the γA chain by its C-terminus and, thus, has no impact on the functional behavior of fibrinogen. The presence of the γA/γ– chain modulates the thrombin functions, the F XIII activity and the fibrin clot architecture and eliminates the platelet binding site. There are associations between γA/γ– fibrinogen and atherothrombosis and VTE.
As an acute phase protein, fibrinogen rises, with a delay of 24–48 h, to high levels in systemic inflammation.Pronounced fibrinogen deficiency may be present in severe liver parenchymal injury as a result of defective synthesis. Usually, however, fibrinogen deficiency is based on increased consumption (e.g., due to disseminated intravascular coagulation, consumption coagulopathy and hyperfibrinolysis).
As a hemostatic component, fibrinogen is the substrate of the coagulation cascade. Following the activation of the coagulation system and the thrombin-catalyzed cleavage of fibrinopeptides A and B from fibrinogen, the remainder of the molecule polymerizes into fibrin. Subsequently, F XIII catalyzes the cross linking of the fibrin polymers, thus leading to the formation of a crosslinked fibrin clot which, together with other ongoing processes, causes the bleeding to cease by closing off the blood vessel.
8. Halbmayer WM, Haushofer A, Schön R, Radek J, Fischer M. Comparison of a new automated kinetically determined fibrinogen assay with the 3 most used fibrinogen assays (functional, derived and nephelometric) in Austrian laboratories in several clinical populations and healthy controls. Haemostasis 1995; 25: 114–23.
10. Miesbach W, Schenk J, Alesci S, Lindhoff-Last E. Comparison of the fibrinogen Clauss assay and the fibrinogen PT derived method in patients with dysfibrinogenemia. Thrombosis Res 2010; 126: e428–e433.
11. Kemkes-Matthes B, Fischer R, Peetz D. Influence of 8 and 24-h storage of whole blood at ambient temperature on prothrombin time, activated partial thromboplastin time, fibrinogen, thrombin time, antithrombin and D-dimer. Coagulation and fibrinolysis 2011; 22: 215–20.
16. Morris TA, Marsh JJ, Chiles PG, Magana MM, Liang NC, Soler X, et al. High prevalence of dysfibrinogenemia among patients with chronic thromboembolic pulmonary hypertension. Blood 2009; 114: 1929–36.
19. Simon G, Thompson MA, Kienast J, Pyke SDM, Haverkate F, van de Loo JCW. Hemostatic factors and the risk of myocardial infarction or sudden death in patients with angina pectoris. N Engl J Med 1995; 332: 635–41.
Factor XIII (F XIII) is the fibrin stabilizing factor since clots formed in the absence of activated F XIII lack stability. F XIII plays an important role in the terminal phase of the coagulation system that promotes formation of cross-linked fibrin polymers leading to a stable hemostatic clot. F XIII is converted into an active trans glutaminase (F XIIIa) by fibrin and Ca2+. F XIII stabilizes fibrin mechanically and protects it from fibrinolysis . Patients with congenital deficiency of the factor develop a severe but rare bleeding disorder. F XIII deficiency is not detected by PT, aPTT and TT tests, but can be identified by special assays and by thromboelastography.
F XIII deficiency is suspected in:
- Diagnostic investigation of bleeding episodes
- Disseminated intravascular coagulation (consumptive coagulopathy)
- Diagnostic investigation of impaired healing
- Umbilical cord bleeding and intracranial bleeding.
Kinetic UV test
Principle: F XIII is a pro transglutaminase activated by thrombin to form trans glutaminase (F XIIIa). F XIIIa then links a specific peptide substrate to glycine-ethyl ester, thus producing ammonia. The latter is determined by an enzymatic reaction which is set to run in parallel with the primary reaction. The decrease in NADH is measured kinetically at 340 nm (). Fibrinogen is not removed prior to the measurement since this is associated with a loss of F XIII. Instead, fibrin which is produced under the influence of thrombin is prevented from forming a clot by an aggregation-inhibiting protein (clot inhibitor) and thus remains in a soluble form .
Employment of the Laurell immunoelectrophoresis including the use of antiserum directed against the F XIII subunit A.
F XIII circulates in plasma bound to fibrinogen and is essential for stable wound closure. It induces cross linking and mechanical stabilization of the fibrin polymers formed in the course of blood coagulation. Incorporation of α2-antiplasmin into the fibrin aggregate protects the clot from excessively rapid lysis and facilitates the migration of connective tissue cells into the stabilized fibrin .
- Congenital deficiency. Umbilical cord bleeding occurs in 80% of congenital F XIII deficiency
- Deficiency due to polymorphisms in the gene of F XIII. A risk factor for hemorrhagic shock among younger women and a risk for recurrent pregnancy loss are reported to the presence of Pro564leu and Tyr204Phe polymorphism in F XIII subunit A gene.
- Acquired F XIII deficiency ().
Under normal conditions, a decrease in F XIII level to approximately 10% of normal is not associated with any significant bleeding tendency.
Acquired F XIII deficiency may occur due to increased consumption in impaired synthesis or can be induced by drugs or F XIII antibodies (e.g., in autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis). Except for Henoch- Schönlein purpura, an acute episode of ulcerous colitis and Crohn’s disease, F XIII deficiency is rarely observed as an isolated condition, but mostly occurs in combination with other coagulation disorders.
A decrease in F XIII after surgical procedures can be due to massive loss or dilution of blood. F XIII deficiency is also observed in pro myelocytic leukemia, gynecological tumors, disseminated intravascular coagulation and in the course of severe liver disease /, /. The F XIII concentration in all of these patients should be at least 50% . After major surgery, however, dangerous hemorrhages may occur at concentrations between 10–40% .
The clinical symptoms of F XIII deficiency include umbilical cord bleeding, intracranial bleeding, delayed-onset bleeding episodes after injuries and major surgery, suffusions after blunt trauma, intramuscular bleeding, hemarthroses, delayed wound healing and spontaneous miscarriages .
Elevated F XIII concentrations may be associated with the risk of atherothrombosis. It is assumed that elevated FXIII levels mechanically strengthen the fibrin clot and make it more resistant to shear forces and to fibrinolysis. In a study , F XIII activity in the upper tertile of a cohort (above 120% by activity measurement and/or above 25.5 mg/L by immunochemical method) was associated with a more than two-fold risk of peripheral artery disease in females. This was not the case in males.
Ammonia concentrations above 294 μg/dL (172 μmol/L) in the sample may interfere with the kinetic UV test, resulting in falsely low activity. If necessary, the ammonia concentration must be determined, followed by repeat testing after the sample has been appropriately diluted using normal saline.
In the case of very low (below 0.8 g/L) and very high (above 8 g/L) fibrinogen concentrations, falsely low F XIII activities may be measured. In the case of high fibrinogen concentrations, the sample may also be pre diluted using normal saline.
F XIII belongs to the family of trans glutaminases (protein-glutamine γ-glutamyl-ε-lysyltransferase, EC 188.8.131.52), catalyzing the insertion of ε- (γ-glutamyl)-lysyl bonds between two peptide chains. In the presence of Ca2+ lysis of the fibrin clot is, thus, prevented in a slightly alkaline environment.
F XIII exists as cellular protein and as plasma protein. The plasma protein is a heterotetramer composed of two identical globular A subunits of 83 kDa (F XIII-A) non covalently bound to two long fibrillar units B (F XIII-B) of 73 kDa. F XIII-A is synthesized in platelets and monocytes/macrophages. Concentrations in platelets are around 100 times higher than in plasma. Damaged platelets release their F XIII A into the plasma where it binds to F XIII B which is released by the hepatocytes and functions as carrier and protective protein . The entire F XIII-A2B2 has a molecular weight of 326 kDa. In plasma, the heterotetramer F XIII-A2B2 is converted to its active form by the thrombin-catalyzed hydrolysis of the Arg37–Gly38 peptide bond at the N-terminus of the F XIII-A subunit (). In the presence of Ca2+ the F XIII-A2B2 heterodimer complex dissociates, yielding F XIII-B2 and activated F XIII-A2 . Ca2+ cause small but significant conformational changes in F XIII-A during activation, exposing potential exosites within F XIII-A.. Activated F XIII-A2 stabilizes the forming protofibril by introducing ε-amino(γ-glutamyl)lysine cross-links between carboxyl terminal portions of adjacent fibrin γ chains, before lateral association of the protofibril. See .
The fibrinogen residues αC 242-424 play a major regulatory role in the activation of F XIII-A2B2. Glu396 is the key amino acid residue involved in binding activated F XIII-A2 . Once a fibrin clot has formed, α2-antiplasmin is incorporated preventing premature fibrinolysis of the clot.
F XIII is a multifunctional protein. In addition to its role in hemostasis, it is essential for carrying out pregnancy and its role in wound healing and angiogenesis has also been unequivocally demonstrated. It also plays an important role in maintaining vascular permeability and is involved in the stabilization and mineralization of extracellular matrix in bone and cartilage .
3. van Giezen JJJ, Minkema J, Bouma BN, Jansen JWCM. Cross-linking of α2-antiplasmin to fibrin is a key factor in regulating blood clot lysis: species differences. Blood Coag Fibrinol 1993; 4: 869–75.
17. Perez DL, Diamond EL, Castro CM, Diaz A, Buonanno F, Nogueira RG, et al. Factor XIII deficiency related recurrent spontaneous intracerebral hemorrhage: A case and literature review. Clinical Neurology and Neurosurgery 2011; 113: 142–5.
Reinhard Schneppenheim, Lothar Thomas
The most common of the inherited bleedimg disordes is von Willebrand disease (VWD). It is caused by a qualitative and/or quantitative decrease of von Willebrand factor (VWF) the carrier protein of FVIII. The VWF present in plasma and thrombocytes is synthesized in megakaryocytes and endothelial cells. The VWF antigenic determinant (VWFAg) plays a key role in primary hemostasis and is characterized by two main functions :
- It mediates the thrombocyte adhesion to injured subendothelial tissue
- It serves as carrier protein for F VIII and stabilizes F VIII coagulant activity. The function of VWF in hemostasis is shown in .
The function of VWF in primary hemostasis is mediated by the large multimeres, the size of which and consequently the risk of hyper function is controlled by a VWF-specific metalloprotease (ADAMTS-13). The absence of this protease is associated with the thrombotic thrombocytopenic purpura (TTP).
- Diagnosis and monitoring in congenital or acquired von Willebrand syndrome (VWS)
- Diagnosis and monitoring in hereditary and acquired thrombotic thrombocytopenic purpura
- Exclusion of hemophilia A.
There is no single diagnostic test due to the high variability of the VWS. Screening tests of VWD are a prolonged bleeding time closure time with the use of the platelet function analyzer (PFA).
Platelet function analyzer (PFA 100)
Principle: VWF has a central role in platelet adhesion and aggregation under conditions of high shear stress. The PFA 100 is an automated device for assessing platelet function under high shear conditions . In the PFA-100 instrument these conditions are created by aspirating whole blood through a capillary and an aperture on a membrane coated with equine tendon collagen type 1 and an additional platelet aggregating agent. In cartridge type this agent is ADP and in the other it is epinephrine. Under these circumstances platelets passing through the membrane adhere to the collagen surface, become activated and aggregate. Subsequently a stable platelet plug forms and occludes the aperture. The PFA-100 measures the required time for total occlusion and reports the "closure time”. The closure time is dependent upon VWF and the platelet receptors GPIb and
The laboratory diagnosis of VWD is based on the results of the, VWF:Ag, FVIII and VWF:RCo. The three initial tests are also used for monitoring therapy. The ristocetin induced thrombocyte aggregation (TIPA) and the analysis of multimeres are used for defining and classifying VWD subtypes . Refer to .
Von Willebrand factor antigen (VWF:Ag)
Principle: The VWFAg determination is a quantitative test for the detection of VWD. It measures the amount of VWF circulating in plasma regardless of the function. The most widely used method is the ELISA. Results are expressed in IU/L or % of normal. Typically the level of VWF:Ag is decreased in type I VWD.
F VIII coagulant activity (F VIII:C)
Principle: F VIII is bound to and protected from proteolytic degradation by VWF. Decrease in the level of VWF causes a decline in the amount and activity of FVIII. The activity of F VIII is measured by an aPTT based assay. F VIII:C correlates well with the severity of the disease and also predicts the risk of hemorrhage. The result is expressed in IU/L or % of normal . Typically, the level of FVIII:C is moderately decreased in type 1 and type 2 VWD, but it may be normal or extremely low in type 3 .
Ristocetin cofactor activity (VWF:RCo)
Several methods are used to assess the thrombocyte agglutination and aggregation that result from the binding of VWF to thrombocyte GPIb induced by ristocetin.
Principle: The assay measures the ability of patient’s plasma to agglutinate formalin-fixed platelets in the presence of 1 g/L ristocetin. Ristocetin is a glycopeptide antibiotic that binds to VWF and induces VWF activation and thrombocyte aggregation. The intensity of aggregation correlates with the activity of the VWF .
Ristocetin-induced platelet aggregation (VWF:RIPA)
Principle: RIPA is carried out as a part of platelet function testing by aggregometry and is carried out in the platelet-rich plasma of patients by different concentrations of ristocetin (usually between 0.5 to 1.5 g/L). Ristocetin binds the VWF to the GP-1b receptor of thrombocytes and induces the aggregation of thrombocytes. The impairment in thrombocyte aggregation correlates with the loss of VWF function. Results are expressed in % of normal . RIPA is only diagnostic in type 2B VWD disease where an enhancement in platelet aggregation is observed.
Principle: The multimeric analysis of plasma VWF is performed by sodium dodecyl sulfate agarose gel electrophoresis, followed by Western blot detection //. Various types and subtypes of VWF are detected ().Only types 2A, 2B and platelet-type VWD have abnormal multimer distributions with relative deficiency of the largest multimers .
GPIbα binding capacity (VWF:GPIbB)
Principle: Binding of VWF in patient plasma to a particle-bound platelet GPIbα fragment containing two gain-of-function mutations. The two mutations mediate a constitutively reacting structure of the receptor enabling the VWF binding to the GPIbα fragment without the addition of ristocetin. The amount of bound VWF is determined using a luminescence immunoassay. Results are expressed in IU/L or % of normal .
Collagen binding capacity (VWF:CB)
Principle: The assay measures the collagen binding activity of VWF by an ELISA. The results are expressed in % of normal . As the binding to collagen is highly dependent upon the multimeric structure, the test mainly detects high molecular weight forms and is especially impaired in type 2A and 2B disease.
F VIII binding capacity (VWF:F8BC)
Principle: VWF is captured to the solid phase of an micro titer plate from patient plasma . Endogenous F VIII is removed by using a high chloride concentration and a known amount of purified F VIII is added. The solid phase bound VWF and the VWF-bound F VIII are separately detected. A disproportionally decreased amount of VWF bound FVIII indicates type 2N vWD. Results are expressed in % of normal . The test is used to detect an impaired ability of the VWF in patient plasma to bind F VIII in type 2N VWD.
FVIII regulates VWF in coagulation. FVIII stabilizes VWF multimers and renders them more susceptible to degradation by the metalloprotease ADAMTS13 a (disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13) while FVIIIa, which is not VWF-bound, does not have this effect .
ADAMTS13 specifically cleaves the VWF subunit at the peptide bond Tyr1605–Met1606, generating fragments of 176 kDa and 140 kDa. A severely deficient ADAMTS13 activity (below 5% of normal) is caused either by mutations of the ADAMTS-13 gene or by antibodies blocking proteolysis of VWF. The ADAMTS13 deficiency causes the accumulation of ultra large VWF multimers in the circulation and the formation of thrombi in the microvasculature under high shear stress conditions. When left untreated thrombotic thrombocytopenia purpura develops. The micro thrombi cause multi-organ failure and can lead to death .
Detection by immunoassay: results are expressed in μg/L.
Principle: Determination of the specific proteolytic destruction of VWF multimers following incubation of recombinant or plasma VWF with patient plasma and denaturation buffer (urea) by addition of BaCl2. The decrease in VWFRCo, VWFCB or the large VWF multimers by multimer analysis are determined. Results are expressed in % of normal .
Fluorescence resonance energy transfer (FRETS)
Principle: Double fluorescent-labeled VWF fragment containing the proteolytic cutting site for ADAMTS13 is used as substrate. Cleavage of the fragment by ADAMTS13 is correlated with the decrease in the quenching effect of both fluorescences. Results are expressed in % of normal.
Principle: Neutralizing autoantibodies are determined based on the decrease in plasma ADAMTS13 activity in the plasma mixing test, whereas non-neutralizing autoantibodies are determined using ELISA. Results are expressed in U/mL.
Principle: Detection of specific genetic VWF defects by the use of polymerase chain reaction and mutation analysis , detection of heterozygous deletions and duplications using multiplex ligation dependent probe amplification (MLPA).
- Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 2 mL
- Platelet-rich citrated plasma for the determination of ristocetin-induced platelet aggregation (RIPA)
- Platelets from platelet-rich citrated plasma for the determination of platelet VWF
- Leukocyte DNA using EDTA whole blood, for molecular genetic diagnostics: 5 mL
Concentration and function: higher than 50 IU/dl
Low dose RIPA: aggregation (0.5 g of ristocetin/L) < 20%.
Multimers: all multimers are detectable, no aberrant electrophoretic bands.
ADAMTS13: > 45% of normal.
Von Willebrand disease (VWD) is a well known inherited bleeding disorder, causing mucous membrane and skin bleeding symptoms, and bleeding with surgical or other hemostatic challenges. In screening populations the prevalence of VWD is 0.6–1.3%. Bleeding is of mild-to-moderate severity for most persons with VWD, reflecting the predominance of type 1 VWD. Factors that increase plasma levels of VWF include age, race (African Americans), acute-phase-response and hormones, particularly those associated with pregnancy and the menstrual cycle. Approximately 12% of women who have menstrual periods have excessive menstrual bleeding. This proportion is much higher among women with VWD /, /. During pregnancy VWF is increased 3–5 fold above the baseline by the third trimester. ABO blood group types have an effect on plasma VWF and FVIII concentrations. The mean VWF level of individuals with blood type 0 is 75 IU/dl and approximately 25% lower than other blood group types. The diagnosis of VWD type 1 occurs more frequently in persons who have blood group type 0 .
Life-threatening bleeding (central nervous system, gastrointestinal tract) can occur in persons either with type 3 VWD and in some patients with type 2 VWD. Women with VWD report a high prevalence of menorrhagia. The sensitivity of menorrhagia as a predictor of VWD may be estimated as 32–100% .
The diagnosis of vWD depends on clinical and laboratory criteria. The clinical criteria include personal and/or family history and/or physical evidence of mucocutaneous bleeding. The Expert Panel recommends that 30 IU/dL be used as a the cutoff level for supporting the definite diagnosis of VWD for the following reasons:
- There is a high frequency of blood type 0 and it is associated with low VWF levels
- Bleeding symptoms are reported by a significant proportion of normal individuals
- No abnormality in the VWF gene has been identified in many individuals who have mildly to moderately low VWF:RCo levels.
Type I VWD
These persons have partial quantitative deficiency of VWF. A concordant decrease in the concentration and activity of the VWF points to VWD type 1. The remaining VWF binds FVIII normally and mediates thrombocyte adhesion normally. The results of laboratory tests are as follows /, /:
- Von Willebrand factor antigen (VWF:Ag) is reduced
- FVIII coagulant activity (FVIII:C) decreased
- Ristocetin Cofactor (VWF:CRo) pathologic
- The FVIII/VWF:Ag ratio 1.5–2.0
- Multimer analysis: normal pattern.
Individuals who have very low VWF levels (< 20 IU/dL) are likely to have VWF gene mutations, mucocutneous bleeding symptoms and a strongly positive family history.
Type 2 VWD
The results of VWF:Ag, FVIII:C and VWF:CRo are variable or may be normal, the multimer pattern is abnormal. Critical tests include the VWF:RIPA, the collagen binding assay, the F VIII binding assay and multimer analysis /, /.
Type 2A VWD: This type of VWD may be caused by mutations that interfere with the assembly or secretion of large multimers or by mutations that increase the susceptibility of VWF multimers to proteolytic degradation in the circulation . VWF:Ag and FVIII are low normal or decreased, VWF:RCo and RIPA are decreased, and the VWF multimer pattern is abnormal.
Type 2B VWD: Mutations occur within or adjacent to VWF domain A1, which changes conformation when it binds to thrombocyte GPIb. Patients typically have thrombocytopenia that is exacerbated by surgery, pregnancy or other stress. The diagnosis depends on finding abnormally increased ristocetin-induced thrombocyte aggregation (RIPA) at low concentrations of ristocetin.
Type 2N VWD: This type of VWD is caused by mutations in the F VIII binding site of VWF and impairs binding of FV III to VWF. The level of F VIII is low (< 10%) with a normal VWF:Ag and VWF:RCo. The type masquerades as an autosomal recessive form of hemophilia A. Discrimination may require assays of F VIII binding. In low F VIII concentrations, the recessively inherited type 2N can be associated with normal VWF:Ag, VWF:RCo and VWF:CB (homozygous or compound-heterozygous F VIII binding mutation of the VWF) or manifest with low VWF:Ag (compound-heterozygous F VIII binding defect with a VWF null allele) . This type is the key differential diagnosis regarding hemophilia A. VWD type 2N must be excluded in cases of unclear inheritance, inadequate response to especially pure or recombinant F VIII concentrates and always in female hemophilia A .
Molecular investigations are a way to circumnavigate the problems encountered in phenotype analysis since the phenotype-genotype correlations are clear in many cases. Thus, it is possible in many unclear cases to obtain a diagnosis based on gene analysis () .
Type 3 VWD
The complete absence of the VWF protein and activity points to type 3 VWD. FVIII levels usually are very low (1–19 IU/dL). Truncated mutations of the VWF gene (e.g., nonsense, splicing and nonsense mutations) and minor and major deletions and insertions as well as missense mutations are the cause .
Platelet-type VWD is an inherited platelet disorder characterized by thrombocytopenia with large platelets caused by gain-of-function variants in GP1BA leading to enhanced GPIa VWF interaction. GPIa and vWF play a role in megakaryopoiesis. Thrombocytopenia in VWD is due to a combination of different pathogenic mechanisms, i.e., the formation of a reduced number of platelets by megakaryocytes, the ectopic release of platelets in the bone marrow, and the increased clearance of platelet/VWD complexes .
Acquired VWD is a rare bleeding disorder with laboratory findings similar to those of inherited disease. However, unlike the inherited disease acquired VWD occurs in persons with no family history of bleeding. The disease is often associated with a variety of diseases, most frequently lymphoproliferative, myeloproliferative and cardiovascular disorders. Laboratory findings in acquired VWD measure defects in VWF concentration similar to those in VWD. The results include decreased values of VWF:Ag, VWF:RCo or FVIII. The VWF multimer pattern often shows a decrease in large multimers similar to that seen in type 2 VWF or a normal pattern .
This observation is supported by the finding that in the hereditary form of the disease administration of a standard dose of fresh frozen plasma is sufficient to prevent TTP . Concentrations below the detection limit (< 2–5% depending on the method) always indicate a corresponding predisposition . The differentiation between the hereditary and the acquired form of the disease is crucial for therapy selection.
In the acquired form of TTP anti-ADAMTS13 autoantibodies block the proteolysis of VWF and/or induce ADAMTS13 clearance from the circulation. The mechanisms leading to the loss of tolerance of the immune system towards ADAMTS13 involve the predisposing genetic factor of the human leukocyte antigen class II locus DRB1* 11 and DQB1*03 alleles as well as the protective allele DRB1*04 and modifying factors such as ethnicity, sex and obesity .
Single administrations of fresh frozen plasma are sufficient in the hereditary form (every 14 days for prophylaxis), whereas the autoantibody-mediated form can only be treated by repeated plasma exchange and immunosuppressive therapy.
Blood sample collection
The setting of phlebotomy should be as calm as possible. Anxiety of the patient may falsely elevate the F VIII and VWF levels.
VWF is an adhesive protein with binding sites for circulating proteins (F VIII), insoluble structures of the sub endothelium (collagen) as well as cellular surface structures (platelet surface glycoproteins GPIb, GPIIb/ IIIa). This accounts for the key role which VWF plays in primary hemostasis as a mediator of platelet adhesion to the injured sub endothelium and subsequent platelet aggregation. This process takes place primarily under strong shear stress typical of the arterial system and the micro circulation (). The large VWF multimers are essential for this function .
The second important function is the binding of F VIII thereby protecting this factor from premature degradation, for example by activated protein C (). In severe VWD (type 3), all these functions are impaired. Accordingly, beside a primary hemostatic defect, there is also a defect in secondary hemostasis due to a pronounced reduction in F VIII (below 0.05 U/L). This must be taken into account for the purpose of adequate therapy. In the mild form of VWD (type 1) and in types 2A, 2B and 2M with a VWF:Ag above 30 IU/dL, there is only little impairment in secondary hemostasis.
In various other subtypes of type 2 VWD, only some particular functions of VWF may be reduced. In particular, type 2 N VWD is of interest because this type becomes evident only because of a concomitant reduction in F VIII:C on the basis of the patient’s VWF which displays impaired F VIII binding capacity; clinically, this defect resembles hemophilia A (pseudohemophilia).
Isolated particular dysfunctions of the VWF can be attributed to defined defects that may be correlated with mutations in the different functional regions of the VWF (). The marked heterogeneity of the VWD can be explained by the multi functionality of the VWF, its domain structure and the numerous options of combination of the different defects /, /.
Hyper function of the VWF leads to the clinical picture of thrombotic thrombocytopenic purpura, a propensity to micro angiopathic thrombosis caused by the persistence of ultra-large VWF multimers in the absence of the specific VWF metalloprotease ADAMTS13 and, consequently, the lack of VWF size regulation /, , /. This disease can be autosomal recessively inherited or caused by autoantibodies . Accordingly, there may be homozygous or compound-heterozygous mutations of the ADAMTS13 gene or specific ADAMTS13 autoantibodies with different consequences on therapy /, /.
The von VWD and the thrombotic thrombocytopenic purpura are the two opposing manifestations of VWF disorders, thus underscoring the factor’s key role in hemostatic balance.
7. Schneppenheim R, Budde U, Krey S, Drewke E, Bergmann F, Lechler E, Oldenburg J, Schwaab R. Results of a screening for von Willebrand disease type 2N in patients with suspected haemophilia A or von Willebrand disease type 1. Thromb Haemostas 1996; 76: 598–602.
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11. Schneppenheim R, Brassard J, Krey S, Budde U, Kunicki TJ, Holmberg L, Ware J, Ruggeri ZM. Defective dimerization of von Willebrand factor subunits due to a Cys –> Arg mutation in type IID von Willebrand disease. Proc Natl Acad Sci USA 1996; 938: 3581–6.
15. Zhang ZP, Blomback M, Egberg N, Falk G, Anvret M. Characterization of the von Willebrand factor gene (VWF) in von Willebrand disease type III patients from 24 families of Swedish and Finnish origin. Genomics 1994; 211: 188–93.
16. Schneppenheim R, Krey S, Bergmann F, Bock D, Budde U, Lange M, Linde R, Mittler U, Meili E, Mertes G, Olek K, Plendl H, Simeoni E. Genetic heterogeneity of severe von Willebrand disease type III in the German population. Human Genetics 1994; 94: 640–52.
18.. Vesely SK, George JN, Lammle B, Studt JD, Alberio L, El-Harake MA, Raskob GE. ADAMTS13 activity in thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: relation to presenting features and clinical outcomes in a prospective cohort of 142 patients. Blood 2003; 102: 60–8
19. Hrdinova J, D’Angelo S, Graca NAG, Ercig B, Vanhoorenbeke K, Veyradier A, et al. Dissecting the pathophysiology of immune thrombotic thrombocytopenic purpura: interplay between genes and environmental triggers. Haematologica 2018; 103: 1099–1109.
20. Moake JL, Rudy CK, Troll JH, Weinstein MJ, Colannino NM, Azocar J, et al. Unusually large plasma factor VIII: von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura. N Engl J Med 1982; 307: 1432–5.
22. Furlan M, Robles R, Lämmle B. Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis. Blood 1996; 87: 4223–34.
Venous thromboembolism (VTE), consisting of deep vein thrombosis and pulmonary embolism, is a multi causal disease associated with substantial morbidity and mortality. VTE has been described as hemostasis in the wrong place and is a time-limited acute disease. VTE can occur spontaneously after trauma and major surgery or in association with malignancies or following immobilization. Patients who suffer idiopathic VTE have a high risk of recurrence, exceeding 50% over 10 years, if not treated with extended-duration anticoagulation.
VTE is age-dependent, the annual incidence increases from 1 : 10,000 at an age ≤ 40 years to almost 1% at an age ≥ 75 years (). More than 70% of the cases are seen at an age > 60 years. On average, 1 in 1,000 individuals suffer from VTE every year, of whom 20% develop post thrombotic syndrome and 1% die of pulmonary embolism . The incidence of VTE in the asymptomatic population is 0.1 –0.2% per year. The relative risk of a relapse is 2–5% per year. Compared to this baseline incidence, women have a markedly higher rate postpartum. According to a study , among 1,687,930 women with a first recorded delivery, 411 had a thrombotic event in the first 6 weeks of the postpartum period, as compared to 38 with a similar time period 1 year later.
- Dispositional risk factors (prothrombotic disorders). This refers to individuals with a genetically defined risk (F V Leiden mutation, prothrombin gene mutation G20210A, deficiency in antithrombin, protein C, protein S, increase in F VIII and fibrinogen) and a non-genetic risk such as the antiphospholipid syndrome.
- Exposure risk factors either cause VTE by itself or increase the risk of dispositional factors () . The risk is especially high in individuals who already had VTE or patients with a malignant tumor. The risk of acute VTE is high in immobilized patients with acute disease and in intensive care unit patients who have not received prophylactic medication or other preventive care. () . According to a metaanalysis , anticoagulant prophylaxis prevents approximately half of the expected events.
- Dispositional risk factors (prothrombotic disorders) combined with environmental risk factors . Heterozygous factor V Leiden, present in about 5% of whites, increases the incidence rate of VTE in young women from 1 in 10,000 annually to 4–7 in 10,000. If a young women with factor V Leiden is obese, the incidence increases to 8–14 in 10,000. Women in their 50s with factor V Leiden have about a 4 in 1,000 annual risk of VTE, which rises to about 1% annually with the use of postmenopausal estrogen plus progestin and about 3% if there is a family history of thrombosis.
- Invasive procedures in patients undergoing long-term anticoagulant therapy if therapy is interrupted or modified.
Comprehensive diagnostic investigation of thrombophilia is not necessary in acute VTE because it is therapeutically irrelevant whether the VTE has genetic or acquired causes. The antiphospholipid syndrome where early adequate therapy can achieve good therapeutic success is an exception .
VTE is a chronic disease with a recurrence rate of 30% within 5–8 years. Approximately 5% of affected patients die of pulmonary embolism. The rate of recurrence is high in the first weeks after a thrombosis and decreases pronouncedly afterwards . Patients with pulmonary embolism have a higher recurrence rate and frequent episodes will also be pulmonary embolisms. The recurrence rate in patients with inborn disposition of VTE is higher than in those with acute VTE after major surgery. The following risk factors for recurrent VTE besides male gender are shown in .
Thrombophilia is the propensity to develop VTE based on certain factors. These factors can be genetic, acquired or both. Thrombosis occurs in the veins and arteries, whereas thrombophilia is associated with VTE. In approximately 15% of the population in the Western world and in up to 50% of individuals with VTE thrombophilic disposition is detectable by laboratory investigations. Positive familial history is especially significant in the assessment of the risk of VTE because a VTE episode in the family indicates a thrombophilic risk that may lead to thrombosis in relatives ().
Familial, genetically determined predisposition to thrombosis can be detected by thrombophilia screening. Approximately 15% of the population are carriers of genetically determined thrombophilia markers. Manifestations of hereditary thrombophilia can be due to :
- Dysregulation of plasmatic coagulation as a result of reduced counter regulation (e.g., reduction in inhibitors) or constitutive hyperactivity (e.g., of F VIII)
- Absolute reduction in the activity of proteins of the fibrinolytic pathway.
The presence of a genetic risk factor is relatively common in adults without VTE and is assumed to lead to VTE only in combination with one or several other risk factors. In children, VTE is usually only seen if there is concurrent evidence of 3 or more risk factors. The assessment of the finding of a genetic risk factor is problematic without positive familial history. In many cases, VTE is triggered by the combination of a hereditary defect and an acute disease. In addition, exogenous factors may act as triggers leading to a transient disruption of the balance between coagulation, inhibitors of coagulation and fibrinolysis. For instance, major surgery, trauma and oral contraceptives trigger VTE in individuals with thrombophilic diathesis. Approximately 10–20% of VTE patients have more than one of the genetic risk factors. This concurrence of several risk factors partly explains the different forms of propensity to thrombosis in different individuals and families with the same genetic defect. Refer to .
A genetically determined elevated risk of thrombophilia will only cause VTE in the presence of additionally acquired (environmental) risk factors. Moreover, the absence of genetic risk factors does not reliably rule out hyper coagulability.
F VIII activity
Increased F VIII activity is a risk factor of thrombophilia. F VIII is an acute phase protein that is increased in inflammation and regulated by the genes AB0. In the Leiden Thrombophilia Study, patients with F VIII concentrations > 150 IU/dL had a 5-fold increased risk of VTE (see also ).
Beside the hereditary thrombophilic diatheses listed in , numerous other congenital and acquired disorders are associated with thrombophilia, for example deficiency in heparin cofactor and plasminogen, dysplasminogenemia, F XII deficiency, increase in histidin-rich glycoprotein, mutations in thrombomodulin, mutations in platelet GP IIb/IIIa receptor, increase in fibrinogen, VWF and F VII, impaired release of tissue plasminogen activator (t-PA), increase in t-PA inhibitor and others. At the present time, there is insufficient evidence to recommend the routine determination of these disorders for association with increased propensity to thrombophilia.
Associations of coagulation factors IX and XIII with the risk of VTE were investigated in a study . Elevated levels of both factors were associated with increased rate of thrombosis, with the odds ratio being 1.4 for F IX and 2.0 for F XIII.
Thrombosis in thrombocytopenia
Causes can be:
- Disseminated intravascular coagulation
- Thrombotic thrombocytopenic purpura
- Thrombotic micro angiopathy
- Heparin induced hemoglobinuria
- Hemolytic uremic syndrome
- Paroxysmal nocturnal hemoglobinuria
- Idiopathic thrombocytopenia e.g., lupus erythematosus, antiphospholipid syndrome.
Antiphospholipid syndrome (APLS) is characterized by:
- VTE in pregnancy and further complications
- The finding of lupus anticoagulant (LA) and/or elevated titers of anti-cardiolipin antibodies (ACA) or anti-β2-glycoprotein (anti-β2-GP) antibodies. See )
LA, ACA and anti-β2-GP are not necessarily, but can be associated with thrombophilia and disorders in pregnancy. Transient LA sometimes seen in children in association with viral infections is not associated with thrombophilia.
The laboratory findings by themselves do not allow a prediction of clinical consequences. Practical experience suggests that the risk of clinical APLS is low in patients who are asymptomatic at the detection of LA or ACA. In patients where APLS was associated with thrombophilia, VTE was seen in 2/3 and arterial thrombosis, especially cerebral thrombosis, was seen in 1/3 of the cases. VTE has a pronounced tendency to recur. There is no bleeding tendency despite prolonged aPTT, except in those cases where LA is present in association with severe prothrombin deficiency.
Approximately half of the APLS patients have no underlying disease (primary APLS). The other half suffer from autoimmune diseases such as systemic lupus erythematosus, monoclonal gammopathies, infections (in children) or drug reactions.
Invasive procedures in patients undergoing long-term anticoagulant therapy imply the risk of VTE if the anticoagulant therapy is interrupted or no suitable bridging anticoagulant therapy is administered. For further information, see Ref. .
- Thrombosis at a young age (< 50 years), especially in association with weak provoking factors (minor surgery, combination of oral contraceptives, or immobility) or unprovoked VTE
- Strong family history of VTE (first-degree family members affected at young age)
- Recurrent VTE events, especially at a young age
- VTE in unusual sites such as splanchnic or cerebral veins.
Well defined VTE risk factors include:
- Activated protein C, factor V Leiden mutation
- Prothrombin gene mutation (FIIG20210A)
- Deficiency in natural anticoagulants (antithrombin, protein C, protein S)
- Presence of lupus anticoagulant. No hereditary risk factor. Refer to .
Tests not conclusively associated with risk of VTE or require further validation are : elevated activity of F VIII, F IX, F XI, plasminogen activator inhibitor type 1 (PAI-1), and the 4G/5G PAI-1 promoter polymorphism.
Not associated with an increased risk of either first VTE or recurrence is the methylene tetrahydrofolate reductase polymorphisms (677C-T, 1298A-C), which are present in up to 45% of the population worldwide .
Thrombophilia testing detects genetically determined risk factors in approximately half of the patients with VTE. Target groups especially include patients with juvenile thrombosis before 45 years of age or a positive familial history . The best time for thrombophilia testing is shortly after the end of anticoagulant therapy as soon as the PT has returned to normal. This timing is selected because a deficiency in proteins C and S is difficult to diagnose in patients under oral anticoagulant therapy (both proteins are dependent on vitamin K) and the AT activity decreases under heparin and slightly increases under oral anticoagulation. Testing shortly after the end of anticoagulant therapy may warrant to quickly revoke the decision regarding termination of the anticoagulant therapy. The determination of D-dimers after termination oral anticoagulants is a non-specific marker of risk of recurrence.
In the acute situation, immediate testing is indicated in neonatal fulminate purpura. This disease is due to homozygous PC and PS mutations. In acute VTE, the determination of antithrombin is advisable in patients who are resistant to heparin (usually for other reasons) or belong to a family with AT deficiency.
It is advisable to test relatives of patients with symptomatic thrombophilia disorders. This especially applies to deficiency of antithrombin, PC or PS, but only to a very limited extent to patients with F V Leiden mutation or prothrombin gene mutation. In many cases, the decision-making process regarding therapy selection can be enhanced by evidence of a thrombophilic defect. On the other hand, the certainty about the absence of a defect is psychologically important in normal findings.
A patient with an acute VTE requires anticoagulant therapy. For patients with provoked VTE a 3-month course of anticoagulant therapy is sufficient. Lifelong therapy may be indicated in patients with unprovoked events.
Patients with VTE and transient provoking factors (major surgery, trauma, hospitalization for acute medical illness, immobility) have of low risk of recurrent VTE. A 3-month course of anticoagulant therapy is usually sufficient. In nonsurgical patients, the risk of recurrence is low and similar for patients with and those without thrombophilia. Even patients with well defined hereditary risk factors do not need lifelong anticoagulant therapy after VTE .
Patients with unprovoked VTE have a significantly increased risk of recurrence. After anticoagulant therapy is stopped the risk is approximately 10% in the first year with a cumulative risk of 40% at 5 years and more than 50% at 10 years. The risk of recurrence is not influenced by factor V Leiden and the prothrombin gene mutation. The risk does not differ significantly among patients with deficiencies of protein C, protein S, and antithrombin. In patients with unprovoked VTE, lifelong or indefinite anticoagulant therapy may be indicated. Patients with unprovoked VTE and inherited thrombophilia have no greater risk of recurrent VTE while receiving standard dose anticoagulant therapy than that without inherited thrombophilia .
Before testing patients should have completed anticoagulant therapy. Vitamin K antagonists should be withheld for a minimum of 2 weeks and direct oral anticoagulants for at least 5 half-lifes, generally a minimum of 2–3 days .
5. Geerts WH, Bergqvist D, Pineo GF, Heit JA, Samana CM, Lassen MR, et al. Prevention of venous thromboembolism: American College of Chest Physicians evidence-based clinical practical guidelines. Chest 2008; 133: 381S–435S.
6. Själander A, Jansson JH, Bergqvist D, Eriksson H, Carlberg B, Svensson P. Efficacy and safety of anticoagulant prophylaxis to prevent venous thromboembolism in acutely ill medical patients: a meta analysis. J Intern Med 2008; 263: 52–60.
13. Cushman M, O’Meara ES, Folsom AR, Heckbert SR. Coagulation factors IX through XIII and the risk of future venous thrombosis: the longitudinal investigation of thromboembolism etiology. Blood 2009; 114: 2878–83.
19. Lijfering WM, Mulder R, ten Kate MK, Veeger NJGM, Mulder AB, van der Meer J. Clinical relevance of decreased free protein S levels: results from a retrospective family cohort study involving 1143 relatives. Blood 2009; 113: 1225–30.
Antithrombin (AT) belongs to the family of serine protease inhibitors (serpines) and is the most important inhibitor of the coagulation system. AT inhibits all of the proteinases of the intrinsic pathway, especially thrombin, F Xa, F IXa, and to a lesser extent the fibrinolytic enzyme plasmin . In contrast, heparin cofactor II is a relatively specific inhibitor of thrombin . In the presence of heparin, the reaction between AT and thrombin and F Xa is drastically accelerated and leads to an effective inhibition of coagulation. The clinically significant AT deficiency is differentiated into the hereditary and the acquired forms. AT has an importance in the pathogenesis of thromboembolism.
- Suspected congenital AT deficiency, especially in the presence of a thromboembolic disease
- In pre term births
- Suspected acquired AT deficiency (e.g., postoperatively, in sepsis and disseminated intravascular coagulation)
- Monitoring of AT replacement therapy
- Suspected heparin resistance.
The plasma AT activity is primarily determined by functional tests. If the activity is reduced, the AT defect is in a secondary step classified into types I (quantitative deficiency) and II type (qualitative deficiency) by immunological methods. Rare variants of hereditary deficiency are detected by molecular biological methods.
Chromogenic amidolytic assay
Principle: the determination of AT activity is carried out on automated platforms using chromogenic assays in which patient plasma is incubated with an excess of thrombin (F IIa) or F Xa /, /. In the presence of heparin, a proportion of the activated thrombin or F Xa is inactivated by the patients endogenous AT of the sample. The residual amount of thrombin or F Xa cleaves a chromogenic peptide substrate, releasing a dye. The concentration of this dye is proportional to the AT activity and is spectrophotometrically measured at 405 nm.
Assays utilizing human F IIa are prone to interferences by heparin cofactor II, especially in patients with heparin therapy. Its effect is small in assays using bovine thrombin. F Xa based assays are not affected by heparin cofactor I, but are influenced by direct F Xa inhibitors such as rivaroxaban.
Progressive activity assay
Principle: this assay is a variant of the chromogenic amidolytic assay. Instead of using heparin, the incubation time is extended to 300 sec. in this assay. Thus, the AT activity is measured independently of the heparin binding site, allowing the differentiation of qualitative AT deficiency into type II RS (RS, reactive site) and type II HBS (HBS, heparin binding site).
The immunochemical assays are designed to measure the quantity of AT protein regardless of the AT’s ability to function. Methods used are immunoelectrophoresis, immunonephelometric and immunoturbidimetric assays.
AT is the essential anticoagulant in plasma and is a key regulator of the coagulation system. The absence of AT is incompatible with life.
- Control of low-level thrombin formation by inhibiting F Xa
- Inhibition of thrombin-mediated fibrin clot formation
- Inhibition of activated clotting factors in the extrinsic pathway (F VIIa-tissue factor complex) and intrinsic pathway (F IXa, F XIa and F XIIa) pathways. The inhibition of these factors is less efficient than that of thrombin and F Xa.
AT deficiency is clinically relevant, whereas an increase in AT in plasma is not. However, therapy with direct thrombin inhibitors such as hirudin and argatroban is an exception because these inhibitors cause falsely elevated AT activity in the thrombin-based AT activity assay, but not in the F Xa-based assay.
Oral anticoagulant therapy with vitamin K antagonists such as warfarin or coumarin may lead to an increase in AT activity.
AT activity below 50–70% indicates an inhibitor deficiency which will lead to inadequate compensation should the levels of pro coagulants or the activities of the pro coagulant factors be increased. Therefore, this results in a shift of the hemostatic equilibrium and the risk of venous thromboembolism (VTE). AT deficiencies can be of hereditary or acquired origin. Hereditary AT deficiency is a hyper coagulable state associated with an increased risk of venous thromboembolism (VTE). The causes of acquired AT deficiency are much more common than hereditary deficiency. AT deficient states are depicted in .
- Type I deficiency that is characterized by decreased AT activity and antigen concentration. Typically they both are below 70% of normal. Homozygous type I deficiency of AT has not been described. Heterozygous AT deficiency occurs in 0.02–0.17% of the general population and in 0.5–4.9% in patients with VTE.
- Type II deficiency is a qualitative defect, resulting in the production of a variant protein with decreased function. The type II is further subdivided into three subtypes:
a) Subtype IIa. Mutations in thrombin binding domain are associated with decreased activity and normal antigen concentration.
b) Subtype IIb. Mutations in heparin-binding domain are associated with decreased activity and normal antigen concentration. This type exhibits progressive activity (i.e., increased activity with prolonged incubation time). The prevalence of subtype II mutations in the general population is 0.03–0.04% and is associated with a low risk of thrombosis in heterozygous carriers.
c) Subtype IIc. Pleiotropic defects result from a genetic mutation in the s1C-s4B region and are associated with a moderate decrease in both AT activity and antigen levels. AT activity is lower than antigen concentration.
Further informations about hereditary AT deficiency are presented in:
The chromogenic assay of AT activity involves heparin and identifies all types of AT deficiency, however, the assay is not able to distinguish type II HBS from other type II defects. A variant of this assay, the progressive activity assay, measures activity independent of the HBS.
Since acquired AT deficiency is associated with other disorders of the hemostatic system, its clinical effects and causes are not clearly definable. Although the plasma AT activity is reduced in patients suffering from nephrotic syndrome, it remains unclear whether AT deficiency is involved in causing an increased incidence of thrombosis. Reduced AT synthesis does not result in increased incidence of thrombosis in patients with liver cirrhosis.
Venous thrombosis is the usual mode of clinical presentation in AT deficient individuals. The common sites for thromboses are the deep leg veins, the iliac, femoral and superficial leg veins. Arterial thrombosis is uncommon, but has been reported. In the European Prospective Cohort on Thrombophilia (EPCOT) study . The risk of first occurrence of VTE in individuals with hereditary deficiency of AT, PT, PS or F V Leiden was analyzed. In the 5.7-year course of the study, 4.5% of the patients developed VTE. The annual incidence was highest (1.7%) in patients with AT deficiency. The relative risk of VTE in AT deficiency was 25–50-fold higher than in individuals with normal AT.
In the case of AT therapy, an AT activity of 80% should be maintained. One unit of AT per kg of body weight increases the plasma AT activity by 1–2%. The half-life of replaced AT is 1.5–2.5 days unless heparin therapy or a major inflammatory response are present in which case the half-life is reduced to less than 1 day. The following markers should be determined beforehand to ensure the efficacy of replacement therapy: PT, aPTT, AT activity, thrombocyte count, fibrinogen, D-dimers .
AT should always be measured in plasma and not in serum because the process of coagulation consumes approximately 30% of AT.
Method of determination
The method used for testing AT deficiency is important to ensure correct diagnosis and treatment of the patient.
- Assays using human thrombin are subject to interference by heparin cofactor, resulting in a falsely high value, especially under heparin therapy. This is not the case in F Xa-based assays.
- Direct thrombin inhibitors such as hirudin and argatroban simulate falsely high AT activity if thrombin-based assays are used
- Some types of hereditary AT deficiency are not detected by thrombin-based assays (e.g., Hamilton mutation A382T) or several mutations in connection with type II HBS
- F Xa-based assays are interfered by direct F Xa inhibitors such as rivaroxaban, but not by heparin cofactor II or direct thrombin inhibitors .
- F Xa based assays do not detect all AT variants, such as the Stockholm mutation (G392D), which can be measured using thrombin-based assays, or the Cambridge II mutation (A384S) , which is in part not detectable by thrombin-based assays. F Xa-based chromogenic assays have a lower detection limit for AT deficiency than thrombin-based assays .
There is no difference in reference intervals for thrombin-based and F Xa-based assays.
AT is a single-chain glycoprotein with a molecular weight of 58 kDa and belongs to the family of serine protease inhibitors. In a two-step reaction, AT initially binds to the proteases much like a substrate. Subsequently, after proteolytic cleavage, AT binds firmly to the serine located in the active center of the protease (). AT inhibits thrombin, factors F Xa and F IXa at about the same level of efficiency. F XIa, F XIIa and kallikrein are inhibited to a lesser degree. All inhibitory reactions are accelerated by heparin. Whereas the reaction with thrombin and F Xa is accelerated 1,000–2,000-fold in the presence of heparin, the reactions of AT with the contact phase factors are only slightly faster. Under physiological conditions, the heparan sulfate proteoglycans incorporated into the surface membrane of endothelial cells exert an accelerating effect on the binding capacity of AT . The half-life of AT is 65 hours and thus longer than that of replaced AT.
During states of enhanced activation of intravascular coagulation connected with an increased release of thrombin, the consumption rate of AT is more rapid than its production rate. The decrease in AT and the increase in the concentration of thrombin-antithrombin complex resulting from inhibition of thrombin are indicators of abnormal fibrin formation.
The decrease in AT and the formation of thrombin-antithrombin complex resulting from the inhibition of thrombin are indicators of abnormal thrombin formation. Parallel to the processes of disseminated intravascular coagulation (DIC), an impairment in the liver synthetic capacity often develops as well. The impaired AT synthesis thus contributes further to the enhancement of DIC.
The assessment of homeostasis requires that, besides the coagulation inhibitors (AT, protein C) and the indicators of activation and consumption (TAT, F1+2, fibrin monomer, D-dimer), the pro coagulant factors are taken into account as well (e.g., by measuring PT and aPTT). Furthermore, in order to interpret the in vivo activity of AT, the actual pH of the blood must be taken into consideration, since acidosis is quantitatively associated with increasing functional deficits of the inhibitor. Accordingly, an AT activity of 60%, for example, measured in vitro under optimal conditions corresponds to an in vivo activity of only 10%, given a pH of 7.05. The AT activity will return to 60% if acidosis is eliminated.
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12. Vossen CY, Conard J, Fontcuberta J, Markis M, van der Meer FJ, Pabinger I, et al. Risk of a first venous thrombotic event in carriers of familial thrombophilic defect. The European Prospective Cohort on Thrombophilia (EPCOT). J Thromb Haemost 2005; 3: 459–64.
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24. Vossen CY, Conard J, Fontcuberta J, Markis M, van der Meer FJ, Pabinger I, et al. Risk of a first venous thrombotic event in carriers of familial thrombophilic defect. The European Prospective Cohort on Thrombophilia (EPCOT). J Thromb Haemost 2005; 3: 459–64.
Environmental risk factors and genetic predisposition play an important role in the development of venous thromboembolic events (VTE). Genetic risk factors, primarily related to the hemostatic system, trigger venous thromboembolic events (VTE).
- Loss of function mutations involving natural anticoagulants antithrombin, protein C and protein S
- Gain of function mutations in pro coagulant factor V (F V Leiden) and F II (prothrombin G20210A9).
The association between genetic risk factors and VTE can be classified as:
- Strong: deficiencies in antithrombin, protein S and protein C (risk of VTE is 5–10-fold increased)
- Moderate: presence of F V Leiden or prothrombin G20210A9 (risk of VTE is 2–5-fold increased)
- Weak: for instance, His95Arg replacement in the B-subunit of F XIII (risk of VTE is approximately 1.5-fold increased).
Protein C (PC) and protein S are natural anticoagulants and play an important role in the regulation of the coagulation system . PC is activated by thrombin in the presence of thrombomodulin (T M). T M is an endothelial cell surface receptor, which plays an important modulating role in the anticoagulant response after vascular injury.
After vascular injury binding of thrombin to
- F Va is cleaved at Arg 506, which is the preferred cleavage site, however, full inactivation also requires cleavage at Arg 306
- F VIIIa is cleaved at Arg 336 and Arg 562.
Refer also to
Protein S (PS) is present in bound and free form . The free form of PS is an important cofactor of APC, enhancing its affinity to negative charged phospholipid surfaces. Free PS is able to displace F Xa from its complex with F Va, allowing APC to cleave F Va at Arg506. In the process of F VIIIa inactivation, APC activity is synergistically stimulated by PS. PS is encoded by the gene PROS 1 consisting of 15 exons. Free PS is the only cofactor of APC. Bound PS forms a complex with the complement 4b binding protein (C4bBP); this complex lacks APC cofactor activity.
Activated PC resistance is caused by a single point mutation in the F V gene, also known as F V Leiden, resulting in insufficient inactivation of F Va by APC.
In the absence of F V Leiden, an acquired activated PC resistant phenotype may be present during pregnancy, with the use of oral contraceptives, in the presence of lupus anticoagulant, in cases with increased F VIII concentration and in patients with multiple myeloma.
Factor V functions as a synergistic cofactor with PS in the degradation of F VIII when this factor is part of the tenase complex (F Xa/F Va) . The genetic background for the APC resistance phenotype is a single G to A nucleotide substitution at position 1691 in the F V gene (G1691A or F V Leiden), resulting in the replacement of Arg 506 by Gln.
A mutation in the Prothrombin gene is the second most common cause of inherited thrombophilia . This mutation involves a single base-pair substitution (guanine to adenine) at nucleotide 20210 in the 3’-untranslated region of the Prothrombin gene. Heterozygous carriers have higher prothrombin plasma levels, with a 2 to 5-fold increased risk of VTE without concomitant risk factors. There is a more than additive synergistic risk of VTE when other thrombophilic factors, particularly factor V Leiden are simultaneously present .
Testing can be considered in patients with VTE suggestive of inherited thrombophilia:
- Thrombosis at a young age (< 50 years), especially in association with weak provoking factors (minor surgery, combination of oral contraceptives, or immobility) or unprovoked VTE
- Strong family history of VTE (first-degree family members affected at young age)
- Recurrent VTE events, especially at a young age
- VTE in unusual sites such as splanchnic or cerebral veins.
- Primarily a functional coagulation assay for identifying APC resistance
- Secondarily, in the case of APC resistance, molecular screening for factor V Leiden
- Tertiarily, if molecular screening is negative, the determination of PC and PS and, possibly, molecular genetic analysis of the gene PROS 1.
The protein C activity dependent clotting time (PCAT) is determined using the aPTT-based assay. The second-generation aPTT method uses patient plasma with pre dilution of F V deficient plasma (1+4). This modification is highly sensitive to F V Leiden and can also be used in patients undergoing anticoagulation therapy with vitamin K antagonists (coumarins).
The aPTT is determined in the presence of a PC activator, the snake venom Agkistrodon contortix (PCAT sample), and in the absence of this activator (PCAT 0 sample). Normal plasma coagulates in an aPTT assay in 28–35 seconds. If activated PC (APC) is added, the aPTT clotting time is prolonged 2-fold or more to 60–100 seconds. This plasma is sensitive to APC. Lower sensitivity of the patient plasma to APC (clotting time only prolonged 1.5–1.7-fold; typical of heterozygous F V Leiden carriers) indicates that the plasma is resistant to APC.
The ratios PCAT/PCAT 0 are obtained by transformation of the clotting times in normalized ratios (NR). NR below 0.80 indicates the presence of F V Leiden.
This assay also detects cooperative effects between the involved factors and, thus, also recognizes PC deficiency.
A differentiation must be made between determination of PC activity (functional assay) and that of PC antigen concentration (immunochemical assay) . There are conditions where the two assays may not provide identical results.
Coagulation assay (functional assay)
Principle: the majority of methods are based on the aPTT which depends on F V and F VIII. The activated factors F Va and F VIIIa are inactivated by activation of PC. Prolongation of the aPTT occurs which is a measure of the anticoagulant activity of PC. After the addition of aPTT reagent, an enzyme, usually extracted from the snake venom of Agkistrodon contortrix, is added which activates PC. Since the reaction mixture contains an excess of all of the coagulation factors, the measured clotting time depends on the PC activity. The result is read off a standard curve as the clotting time and is expressed in % of normal. Lupus anticoagulant and FVIII concentrations above 150% interfere with coagulation assays.
Amidolytic assay (functional assay)
The enzyme activity of PC is determined using a chromogenic peptide substrate. Contrary to the coagulation assay, a change in the phospholipid binding domain, for example under oral anticoagulant therapy, has no effect.
Principle: PC is activated by a PC activator (snake venom); subsequently, the cleavage of the chromogenic substrate is determined kinetically using spectrophotometric measurement. The activity is expressed in % of normal.
The concentration of the PC antigen is determined. There is currently no need to differentiate between type I and type II. Therefore, the enzyme immunoassay is used for confirmation of decreased activity measured in the functional assay.
Principle: antibodies to PC are bound on the surface of micro titer wells; these bind PC contained in the plasma sample. Subsequently, a peroxidase-labeled antibody to PC binds to the immune-complex-bound PC. The amount of bound peroxidase which is proportional to the quantity of PC is measured by the addition of substrate. The result is expressed in % of normal or in mg/L.
Approximately 60% of PS in the plasma is bound to the C4b-complement-binding protein (C4bBP) while 40% are present in a free circulating form . Only free PS can exert its anticoagulant effect as a cofactor of APC. A functional assay as well as an immunochemical assay should be used. The functional assay is the screening test . The analysis of the gene PROS 1 includes the investigation of all 15 exons for sequence variants.
The functional activity of free PS (i.e., the capacity as an APC cofactor to degrade F Va and F VIIIa) and, thus, to prolong the clotting time, is measured by the prolongation of the aPTT or the PT or the Russell’s viper venom time (RVVT) . Analogously to the PC determination by a coagulation assay, the sample is diluted and mixed with plasma deficient in PS. The clotting time is measured after the addition of a coagulation activator and already activated PC. The result is expressed in % of normal.
Using an enzyme immunoassay, the total and the free PS concentrations can be determined. In order to determine the free proportion, bound PS is precipitated using polyethylene glycol, followed by the determination of the remaining portion in the supernatant. Newer methods use monoclonal antibodies which bind to the PS region covered by C4bBP. This makes the precipitation of bound PS of the sample unnecessary.
Molecular genetic analysis
A fragment of exon 10 of the FV gene is amplified by PCR (DNA polymerase chain reaction). The mutation of the Leiden variant of F V is located in this fragment. The amplified fragment is cut at two sites by the restriction enzyme Mnl. The mutation results in the loss of one of the two interface sites for the restriction enzyme, thus making a higher-molecular-weight-DNA band visible during the gel electrophoresis of samples belonging to individuals afflicted with this defect. The differentiation between heterozygous and homozygous carriers of this defect is only possible by using this method.
Molecular genetic analysis with differentiation between homozygous and heterozygous variations.
APC resistance, PC and PS: citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 1 mL
Molecular genetic analysis for F V Leiden, prothrombin G20210A variation: EDTA blood: 3–5 mL
- All patients are diagnosed with F V Leiden. Relevant studies report a diagnostic sensitivity of 100% with a specificity of 95%. One study reported a specificity of only 72%.
- Impaired functional activity of PC is detected with a diagnostic sensitivity of 82–100%
- PS deficiency is diagnosed with a sensitivity of 87–97% and a specificity of only 30% .
The functional coagulation assay is a screening test used to determine F V Leiden and PC deficiency whereas detection of PS deficiency is problematic. The assay is clinically useful to detect defects as well as to assess the capacity of the PC pathway and, thus, to a certain extent predict a future risk of VTE. In patients with PS deficiency, the screening assay is a good tool for assessing the risk of a thrombotic event. Borderline or positive findings should undergo molecular genetic analysis for F V Leiden.
Factor V Leiden mutation is one of the most common inherited risk factors for VTE. The prevalence of heterozygous carriers in the general population is 2–15%. This mutation is limited to populations of Caucasian origin and their descent in North and South America. The incidence of VTE is 1 in 10,000 per year in young women and is increased to 4–7 in female heterozygous carriers of F V Leiden. If a young women with F V Leiden is obese, the incidence increases to 8–14 in 10,000, and if she is taking oral contraceptives, the incidence is even 34 in 10,000 . Related to both sexes, the probability of VTE is increased 3–7-fold by a heterozygous defect and 50–100-fold by a homozygous defect. About 25% of family members with F V Leiden suffer their first VTE event before the age of 45.
VTE after general surgery does not appear to be increased in the presence of F V Leiden heterozygosity when patients are under prophylactic anticoagulation. There may be an increased risk of arterial thrombosis and myocardial infarction after vascular surgery. Coronary bypass graft occlusion within the first few months after surgery may be increased in the presence of F V Leiden.
The annual incidence of VTE in women above 50 years of age is 4 in 10,000. This rate increases to 10 in 10,000 if these women are taking postmenopausal estrogens and progesterone. In women with a familial history of VTE, the annual incidence increases to 30 in 10,000.
In hereditary PC deficiency, a clinically irrelevant differentiation is made between two types:
- Type I; both the protein concentration as well as the activity are decreased due to a defect in synthesis
- Type II; a dysfunctional protein is present (i.e., the protein concentration is within the reference interval whereas the activity is significantly reduced).
The majority of patients heterozygous for PC deficiency have PC activities of 30–65% . Homozygous carriers of the defect have almost undetectable PC activity. Most of the about 250 mutations detected to date are causative of type I deficiency. Approximately 70% of these patients have a single mutation in the encoding region of the gene PROC, leading to amino acid changes in PC. For acquired PC deficiency, see .
- Type I; both the antigen concentration as well as the activity are decreased due to a defect in synthesis
- Type II; a dysfunctional protein is present (i.e., the antigen concentration is within the reference interval whereas the activity is significantly reduced)
- Type III; the concentration of free PS is decreased due to an elevated concentration of C4bBP (i.e., the antigen concentration is within the reference interval although the activity and the concentration of free PS antigen are reduced). A causal relation between this type and VTE has not been established to date.
Acquired PS deficiency is much more common than the hereditary type. Moreover, the PS concentrations and activities of healthy individuals overlap significantly with those of carriers of the hereditary defect. Therefore, the PS reference values are not suited for diagnosing a hereditary defect. The classification of hereditary PS deficiency is shown in .
Hereditary PS deficiency
PS deficiency types I and II are defects associated with decreased PS activity and account for 95% of the cases . Investigations for PS deficiency should always include a functional assay and the determination of the PS concentration. The PS values of a study are shown in . Only PS values below 45% generally point to a disease defined by a mutation in the gene PROS 1 . In these cases, a molecular genetic analysis of the 15 exons of PROS 1 should be performed. A diagnostic algorithm is recommended .
Heterozygous PC and PS deficiency present with the same clinical features. Only sporadic cases of homozygous deficiency have been known to date. In patients below 45 years with venous thrombosis, heterozygous PS deficiency type I was found in approximately in 2–3% of cases. See also .
Acquired protein S deficiency
Liver disease causes but a moderate decrease in PS, and DIC is associated with normal PS levels. The decline in PS in the presence of acute respiratory distress syndrome affects the free PS portion significantly more than the bound form .
Estrogens result in a decreased release of PS, thus explaining why premenopausal women have physiologically lower levels in comparison to men (median of approximately 80%). Hormone therapy or the intake of oral contraceptives also cause a significant decrease in PS activity.
The Prothrombin G20210A mutation involves a single base-pair substitution (guanine to adenine) at nucleotide 20210 in the 3’ un translated region of the Prothrombin gene. Carriers of this mutation have a higher endogenous thrombin potential compared to those with the wild type genotype. Prothrombin is increased by 30% in heterozygous carriers and by 70% in homozygous carriers . This mutation is associated with a hyper coagulable state and represents the second highest risk of hereditary thrombophilia . Heterozygous carriers among the Caucasian population:
- Have a prevalence of 2%
- Have a 2–5-fold risk of thrombosis (10-fold in homozygous carriers)
- Account for 20% of VTE patients.
Women with the Prothrombin G20210A mutation undergoing oral anticoagulation have a 150-fold increased risk of cerebral venous sinus thrombosis and a 16–59-fold increased risk of other deep venous thromboses compared to those without oral anticoagulation .
However, the Prothrombin G20210A mutation may not be the only cause of a higher endogenous thrombin potential and VTE in affected carriers. Increased thrombin generation is thought to be associated with other genetic and/or environmental factors .
The homozygous genotype of MTHFR gene polymorphism at position C677T is linked to the Caucasian population. This mutation is associated with an increase in plasma homocysteine, depending on the diet. Carriers among the Caucasian population:
- Have a prevalence of 10%
- Have a 2–3-fold risk of thrombosis
- Account for 15–20% of VTE patients.
The risk of cerebral venous sinus thrombosis is 20-fold increased in women with the MTHFR C677T mutation and hyper homocysteinemia undergoing hormonal anticoagulation compared to those without oral anticoagulation . The association between the MTHFR-C677T mutation and venous thromboembolism has been discussed controversially.
Preanalytics: since the vitamin K+-dependent coagulation factors and protein S are decreased, patients should not be on oral anticoagulant treatment. APC resistance is simulated. Vitamin K inhibitors do not interfere with assays which use samples diluted with F V deficient plasma . The investigation can be performed on patients on heparin treatment if the plasma heparin concentration is below 1 IU/mL because the reagents contain heparin-neutralizing polybrenes.
Influence factors: despite using patient plasma diluted with F V deficient plasma, the presence of lupus anticoagulant, direct thrombin and F Xa inhibitors and heparin values higher than 1 IU/mL, all of which prolong the clotting time, may result in a falsely elevated APC, leading to missed detection of APC resistance. The presence of argatroban, a direct thrombin inhibitor, should be kept in mind if the APC resistance assay yields unexpectedly prolonged clotting times in samples with and without APC . The sensitivity of patient plasma to APC is lower in the presence of elevated F VIII seen, for example, in inflammatory conditions and pregnancy. This results in shorter clotting times and simulated APC resistance.
F VIII activities higher than 150% shorten the clotting time and are connected with falsely low protein C activities in the functional assay. Anticoagulation in the patient does not interfere with the determination.
Amidolytic assays for PC determination are not sensitive to lupus anticoagulant, high F VIII concentration and F V Leiden. Chromogenic peptide substrates which have no high specificity for APC may overestimate PC in the presence of other proteolytic enzymes, such as plasmin, kallikrein and thrombin. Moreover, they are insensitive to a certain type of qualitative PC deficiency.
Platelets cause falsely low activities. Therefore, platelet-poor plasma samples should be used for analysis.
Direct oral anticoagulant interference on thrombophilia testing
In a study the introduction of DOACs into the US marketplace has caused significant interference in thrombophilia assays. Approximately 5-10% of tests for thrombophilia risk have potential DOAC interference. False positive test results for lupus anticoagulant, falsely increase protein S and protein C activities, and interference with FV dependent prothrombin venom based, activated protein C resistance were registered. Laboratories trying to determine whether samples submitted for thrombophilia testing have potential DOAC interference, should perform a thrombin time and a test for the anti-Xa activity, if test results as described above, are present.
PC is a vitamin K dependent glycoprotein synthesized as proenzyme in the liver. The mature protein has a molecular weight of 62 kDa and consists of a heavy chain (41 kDa) and a light chain (21 kDa) connected by a disulfide bond between Cys 141 and Cys 265.
Circulating plasma PC is a proenzyme that needs to be activated for anticoagulatory action. It is activated by thrombin bound in a thrombomodulin-thrombin complex on the vascular endothelial cells, in the presence of Ca2+ ().
APC causes proteolysis at the phospholipid surface and, thus, inactivates F Va and F VIIIa. This process depends on the presence of Ca2+ and is accelerated by protein S. APC is regulated by the serine proteinase inhibitors α1-antitrypsin, α2-antiplasmin and the heparin-stimulated PC inhibitor as well as by α2-macroglobulin.
PS has a molecular weight of 69 kDa and is synthesized in the liver and vascular endothelial cells. In comparison to PC with a half-life of only 8 h, the half-life of PS is 60 h. About 40% of PS are present in a free form while 60% are bound to C4bBP. Free PS circulates as cofactor of PC and is present in its active form.
The gene encoding PC (PROC) is located at position 2q13–q14 and contains 9 exons and 8 introns. The gene encoding PS (PROS1) is located at position 3q11.2 and contains 15 exons and 14 introns.
A defect in the synthesis or a functional defect of PC or PS cause a reduction in the anticoagulant potential and therefore can result in hyper coagulability which is characterized by an increased susceptibility to venous thromboembolic events.
The variant Leiden of F V is another defect which counteracts the PC pathway. Under normal conditions, F V circulates in plasma and shows no notable pro- or anticoagulant activity.
However, the pro coagulant activity of F Xa and thrombin and the anticoagulant activity of APC have an influence on F V:
- Enhanced pro coagulant activity is achieved upon activation of the coagulation system in the event of vascular injury. In this case, F Xa or thrombin cleave three arginine-associated peptide bonds in F V, thus detaching domain B in the F V molecule and causing F V to become the activated pro coagulant F Va (). F Va is an important cofactor of F Xa in prothrombin activation. Both activated pro coagulators form the pro thrombokinase complex (F IXa, F VIIIa, F Xa, F IIa) on the surface of phospholipids.
- F V follows a different direction, however, if the protein C pathway is activated. APC regulates the pro coagulant activity of the pro thrombokinase complex by proteolytic degradation of F V, cleaving the F V molecule at positions Arg 306 and Arg 506. Cleavage at position 306 leads to the complete loss of F V activity, whereas cleavage at position 506 results in an intermediate retaining F Xa cofactor activity. The F V intermediate cleaved off by APC forms an anticoagulant complex with protein S which cleaves F VIII present in the tenase complex, thus inactivating the tenase complex. The tenase complex consists of F IXa and F VIIIa bound to the phospholipid surface. It is efficient in F Xa activation and cannot be down regulated by APC or PC.
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19. Lavigne-Lissalde G, Sanchez C, Castelli C, Alonso S, Mazoyer E, Bal dit Sollier C, et al. Prothrombin G20210A carriers the genetic mutation and a history of venous thrombosis contributes to thrombin generation independently of factor II plasma levels. J Thrombos Haemost 2010; 8: 942–9.
20. Seitz R, Rappe N, Wolf M, Heidtmann HH, Maasberg M, Immel A, Kraus M, Egbring R, Pfab R, Havemann K. Impaired anticoagulant activity of protein C and activation of neutrophils in extensive lung cancer. Clin Appl Thromb Haemostas 1995; 1: 131–4.
21. Quincampoix JC, Legarff M, Rittling C, Andiva S, Toulon P. Modification of the Pro C Global assay using dilution of patient plasma in factor V-depleted plasma as a screening assay for factor V Leiden mutation. Blood Coagul Fibrinolysis 2001; 12: 569–76.
25. Wong CC, Worfolk LA, Sahud M, Dlott J.S. Prevalence of suspected direct oral anticoagulant interference on thrombophilia testing at US National Reference Laboratory. Clin Chem 2020; 66 (4): 618–20.
The antiphospholipid antibody syndrome (APS) has a broad spectrum of thrombotic, non thrombotic and obstetrical manifestations. The APS is generally considered to confer a high risk of recurrent venous thromboembolism (VTE), arterial and microvascular thrombosis. Tests for antiphospholipid antibodies are generally included in the workup for hyper coagulable state. Obstetrical APS is characterized by fetal loss after the 10th week of gestation, recurrent early miscarriages, intrauterine growth restriction, or severe preeclampsia /, /.
- Venous thromboembolism (VTE)
- Arterial thrombosis (myocardial infarction, stroke)
- Pregnancy disorders
- Autoimmune disorders, especially systemic lupus erythematosus
- APTT prolongation of unknown origin
- Thrombocytopenia of unknown origin.
- Lupus anticoagulant
- Anti cardiolipin immunoglobulin isotye G (IgG) and M (IgM ) or IgG and IgM anti-β2 gycoprotein-I (anti-β2GPI).
- Coagulation assays; they indirectly identify interference by antibodies based on prolonged clotting time. Antibodies directed against phospholipids that prolong the clotting time are also referred to as lupus anticoagulants.
- Immunoassays (ELISA) for the direct determination of anti-β2-glycoprotein I and anti-cardiolipin.
Results of first-line tests that are suggestive of antiphospholipid syndrome:
- Prolongation of aPTT clotting time
- Thrombocyte count below 100 × 109/L.
Lupus anticoagulants (LAs) are antibodies directed against phospholipids and/or phospholipid-protein complexes. They prolong the clotting time of phospholipid dependent coagulation assays in vitro, but are not associated with thrombophilia. The term lupus anticoagulant results from the year 1952 when a prolonged aPTT was for the first time determined in patients with systemic lupus erythematosus.
1. Demonstration of a prolonged phospholipid-dependent coagulation screening test based on the local clotting time threshold value.
2. Plasma mixing test to confirm the presence of an inhibitor and exclusion of coagulation factor deficiency.
3. Confirmatory test to demonstrate that the inhibitor is phospholipid-dependent and is not directed against an individual coagulation factor.
- A test based on F X activation (diluted Russel viper venom time)
- A second test based on intrinsic coagulation activation (aPTT or kaolin clotting time).
LA is suspected when the 99th percentile of the clotting time of healthy individuals is exceeded. lists recommendations for optimal laboratory detection. For streamlining the test procedure, the Sidney criteria recommend the integration of screening and confirmation into a single assay. These tests do not require performance of the plasma mixing test.
Diluted Russel viper venom time (dRVVT) assay
Principle: the snake venom Russel’s viper venom contained in the reagent activates F X causing prothrombin activation in the presence of F V, Ca2+ and phospholipids. The high sensitivity of the assay is achieved by low phospholipid concentration in the reagent .
Activated partial thromboplastin time assay (aPTT)
Principle: see . The different commercially available reagents differ by the type of activator and the type, composition and concentration of phospholipids. The kaolin clotting time (KCT) assay is recommended. Kaolin consists of silicic acid the microcrystalline form of which has a large negatively charged surface. Thus, the process of contact activation of the intrinsic pathway is initiated and F X is activated. The F VIII activity may be increased during acute phase response or during pregnancy, leading to a shortened clotting time. Therefore, specific lupus-sensitive reagents are provided for LA detection .
Plasma mixing test
Perform testing on plasmas from healthy donors mixed with pooled normal plasma (PNP) at 1 : 1 proportion. Testing should be performed without pre incubation. The PNP should be prepared ad hoc (home-made) to ensure that the PNP contains less than 10 × 109 platelets/L and to ensure approximately 100% activity for all clotting factors. The thrombin time performed on patient plasma or an anti-F Xa test will help to identify heparin or specific inhibitors to clotting factors .
A prolonged clotting time in the plasma mixing test indicates the presence of LA in the patient plasma, whereas shortening of the clotting time points to factor deficiency that should be confirmed by determination of the relevant individual factor. In many cases, the concurrent decrease in several coagulation factors indicates the presence of LA.
Integrated tests include screening and confirmation testing in a single procedure. Such tests consist of testing the patient plasma twice by means of the dRVVT or aPTT performed in parallel at low (screen) and high (confirm) phospholipid concentrations. In the presence of LA, the clotting time should be prolonged in the screening plasma sample and normal in the confirmation plasma sample. The results should be interpreted by calculating the LA ratio (screen minus confirm) or the percentage correction.
Correction (%) = (screen – confirm)/screen × 100
The presence of LA induces normalization of the clotting time in the confirmation plasma sample. The presence of coagulation factor inhibitors and unfractionated heparin may lead to falsely positive results.
Threshold values for lupus anticoagulant detection
Results of screening tests and the plasma mixing test are potentially suggestive of LA when their clotting times are longer than the 99th percentile of the distribution of at least 40 PNP patients below 50 years of age. Alternatively, the cutoff in the plasma mixing study may be the value of the inhibitory coagulation activity (ICA) defined according to the equation:
ICA = [(b – c/a)] × 100
a, b and c are the clotting times of the patient plasma, mixture and normal plasma, respectively.
The clotting time of the confirmatory test in LA positive samples is not always shortened to within the normal interval of controls. It is, therefore, recommended to perform confirmatory tests in all normal controls and to use the mean of obtained clotting times to calculate the threshold value. Patient plasmas below this threshold are LA positive.
APA are directed against phospholipid-protein complexes and are determined using ELISA methodology. The results of the anti-β2-glycoprotein-I test and the anti-cardiolipin test should always be interpreted in the context of the LA test. In a prospective study , it was shown that the sensitivity of IgG antibody determination is sufficient to diagnose APS associated thrombosis, whereas the determination of IgM or IgA antibodies is not advantageous.
Antibodies to β2-glycoprotein I (anti-β2-GPI)
β2-glycoprotein I (β2-GPI) is a 50 kDa-glycoprotein which is composed of a polypeptide chain possessing 5 domains. It binds anionic phospholipids and is a cofactor of cardiolipin. The circulating β2-GPI protein is not able to interact with cellular receptors until after its dimerization by autoantibodies. Receptor-bound APA complexes induce an intracellular signaling that leads to the deregulated activation of endothelial cells, monocytes and platelets, thus providing a possible explanation for the thrombotic predisposition in APS patients .
Anti-β2-GPI antibodies are primarily directed against the amino acids Gly40–Arg43 in domain I. Association with thrombosis has not been found for any APA not directed against this domain.
Anti-cardiolipin antibodies (aCL)
Cardiolipins (diphosphatidylglycerins) are a subgroup of phospholipids.
Principle: diluted serum is added to bovine or human serum in a cardiolipin-coated tube and allowed to react. The objective is to detect antibodies to cardiolipin and albumin-bound β2-GPI. The protocol for determination of aCL by ELISA should be observed during testing .
Lupus anticoagulant: platelet-poor citrated plasma
Blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood: 1 mL
Preparation of platelet-poor citrated plasma: centrifugation performed twice to keep the thrombocyte count below 10 × 109/L. Blood collection should be performed prior to anticoagulant therapy or after an adequate period after stopping anticoagulants.
Anti-β2-GPI and aCL
Serum, plasma: 1 mL
The local reference intervals are applied.
Below 10 GPL-U/mL: negative
10–40 GPL-U/mL: weak positive
Higher than 40 GPL-U/mL: positive
Below MPL-U/mL: negative
10–40 MPL-U/mL: weak positive
Higher than 40 MPL-U/mL: positive
Ten percent of healthy blood donors are positive for anti cardiolipin antibodies, and 1% are positive for lupus anticoagulant. However, after 1 year, less than 1% are still positive for these tests . Patients who are positive for antiphospholipid antibodies may present with no related symptoms. Such patients are usually identified during an evaluation for systemic autoimmune disease, early miscarriages, an elevated aPTT, or a false positive result of a syphilis test .
The diagnosis of antiphospholipid syndrome should be considered in patients with persistent, moderate-to-high-risk antiphospholipid antibody profiles and in patients with any antiphospholipid antibody-related finding .
Antiphospholipid syndrome (APS) is a systemic autoimmune disease defined by thrombotic or obstetrical events that occur in patients with persistent antiphospholipid antibodies (APA) . APA are the most common acquired inhibitors of the coagulation system.
- Venous thromboembolism (VTE). Among patients with unprovoked VTE, those with a lupus anticoagulant had a 40% increase in the risk of recurrence, as compared with patients who did not have lupus anticoagulant . For patients with clinically significant, unprovoked thrombotic events, such as large pulmonary embolism or extensive lower-extremity deep vein thrombosis, and persistently high levels of APA, continued anticoagulant therapy is advised .
- Arterial and microvascular thrombosis. Stroke and transient ischemic attack are the most common arterial events in patients with APS. Patients with catastrophic APS present with thrombosis involving multiple organs .
- Obstetrical manifestations are fetal loss after the 10th week of gestation, recurrent early miscarriages, intrauterine growth restriction, or severe preeclampsia . Approximately 1% of pregnant women experience spontaneous abortions and are positive for APS in 10–15% of the cases . In pregnant women who are positive for LA, the annual rate of VTE is 1.46% and that of ischemic stroke is 0.32% . A positive lupus anticoagulant test indicates a higher risk of thrombosis and a negative outcome of pregnancy after gestational week 12 than positive anti-β2-GPI or aCL.
The following clinical findings may be a clue that a patient has the APS: livedo, signs or symptoms of another systemic autoimmune disease, unexplained prolongation of aPTT or mild thrombocytopenia. A thrombocyte count below 20 × 109/L should the clinician consider other causes than APS .
Besides thrombotic the APS has a broad spectrum of non thrombotic clinical manifestations /, /. Among patients with systemic lupus eryrhematosus, valve disease, pulmonary hypertension, livedo reticularis, thrombocytopenia, hemolytic anemia, acute or chronic renal vascular lesions, and moderate or severe cognitive impairment is higher than among patients with APA than among patients who are negative for such antibodies.
The prevalence of lupus anticoagulant positivity among patients with SLE is 30%, and the presence of lupus anticoagulant positivity in such patients is associated with an increased risk of thrombosis (odds ratio 5.6). Forty percent of patients with the APS also have SLE and 37% of patients with SLE have anti-β2-glycoprotein I antibodies. These findings suggest that there is overlap in the pathogenesis of SLE and that of the APS .
The diagnosis of APS is based on clinical manifestation in combination with typical laboratory findings. APS is present if at least one of the clinical criteria and one of the laboratory APA test criteria are met.
- Low; venous or arterial thrombosis, older patients
- Moderate; coincidental evidence of prolonged aPTT in asymptomatic individuals, recurrent spontaneous abortions, predictable thrombosis in young patients
- High; unexplained acute deep vein thrombosis or arterial thrombosis in patients below 50 years of age, thrombosis at unusual sites, miscarriage in late pregnancy, diseases associated with thrombosis or pregnancy in patients with autoimmune disease (systemic lupus erythematosus, rheumatoid arthritis, autoimmune thrombocytopenia, autoimmune hemolytic anemia) .
APA can be only transiently positive, for example in the presence of inflammation and infection. Therefore, a positive finding should be confirmed by testing after an interval ≥ 12 weeks. A negative result of the second investigation indicates that the APA presence was transient.
Laboratory test criteria for APS are shown in . Evidence of APS-induced thrombosis is provided by positive findings for lupus anticoagulant and/or anti-β2 GPI or aCL. The annual risk of a first thrombotic event in asymptomatic individuals who are positive for lupus anticoagulant, anti-β2 GPI and aCL, referred to as triple positive patients, is 5.3%.
Patients who already experienced the clinical event 5 years ago should not undergo laboratory testing for APS.
LA are detected based on their functional activity through interference with phospholipid-dependent steps in the coagulation cascade. High-activity LA assays are highly specific and show strong association with thromboembolic events and obstetric complications . Since a single assay will not cover all APAs possibly present in APS, different methods must be used.
LA activity is reported with quantitative test results to enable numeric differentiation between low and high inhibitory activity. It is recommended to perform laboratory procedures on patients undergoing coumarin therapy 1–2 weeks after dis continuation of treatment or when the INR is below 1.5. Bridging coumarin dis continuation with low molecular weight heparin (LMWH) is recommended with the last dose of LMWH administered more than 12 h before the blood is drawn for LA testing. Alternatively, if the INR is between 1.5 and below 3.0, a 1 : 1 dilution of patient plasma and PNP can be considered.
Isolated LA positivity is significantly more frequent in individuals without clinical APS events or may be false-positive especially if identified as mild in potency, if it is found in elderly patients or if it is diagnosed for the first time .
Anti-β2GPI antibodies are usually detected together with other APAs and are strictly associated with pregnancy complications. Patients with high concentrations of these antibodies are at high risk of thrombosis. Anti-β2GPI antibodies are primarily detected in patients with autoimmune disease . Patients tested positive in all three assays (LA, aCL and anti-β2GPI) usually have a higher concentration of anti-β2GPI than those with positive results in two assays.
Commercial assays for the determination of aCL antibodies have a significant discrepancy regarding their diagnostic sensitivity and specificity. Moreover, the diagnostic sensitivity of aCL assays is high, while specificity is low. Therefore, the evaluation should only take into account results above the 99th percentile of healthy controls or concentrations higher than 40 units/mL of IgG or IgM-aCL antibodies . aCL antibody assays should only be interpreted in the context of clinical findings and considering the LA assay results. According to longitudinal studies, the positivity of aCL ELISA is not associated with the incidence of first event of deep vein thrombosis .
A profile of anti-β2GPI and aCL should always be determined and assessed in the context of LA testing. The presence of medium to high titers of these antibodies of the same isotype (most often IgG) if in agreement with a positive LA identifies patients at high risk for thrombosis .
Lupus anticoagulant (LA) testing
None of the individual assays for determining LA has 100% diagnostic sensitivity and specificity due to the difference in commercially available reagents. For instance, the presence of heparin and coagulation factor inhibitors and deficiency in coagulation factors lead to false-positive results. False-negative results are obtained if the plasma used is not platelet-poor.
Low LA concentrations may not be detectable in the plasma mixing test.
The plasma to be analyzed should contain less than 10 × 109 platelets per liter.
Prior to LA testing, a thrombin time or anti F Xa assay must be performed to determine the presence of heparin in the sample even if the reagent comprises a heparin neutralizer.
The analytic specificity of ELISA can be lower than possible, presumably because when β2-GPI antibodies are transferred to ELISA tubes, neo antigens are formed which preferably bind non-pathogenic antibodies. Hence, fewer pathogenic antibodies are bound, resulting in decreased specificity of the assay. Inter laboratory variation between the assays for anti-β2-GPI is not as high as for aCL, and the specificity for APS diagnosis is higher .
The antiphospholipid syndrome is an autoimmune disorder characterized by persistently elevated concentrations of antiphospholipid antibodies. The antibodies represent a heterogenous group of autoantibodies that recognize various phospholipids, phospholipid-binding plasma proteins, and/or phospholipid-protein complexes . The major target of antiphospholipid antibodies is directed against the plasma protein β2-glycoprotein-I.
The risk of thrombosis correlates more strongly due to anti-β2-glycoprotein autoantibodies than due to lupus anticoagulant. The thrombogenic properties are eliminated when the fraction of anti-β2-glycoprotein-I antibodies is removed as shown in mice studies . In patients with the risk of thrombosis autoantibodies bind the major B-cell epitope on domain I of the β2-glycoprotein-I molecule. The autoantibodies confer lupus-anticoagulant activity.
- In the circular form domain I interacts with domain V. In this form the B-cell epitope is hidden from the immune system
- A fish hook confirmation, exposing the domain I epitope and allowing domain I anti-β2-glycoprotein-I antibodies to bind.
The fish hook confirmation with affinity to phospholipid containing surfaces forms an immunocomplex on cellular surfaces, especially when bound to an β2-glycoprotein-I-antibody. The surface bound complex up regulates the expression of prothrombotic cellular adhesion proteins such as tissue factor and E-selectin. The binding of the immunocomplex suppresses the activity of the tissue factor inhibitor and reduces the activity of protein C in addition.
3. Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, Cervera R, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome. J Thrombos Haemost 2006; 4: 295–306.
6. Brandt JT, Barna LK, Triplett DA. Laboratory identification of lupus anticoagulants: Results of the second international workshop for identification of lupus anticoagulants. On behalf of the subcommittee on lupus anticoagulants/antiphospholipid antibodies of the ISTH. Thromb Haemost 1995; 74: 1597–1603.
9. Forasteiro R, Martinuzzo M, Pombo G, Puente D, Rossi A, Celebrin L, et al. A prospective study of antibodies to beta2-glycoprotein I and prothrombin, and risk of thrombosis. J Thromb Haemost 2005; 3: 1231–8.
10. Ticani A, Allegri F, Balestrieri G, Reber G, Sanmarco M, Meroni P, et al. Minimal requirements for antiphospholipid antibodies ELISAs proposed by the European Forum on antiphospholipid antibodies. Thrombosis Res 2004; 114: 553–8.
13. Asherson RA, Cervera R, de Groot PG, Erkan D, Boffa MC, Piette JC, et al. Catastrophic antiphospholipid syndrome: international consensus statement on classification criteria and treatment guidelines. Lupus 2003; 12: 530–4.
15. Gris JC, Bouvier S, Molinari N, et al. Comparative incidence of a first thrombotic event in purely obstetric antiphospholipid syndrome with pregnancy loss: the NOH-APS observational study. Blood 2012; 119: 2624–32.
16. Ruffatti A, Tonello M, Del Ross T, Cavazzana A, Grava C, Noventa F, et al. Antibody profile and clinical course in primary antiphospholipid syndrome with pregnancy morbidity. Thromb Haemost 2006; 96: 337–41.
22. Arad A, Proulle V, Furie RA, Furie BC, Furie B. β2-glycoprotein-1 autoantibodies from patients with antiphospholipid syndrome are sufficient to potentiate arterial thrombus formation in a mouse model. Blood 2011; 117: 3453–9.
Michael Kraus, Lothar Thomas
Plasminogen is the inactive precursor of the fibrinolytic enzyme plasmin. Plasminogen is activated by activator substances such as tissue activator (t-PA) and urokinase (u-PA) or by the contact phase of coagulation (F XIIa-high-molecular weight kininogen complex). Therapeutically, exogenous activators such as streptokinase are employed as well. When a fibrin clot forms approximately 20% of the plasminogen content of plasma is trapped in the clot. Activators diffuse into the clot and activate plasminogen to plasmin. The lysis of fibrin clots by plasmin results in the formation of fibrinogen-fibrin degradation products. This keeps blood vessels and secretory ducts free.
- Suspected hyper fibrinolysis (increased consumption)
- Thrombolytic therapy monitoring
- Suspected plasminogen deficiency
- Thrombophilia of unknown origin
- Suspected dysplasminogenemia.
Activity measurement using a chromogenic substrate method
Principle: by the action of streptokinase, plasminogen present in the plasma sample is completely transformed into plasminogen activator (streptokinase-plasmin complex). This complex subsequently hydrolyzes a chromogenic substrate. The increase in absorption, measured spectrophotometrically, is proportional to the plasminogen activity .
Immunonephelometry, radial immunodiffusion.
Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of venous blood): 1 mL
* Related to a plasminogen preparation of the U.S.A. National Institute of Health
Fibrinolysis is an efficient protective mechanism to limit the hemostatic process.
Plasminogen deficiency causes a decrease in the ability to react to excess coagulation activity (i.e., the risk of thrombosis is increased). This situation is encountered, for example, in the form of an increased risk of re occlusion after thrombolytic therapy or after surgery. Causes of plasminogen deficiency may include inherited defects, impaired synthesis in the liver, increased consumption (e.g., as seen in DIC), sepsis or thrombolytic therapy ().
Plasminogen activity is a less sensitive indicator of (past) hyper fibrinolysis than the inhibitor α2-antiplasmin since its concentration is subject to more fluctuation. Both parameters, on the basis of consumption, only allow indirect conclusions to be drawn with regard to the actual fibrinolytic activity. For the latter purpose, the determination of the plasmin-α2-antiplasmin complex (PAP) is better suited.
The chromogenic assay is preferable to the immunochemical methods because it is easier and faster to perform. The activity and antigen determinations usually correlate very well except in the case of the rare type II deficiency.
The body is protected against bleeding and thrombosis by the equilibrium between coagulation and fibrinolysis. Plasmin is the key enzyme in fibrinolysis and has a molecular weight of 90 kDa. It is produced by activation of the proenzyme plasminogen initiated by the tissue (tPA) or urinary (uPA; urokinase) plasminogen activator ().
The plasmin thus formed in turn increases the activity of t-PA and the affinity of urokinase for plasminogen by proteolytically cleaving the single-chain plasminogen activators into two-chain structures. Both enzymes then synergistically participate in the activation of plasminogen. t-PA and urokinase are regulated by their inhibitors plasminogen activator inhibitor (PAI) 1 and PAI 2.
The essential role of plasminogen, in its active form plasmin, is to maintain the hemostatic equilibrium by proteolytically dissolving fibrin clots. The fibrin specificity of the plasminogen activators not only supports this process but also localizes it. Under abnormal conditions, a systemic enhancement of plasmin may occur causing the degradation of fibrinogen and thus an increased risk of bleeding .
In the plasma, plasminogen occurs in two modifications which differ with regard to their level of glycosylation and their affinity for fibrin. The binding of plasminogen to fibrin is interfered by the presence of histidine-rich glycoprotein, whereas plasmin in the circulation is inactivated by its inhibitor α2-antiplasmin. Older clots are protected from degradation by plasmin through the incorporation of α2-antiplasmin mediated by F XIIIa. Lipoprotein(a) may competitively inhibit the binding of plasminogen to tPA due to structural similarities (i.e., so-called crinkle regions) ().
Plasmin is a relatively unspecific protease participating in numerous mechanisms at cell level, such as fertilization, cell migration and macrophage activation. These processes are thought to be rather mediated by uPA binding cell receptors. Plasmin especially participates in inflammatory degenerative processes by activating the complement and kallikrein/kinine pathways and metalloproteases playing a key role in tissue degradation, for example in rheumatic joint disease.
Plasmin not bound to fibrin or other extracellular matrices quickly binds to its specific inhibitor α2-antiplasmin forming an irreversible, covalent complex, the plasmin-α2-antiplasmin complex (PAP) which has a half-life of approximately 12 h.
Streptokinase, an activator extracted from streptococci, is employed therapeutically. In contrast to the physiological activators, streptokinase does not activate plasminogen itself but instead forms a 1 : 1 complex with plasminogen. As a result of this configurational alteration of plasminogen, an autocatalytic proteolytic activation into plasmin occurs. Active plasmin may be competitively displaced from its substrate (fibrin) by the administration of ε-aminocaproic acid (6-aminohexanoic acid), thus rendering plasmin more subject to inhibition by α2-antiplasmin. Direct inhibition of plasmin can be achieved by the administration of aprotinin, a protein extracted from the lungs of cattle.
3. Munkvad S. Fibrinolysis in patients with acute ischaemic heart disease. With particular reference to systemic effects of tissue-type plasminogen activator treatment on fibrinolysis, coagulation and complement pathways. Danish Med Bull 1993; 40: 383–408.
5. Sartori MT, Patrassi GM, Theodoridis P, Perin A, Pietrogrande F, Girolami A. Heterozygous type I plasminogen deficiency is associated with an increased risk for thrombosis: a statistical analysis in 20 kindreds. Blood Coag Fibrinol 1994; 5: 889–93.
Michael Kraus, Lothar Thomas
α2-antiplasmin is the physiologically most important inhibitor of the fibrinolytic enzyme plasmin. α2-antiplasmin circulating freely or bound to fibrin clots very rapidly and irreversibly inhibits plasmin by converting it into an inactive complex (plasmin-α2-antiplasmin complex; PAP). Plasmin bound to receptors or substrates such as fibrin, on the other hand, is protected from inhibition by α2-antiplasmin.
- Suspected hyper fibrinolysis (e.g., in conjunction with disseminated intravascular coagulation or surgery involving organs with a high content of plasminogen activators)
- Thrombolytic therapy monitoring (increased risk of bleeding)
- Suspected synthesis defects (liver damage)
- Suspected congenital α2-antiplasmin deficiency.
Activity measurement using a chromogenic substrate method
Principle: a defined excess of plasmin is added to the plasma sample to be tested. The α2-antiplasmin contained in the sample neutralizes equivalent quantities by forming plasmin-α2-antiplasmin complexes. The remaining quantity of plasmin is measured using a chromogenic substrate which is added to the test sample .
Laurell electrophoresis, immunonephelometry.
Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of venous blood): 1 mL
The plasma α2-antiplasmin concentration is usually constant, approximately 1 μmol/L. Hence, a decrease in α2-antiplasmin is a more sensitive biomarker of hyperfibrinolysis than a change in plasminogen activity . A deficiency in α2-antiplasmin causes less regulation of fibrinolytic pathway, resulting in hyperfibrinolysis which may therefore spread systemically and lead to bleeding complications. Causes of α2-antiplasmin deficiency may include: inherited defects, impaired synthesis in the liver, increased consumption (e.g., as seen in DIC), surgery involving organs with an increased plasminogen activator potential (lung, prostate, uterus) or as a result of thrombolytic therapy (). In addition, the α2-antiplasmin concentration may fluctuate due to hormonal influences.
α2-antiplasmin with a molecular weight of 68 kDa is present in plasma in two forms, one bound to plasmin with high affinity and the other one with low affinity . The first form accounts for 70% and the latter for 30% of the total. The half-life of α2-antiplasmin is approximately 2.5 days .
α2-antiplasmin regulates the activity of the key enzyme of fibrinolysis by:
- Rapidly forming an irreversible complex with plasmin in the circulation (plasmin-α2-antiplasmin complex), thus preventing systemic enhancement of active protease
- Competing with fibrin for plasminogen binding, thus decreasing the amount of plasminogen that may be activated on clots
- Forming F XIIIa mediated covalent links with older fibrin clots, thus preventing the clots from degradation by plasmin and enhancing wound healing.
5. Hayashi S, Yamada K. Role of α2-plasmin inhibitor in the appearance of fibrinolytic activity during urokinase administration and an evaluation of the optimal urokinase dosage. Thromb Res 1979; 16: 393–400.
Carola Wagner, Lothar Thomas
The fibrinolytic pathway is activated through the conversion of the proenzyme plasminogen into the active fibrinolytic enzyme plasmin. Plasmin degrades fibrin into soluble fibrin degradation products. The fibrinolytic pathway is inhibited at the PA level by specific plasmin activator inhibitors (PAIs), especially PAI-1 or later by α2-antiplasmin ().
The essential function of the fibrinolytic system is to maintain the circulatory form of the blood. Increased activity of the fibrinolytic pathway may lead to an increased risk of bleeding and decreased activity may cause thrombosis .
- Suspected defects in the fibrinolytic system in the case of thromboembolic disease (e.g., acute myocardial infarction, pulmonary embolism, venous thrombosis or stroke)
- Risk indicator for thromboembolic complications in suspected hereditary or acquired defects (e.g., in patients with hyperinsulinism, diabetes mellitus, cardiovascular disease)
- Inefficacy of thrombolytic therapy in acute myocardial infarction or peripheral arterial occlusion, high-risk pregnancies, neoplasia, sepsis and surgery with a high thrombotic risk.
Determination of the PA and t-PA activity
Active, free PA in the sample, in plasma mainly t-PA, are measured by the conversion of plasminogen into plasmin, followed by the measurement of the resultant plasmin activity through the cleavage of a chromogenic substrate. The t-PA-mediated plasminogen activation requires the addition of an accelerator (e.g., fibrin monomer or fibrin fragments obtained with cyanobromide). In order to avoid any interference by α2-antiplasmin which would immediately bind to the plasmin by forming a complex with it, a reduction in the plasma pH is required .
t-PA antigen measurement
Measurements of the PAI and PAI-1 activity
The plasma PAI-1 activity is measured indirectly by the inhibition of added PA and the chromogenic determination of the residual PA activity via the activation of plasminogen. Both t-PA and u-PA can be used as the target enzyme. Due to the short incubation period of 5–15 min., only the most rapidly active PAI (i.e., PAI-1) is determined. The influence of the α2-antiplasmin is eliminated by acidification of the reaction mixture or by oxidative inactivation .
PAI-1 antigen measurement
Various enzyme immunoassays are available for the immunochemical measurement of the PAI-1 antigen. Depending on the monoclonal antibodies used, the different assays detect the four possible forms of PAI-1 (active, latent, bound to t-PA and bound to u-PA) to a varying extent .
Venous, platelet-poor citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of blood): 1 mL
t-PA activity: to avoid an in vitro reaction of t-PA with its inhibitors, acid citrate solution is necessary for the collection of plasma (final pH ~ 6.0).
Due to the absence of standardization, the reference interval depends on the test system employed. Therefore, every laboratory should establish its own reference interval under local conditions or take into account the reference interval specified in the specific test kit.
The measurement of t-PA and PAI-1, the physiologically most important regulators of the fibrinolytic system in the circulation, serves to estimate the fibrinolytic potential of the blood.
Frequent abnormalities of this system result in a thrombophilic shift of the hemostatic equilibrium due to an increased PAI-1 release or a deficient release of t-PA. Although sporadic cases with an increased risk of bleeding as a result of PAI-1 deficiency or an excessive t-PA release have been described, they are, however, clinically irrelevant for all practical purposes because of their extremely low prevalence .
Plasminogen activator inhibitors
Ten PAI-1 gene polymorphisms have been described to date, but only a few of them seem to influence the inhibitor’s level in plasma. A functional role in PAI-1 synthesis and expression has been attributed to a single guanosine deletion/insertion polymorphism (4G or 5G) located at the promoter region of the PAI-1 gene at 675 bp upstream from the transcription starting site. Both polymorphic alleles may bind a transcription activator, whereas the 5G allele also binds a repressor protein to an overlapping binding site, which consequently interferes with activator binding by steric hindrance. The relationship between the 4G/5G polymorphism and PAI-1 levels, which are higher in 4G allele carriers exists in patients with deep vein thrombosis, cardiovascular and metabolic diseases, while a weaker association has been described in healthy populations. PAI-1 is assumed to play a role in thrombotic vascular disease .
The PAI concentration in the 4G/4G genotype is 25% higher than in the 5G/5G genotype and associated with deep vein thrombosis. It is assumed that the regulation of the 4G allele by the plasma triglyceride concentration influences the involvement of PAI-1 in cardiovascular disease .
Refer to tables:
Due to the pronounced circadian rhythms of PAI-1 release with the highest levels measured in the morning and the lowest in the afternoon, blood for the determination of both PAI-1 and t-PA should be drawn between 8:00 and 10:00 a.m. Prior to blood sampling, the patients should rest for at least 20 min. since increased physical activity can lead to an increased release of t-PA.
In order to prevent the release of latent PAI-1 from platelets during storage, immediate cooling of the sample to 4 °C is necessary after blood collection as well as the preparation of platelet-poor plasma (by centrifugation at 2000 × g for 15 min.).
The PAI-1 activity can be expressed either as a t-PA- or u-PA-inhibiting unit (1 u-PA-inhibiting unit ~ 7.8 t-PA-inhibiting units). The test system used should always be specified because of the common significant variations in the specificity and sensitivity of the immunochemical tests which are employed for the determination of the different forms of t-PA and PAI-1 in the circulation.
The main component of the fibrinolytic system is plasminogen, an inactive proenzyme which, following its conversion into the active enzyme, is responsible for the degradation of fibrin as well as of various other extracellular matrix proteins.
Two physiological PA are known: t-PA, which is found primarily in the circulation where it induces the degradation of fibrin clots, and u-PA, which, through a cellular tissue receptor, leads to the degradation of matrix proteins by means of pericellular, plasmin-mediated proteolysis as well as to the activation of latent proteases and growth factors.
The fibrinolytic activity is regulated:
- At the level of plasmin by the irreversible formation of a complex with α2-antiplasmin
- Via the activation of plasmin as a result of the formation of complexes consisting of t-PA and u-PA, on the one hand, and PAI, on the other hand; PAI-1 plays the most important role in this context ().
t-PA is continuously released from the vascular endothelium; as a result of stimulation due to (e.g., venous occlusion, strenuous physical activity or vasoactive drugs such as catecholamines or bradykinin). A rapid rise in the release of t-PA occurs within a few minutes. The half-life of t-PA in the circulation is only 5 min. due to its rapid hepatic clearance and its efficient inactivation by the formation of a complex with PAI-1.
In contrast to other hemostatic enzymes, t-PA is not released as an inactive proenzyme but instead directly as an active protease. The affinity of t-PA to plasminogen freely circulating in the plasma is too low, however, for its activation and is not sufficient unless fibrin is present /, /.
PAI-1, a serine protease inhibitor with a molecular weight of 52 kDa, is released from vascular endothelial cells into the circulation in its active form and, due to a conformational change, transforms into an active form with a half-life of 30 min. PAI-1 which is contained in the α-granules of platelets and which represents about 90% of PAI-1 circulating in the blood, on the contrary, is present almost entirely in the inactive form. The binding of PAI-1 to protein S or vitronectin stabilizes the active phase of PAI-1 in plasma; however, this does not make it resistant to enzymatic degradation by thrombin or activated protein C.
2. Verheijen JH. Tissue-type plasminogen activator activity assay. In: Jespersen J, Bertina RM, Haverkate F (eds). ECAT assay procedures. A manual of laboratory techniques. Lancaster UK: Kluwer Academic Publishers, 1992: 139–46.
3. Juhan-Vague I, Alessi MC. Tissue type plasminogen activator antigen (t-PA Ag). In: Jespersen J, Bertina RM, Haverkate F (eds). ECAT assay procedures. A manual of laboratory techniques. Lancaster UK: Kluwer Academic Publishers, 1992: 131–7.
4. Kruithof EKO. Plasminogen activator inhibitor activity assay. In: Jespersen J, Bertina RM, Haverkate F (eds). ECAT assay procedures. A manual of laboratory techniques. Lancaster UK: Kluwer Academic Publishers, 1992: 147–50.
7. Sartori MT, Danesin C, Saggiorato G, Tormene D, Simioni P, Spiezia L, et al. The PAI-1 gene 4G/5G polymorphism and deep vein thrombosis in patients with inherited thrombophilia. Clin Appl Thromb Hemost 2003; 9: 299–307.
9. Dossenbach Glaninger A, van Trostenburg M, Dossenbach M, Oberkanis C, Moritz A, Krugluker W, Huber J, Hopmeier P. Plasminogen activator inhibitor I 4G/5G polymorphism and coagulation factor XIII (4G/5G) Val 34/Leu polymorphism: impaired fibrinolysis and early pregnancy loss. Clin Chem 2003; 49: 1081–6.
10. Sobel BE, Woodcock-Mitchell J, Schneider DJ, et al. Increased plasminogen activator inhibitor type 1 in coronary artery atherectomy specimens from type 2 diabetics compared with non diabetic patients: a potential factor predisposing to thrombosis and its persistance. Circulation 1998; 97: 2213–21.
11. Festa A, d’Augustino R, Mykkanen L, et al. Relative contribution of insulin and its precursors to fibrinogen and PAI-1 in a large population with different states of glucose tolerance: The Insulin Resistance Atherosclerotic Study (IRAS). Arterioscer Thromb Vasc Biol 1999; 19: 562–8.
Michael Kraus, Lothar Thomas
Elevated fibrin monomer concentration is a sign of increased thrombin activity (hyper coagulability) and, thus, indicates an acute danger of clot formation (pre thrombotic state).
Detection of consumptive coagulopathy associated with:
- Disseminated intravascular coagulation
- SIRS, sepsis, severe infectious disease, complications of pregnancy
- States of shock (e.g., traumatic, cardiogenic or septic shock)
- Tissue necrosis (e.g., due to poly trauma, burns, acute pancreatitis, malignant tumor, immune hemolysis)
- Transplant rejection.
Recognition of a pre thrombotic state (i.e., the acute risk of developing deep vein thrombosis or pulmonary embolism):
- Postoperatively (with or without heparin prophylaxis)
- In patients with inherited inhibitor defects such as factor V Leiden and antithrombin deficiency
- Monitoring of the thrombotic risk in patients under anticoagulant therapy (e.g., following stent implantation).
Principle: agglutination tests precipitate the soluble fibrin monomer by the addition of reagents, immobilized fibrin monomers or hydrophobic surfaces. The agglutination reaction can be measured macroscopically or turbidimetrically and depends on the concentration of the fibrin monomer present in the sample. The oldest method is based on the precipitation of soluble fibrin by ethanol . In the hemagglutination assay, erythrocytes loaded with fibrin monomer are used as the agglutination partner .
Furthermore, fibrin monomer can be precipitated using uncoated latex particles in the presence of a dye (blue dextran) . Direct precipitation is feasible using protamine sulfate , ristocetin or netropsin .
Principle: the functional tests are based on the characteristic ability of soluble fibrin monomer complexes to stimulate the tissue plasminogen activator (t-PA). t-PA activates plasminogen by converting it into plasmin which in turn leads to the transformation of a chromogenic substrate which is photometrically detected .
Principle: immunochemical assays involve the use of monoclonal antibodies which recognize specific fibrin epitopes. The epitopes are not created until fibrinogen is converted into fibrin (fibrinopeptide cleavage from fibrinogen; des-A-neoepitope) or become accessible only after configurational alterations (t-PA binding sites) . In the event of des-A-neoepitope recognition, the samples must be pretreated using a chaotropic reagent (NaSCN) in order to cleave the fibrin complexes which attach via this epitope .
Platelet-poor citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of venous blood): 1 mL
The specifications of the test kit should be taken into account for each ay used.
An elevated concentration of fibrin monomer (FM) suggests the presence of increased thrombin activity and, thus, is a direct indicator of ongoing increased intravascular coagulation activity (hyper coagulability) . If this process was triggered locally and there is inadequate fibrinolytic activity, this may result in the formation of a localized thrombosis; if this process, on the other hand, is generalized, i.e., disseminated intravascular coagulation is present, consumptive coagulopathy may ensue .
Trauma, sepsis and shock are the major causes of hyper coagulability. In a study involving intensive care patients, the extent of multiple organ failure and the mortality rate were directly correlated with the FM concentration. The course of pregnancy is also characterized by the increasing development of a hyper coagulable state.
Soluble fibrin is in a labile state. At a certain ratio of fibrin to fibrinogen (1–2%), the fibrin molecules begin to spontaneously aggregate. Therefore, the detection of FM is an early indicator of an acute thrombotic risk (e.g., of deep vein thrombosis after surgery). The diagnostic sensitivity of FM for deep vein thrombosis of the lower extremities is 91% with a negative predictive value of 94% .
In some assays, the use of frozen samples is not recommended. The allowed time period between the collection and the processing of the blood sample is also limited in some assays. The instructions of each test kit should be taken into consideration.
Method of determination
In general, the methods do not correlate with each other even if similar methodologies are employed. The specifications in each test kit concerning the reference interval and interpretation must therefore be taken into account.
The agglutination assays are relatively insensitive to fibrinogen. Very high concentrations of fibrin or fibrin(ogen) degradation products may inhibit aggregation by intercalating in-between the fibrin molecules. Overall, these assays are relatively imprecise and, in the case of manual methods, depend heavily on the experience of the person performing the assay.
The functional as well as the immunochemical assays provide reproducible and quantitative results. To a certain extent, the results usually correlate with fibrinogen. This is probably due to the fact that more fibrinogen can keep more fibrin soluble and that fibrinogen is elevated because of an acute phase response which is also associated with hyper coagulability (i.e., increased fibrin formation).
The fibrinogen molecule is a dimer of three different pairs of nonidentical polypeptide chains which are labeled Aα, Bβ and γ. Thrombin initially cleaves fibrinopeptide A and then fibrinopeptide B from the Aα and Bβ chains, resulting in exposure of new N-termini and causing conformational changes and exposure of polymerization sites. This generates fibrin monomer a molecule with strong tendency to polymerize. Because of its tendency to polymerize, fibrin monomer is present physiologically in only very low concentration because it rapidly self-associates to form an insoluble fibrin deposit .
Refer also to
The physiological half-life of fibrin monomer is approximately 10 h.
When small amounts of thrombin are formed, producing minimal fibrin polymer production, fibrin monomers may complex with intact fibrinogen molecules. If some intravascular fibrin is formed and fibrinolysis occurs, complexes may also be formed between the larger fibrin fragments, fibrin monomers and fibrinogen molecules. These soluble complexes are called soluble fibrin monomer complexes and are protective in preventing the further polymerization of fibrin monomers .
9. Lill H, Spannagl M, Trauner A, Schramm W, Schuller D, Ofenloch-Haehnle B, Draeger B, Naser W, Dessauer A. A new immunoassay for soluble fibrin enables a more sensitive detection of the activation state of blood coagulation in vivo. Blood Coag Fibrinol 1993; 4: 97–102.
10. Nieuwenhuizen A, Hoegee-De Nobel E, Laterveer R. A rapid monoclonal antibody-based enzyme immunoassay (EIA) for the quantitative determination of soluble fibrin in plasma. Thromb Haemost 1992; 68: 273–7.
14. Ginsberg JS, Siragusa S, Douketis J, Johnston M, Moffat K, Stevens P, Brill-Edwards P, Panju A, Patel A. Evaluation of a soluble fibrin assay in patients with suspected deep vein thrombosis. Thromb Haemost 1995; 74: 833–6.
15. Korte W, Gabi K, Rohner M, Gähler A, Szadkowski C, Snider DW, et al. Preoperative fibrin monomer measurement allows risk stratification for high intraoperative blood loss in elective surgery. Thromb Haemost 2005; 94: 211–5.
D-dimer is a fibrin degradation product and elevated in clinical situations with hyper coagulability such as venous thromboembolism, disseminated intravascular coagulation and a further spectrum of diseases. D-dimer is an important diagnostic marker because of its high negative predictive value .
- Exclusion of venous thromboembolism (VTE), particularly deep vein thrombosis and pulmonary embolism
- Prediction of recurrent VTE and risk stratification of patients for VTE recurrence
- Diagnosis and monitoring of coagulation activation in suspected disseminated intravascular coagulation
- Suspected preeclampsia.
Principle: the D-dimer assays measure an epitope on degradation products of factor XIIIa crosslinked fibrin by particle-enhanced immunonephelometric and immunoturbidimetric methods . The assays use monoclonal antibodies directed against an epitope that is present in the factor XIIIa crosslinked fragment D domain of fibrin.
Citrated plasma (blood collection: 1 part of sodium citrate solution 0.11 mol/L mixed with 9 parts of venous blood): 1 mL
50–500 Fibrinogen Equivalent Units/Liter (FEU/L = ug/L)
Venous thromboembolism (VTE) consists of deep vein thrombosis (DVT) and pulmonary embolism. The diagnostic management of DVT based on signs and symptoms is non-specific. The diagnosis of DVT can be safely excluded based on the combination of a low to or intermediate clinical probability combined with a negative D-dimer blood test, in which case imaging studies can be avoided . The D-dimer concentration indicates the extent to which fibrin formation is increased. An age-adjusted D-dimer cutoff (patient’s age years × 10 μg/L) is recommended to exclude deep vein thrombosis in patients above 50 years of age .
D-dimer tests are performed as markers of coagulation activation in patients with suspected VTE and to serve as a diagnostic tool in support of clinical findings. The fact that only a small portion of circulating fibrinogen needs to be converted to crosslinked fibrin to generate a detectable D-dimer signal confers the diagnostic sensitivity required . Clinical signs and symptoms (Wells score ) suggesting that patients are clinically unlikely to have VTE and normal results of the D-dimer test indicate that clinical significant intravascular coagulation is not increased.
- Wells score ≤ 1 and D-dimer antigen ≤ 500 μg/L: no DVT present
- Wells score ≤ 1, D-dimer > 500 μg/L and positive compression ultrasound sonography: deep vein thrombosis and therapy
- Wells score > 1, positive compression ultrasound sonography: deep vein thrombosis and therapy
- Wells score > 1, negative compression ultrasound sonography, D-dimer > 500 μg/L: further serial compression ultrasound sonography; positive results of these examinations indicate the presence of a treatable deep vein thrombosis
- Wells score > 1, negative compression ultrasound sonography, D-dimer < 500 μg/L: no deep vein thrombosis.
In many cases, the determination of D-dimer in hospitalized patients is inadequate for VTE diagnosis due to low diagnostic specificity.
The detection of D-dimer in plasma is an indicator of increased presence of fibrin in the blood, degraded to fibrin fragments in the course of secondary hyper fibrinolysis. Elevated D-dimer does not allow any conclusion as to the site and cause of increased fibrin formation.
- Systemic coagulation activation
- Release of thrombin from clots following intravascular fibrin formation
- Release of fibrin complexes from clots
- Release of fibrin degradation products from clots or circulating fibrin complexes
- Release of fibrin formed extravascularly in wounds and hematoma.
A distinction is made between conditions with acutely and permanently elevated D-dimer antigen.
Elevated D-dimer levels are detected in almost all patients with deep venous thrombosis (DVT) of the lower extremities. The diagnostic sensitivity is markedly higher for thrombosis of the thigh than for thrombosis of the lower leg. The use of abnormal D-dimer findings in the diagnosis of lower extremity DVT is controversial due to the fact that diagnostic specificity is acceptable in outpatients, but not in inpatients. For instance, in a study , only 22% of hospitalized patients classified as free of DVT had a D-dimer level below the cutoff value. This is because hospitalized patients often present abnormal conditions associated with increased fibrin formation without a thrombotic event.
Such processes and conditions are:
- Sepsis, pneumonia, erysipelas
- Metastatic malignant tumors
- Liver cirrhosis, particularly in conjunction with shunts
- Surgery or trauma within the past 4 weeks
- Anticoagulant therapy for more than 24 h
- Thrombolytic therapy within the past 7 days.
- Vascular aneurysm, particularly aortic aneurysm
- Portocaval shunt
- Malignant disease (e.g., adenocarcinoma, myeloproliferative disease)
- Vascular malformation such as hemangioma and Kasabach-Merritt syndrome.
Method of determination
The commercially available tests do not yield identical results because the D-dimer antigen is present on fibrin degradation products of different size and the used antibodies have a different epitope specificity. Thus, a generally valid reference interval for excluding VTE events cannot be specified because of:
- The different specificity of the antibodies used
- The composition of the antigen used for calibration
- The differences in the method of determination. For instance, an enzyme immunoassay requires only one epitope for binding crosslinked degradation products, whereas in the agglutination test this epitope needs to be present at least twice
The D-dimer is reported as in fibrinogen-equivalent units (FEU) and expressed in μg/L. Because of the different assays and standardization, a test with a cutoff value of 150 μg FEU/L is not necessarily more sensitive than a test with a cutoff value of 500 μg FEU/L.
The end of the coagulation cascade is characterized by the thrombin-mediated cleavage of fibrinopeptide A and B from fibrinogen. This results in the formation of fibrin monomers. These monomers associate to form protofibrils or associate with fibrin or fibrinogen. In addition fibrin monomer rapidly self-associates to form insoluble fibrin deposits on vessel walls. Thrombin also activates F XIII as fibrin is formed, and F XIIIa rapidly cross-links adjacent γ-chains in fibrin polymers. They are held together by non-covalent forces between the intermolecular D-domain and DE-domain. The protofibrils are stabilized by the action of F XIIIa. The formation of a crosslinked fibrin clot occurs.
Plasmic degradation of fibrinogen and non-cross-linked fibrin results in formation of fragments X and Y and the terminal degradation products D an E. However, the formation of γ-chain cross-links in fibrin, that are resistant to degradation leads to formation of unique degradation products that differ from those of fibrinogen. Consequently, soluble products are produced with γ-chain cross-link intact, and this results in formation of a large number of polymeric forms of defined structure. The smallest is D-dimer, which consists of the fragment D domains of two adjacent monomers joined by the γ-chain cross-link . Refer to .
3. Douma RA, Tan M, Schutgens REG, Bates SM, Perrier A, Legnani C, et al. Using an age-dependent D-dimer cut-off value increases the number of older patients in whom deep vein thrombosis can be safely excluded. Haematologica 2012; 97: 1507–13.
8. Kristoffersen AH, Petersen PH, Sandberg S. A model for calculating the within-subject biological variation and likelihood ratios for analytes with a time-dependent change in concentrations; exemplified with the use of D-dimer in suspected venous thromboembolism in healthy pregnant women. Ann Clin Biochem 2012; 49: 561–9.
9. Geersing GJ, Toll DB, Janssen KJM, Oudega R, Blikman MJC, Wijland R, et al. Diagnostic accuracy and user-friendliness of 5 point-of-care D-dimer tests for the exclusion of deep vein thrombosis. Clin Chem 2010: 56: 1758–66.
10. Kemkes-Matthes B, Fischer R, Peetz D. Influence of 8 and 24-h storage of whole blood at ambient temperature on prothrombin time, fibrinogen, thrombin time, antithrombin and D-dimer. Blood Coagulation and Fibrinolysis 2011; 22: 215–20.
13. Douma RA, Kamphuisen PW, Huisman MV, Buller HR. False normal results on multidetector-row spiral computed tomography in patients with high clinical probability of pulmonary embolism. J Thromb Haemost 2008; 6: 1978–9.
17. Taylor jr FB, Toh C, Hoots WK, Wada H, Levi M. Towards definition, clinical and laboratory criteria, and scoring system for disseminated intravascular coagulation. Thromb Haemos 2001; 86: 1327–30.
18. McDermott MM, Greenland P, Green D, Guralnik JM, Criqui MH, Lui K, Chan C, et al. D-dimer, inflammatory markers, and lower extremity functioning in patients with and without peripheral arterial disease. Circulation 2003; 107: 3191–8.
22. Mahe I, Drouet L, Chassany O, Mazoyer E, Simoneau G, Knellwolf AL, et al. D-dimer: a characteristic of the coagulation state of each patient with chronic atrial fibrillation. Thromb Res 2002; 107: 1–6.
23. Shorr AF, Trotta RF, Alkins SA, Hanzel GS, Diehl LF. D-Dimer assay predicts mortality in critically ill patients without disseminated intravascular coagulation or venous thromboembolic disease. Intensive Care Med 1999; 25: 207–10.
The pathogenesis of thrombosis is described by Virchow’s triad. According to this concept, there are mainly three general factors that contribute to thrombosis:
- Endothelial alterations (vascular injury)
- Hemodynamic changes (stasis or turbulence of blood stream)
- Increase in blood viscosity (polyglobulinemia and exsiccosis).
Hyper coagulability of the blood can be essentially promoted by:
- Deficiency in inhibitors of coagulation
- Excess of coagulation activators
- Increased platelet aggregation
- Dysfunctional fibrinolysis.
Anti thrombotic therapy plays a key role in the primary and secondary prophylaxis of cardiovascular and cerebrovascular disease and venous thromboembolism.
Arterial blood clots form due to shear-induced platelet aggregration in the presence of small amounts of fibrin, as a rule based on atherosclerosis as the main underlying condition. The rupture of an atheromatous plaque leads to the exposure of a strongly thrombogenic lipid core. This is likely to result in subsequent platelet adhesion, activation and aggregation. Hence, anti-platelet therapy is the main focus of anti thrombotic therapy in arterial thrombosis.
However, anticoagulation under inhibition of the plasmatic coagulation is also employed for the prophylaxis of cardiac arterial clots and cardioembolic events in valvular or structural cardiac disease. Fibrinolytic substances can be administered to rapidly restore the blood flow in patients with acute myocardial infarction in cases where percutaneous coronary intervention is not immediately possible. The same approach can also be followed in acute ischemic stroke or acute pulmonary embolism in conjunction with hemodynamic instability.
Venous clots form under low shear stress. They are fibrin-rich and contain captured red blood cells and relatively small amounts of platelets. Although the local activation of platelets leads to the concentration of the entire coagulation process, for example, on the site of a vessel wall injury, the plasmatic coagulation system plays a key role in the formation of venous thromboembolism (VTE). Considering the predominance of fibrin clot formation, anticoagulants are the substances of choice in the prophylaxis and therapy of VTE, whereas anti thrombocytic strategies play almost no role. Drug-induced fibrinolysis is employed in the venous system in exceptional cases, only.
VTE is one of the leading causes of morbidity and mortality in the Western world. More than half of all hospitalized patients are at risk of VTE complications . Therefore, anticoagulant thromboprophylaxis is recommended for almost every inpatient to prevent VTE /, /. Prophylactic anticoagulation should also be considered for outpatients in the presence of thromboembolic risk factors .
Indications for anticoagulation at therapeutic doses include deep vein thrombosis as well as absolute arrhythmia in atrial fibrillation, acute myocardial infarction, extra corporeal circulation, disseminated intravascular coagulation, arterial thrombosis and embolism, severe cardiac failure, condition following aortocoronary venous bypass and implantation of heart valve or arterial vascular prostheses . The decision in favor of full anticoagulation therapy should always consider potential contra indications and reflect the balancing of the risk of bleeding against the benefit to the patient. For instance, it is possible to assess the risk of a thromboembolic event in atrial fibrillation using the CHADS score and the risk of bleeding using the HAS-BLED score.
The CHADS2 score is used to initially assess the risk of a thromboembolic event in atrial fibrillation.
In the absence of contra indications, a CHADS2 ≥ 2 (high rate of thromboembolism (e.g., CHADS2 score 2 = 4%/year; CHADS2 score 4 = 8.5%/year; CHADS2 score 8 = 18.2%/year) warrants oral anticoagulation with vitamin K antagonists at therapeutic doses with a target INR of 2.5 (2.0–3.0).
However, patients classified in the categories CHADS2 score = 1 (moderate risk = 2.8%/year) and CHADS2 score = 0 (low risk = 1.9%/year) can still be clinically at risk of developing thromboembolism.
A CHA2DS2-VASc ≥ 2 (e.g., CHA2DS2-VASc score 2 = 2.2%/year; CHA2DS2-VASc score 4 = 4%/year; CHA2DS2-VASc score 7 = 9.6%/year; CHA2DS2-VASc score 9 = 15.2%/year) warrants oral anticoagulation with vitamin K antagonists at therapeutic doses with a target INR of 2.5 (2.0–3.0).
A CHA2DS2-VASc score = 1 (risk of stroke = 1.3%/year requires individual evaluation of the preference of vitamin K antagonist treatment and the concomitant use of acetylsalicylic acid (75–325 mg per day). At a score = 1, the administration of dabigatran (2 × 110 mg/day) should be considered due to the decreased rate of intracranial hemorrhage and major bleeding and similar efficiency in the prevention of embolism compared to warfarin .
A CHA2DS2-VASc score = 0 requires the individual balancing of the preference of non-medication management against the administration of ASA (75–325 mg per day), which is associated with a risk of bleeding.
HAS-BLED ≥ 3 indicates a risk of major bleeding. The indication of anticoagulation with vitamin K antagonists and the administration of acetylsalicylic acid must be strictly verified. Close-meshed monitoring of the patient is required.
Provided the indication is confirmed, patients with HAS-BLED score = 2 (moderate risk of bleeding) or HAS-BLED score = 0 or 1 (no risk of bleeding) clearly benefit from oral anticoagulation. The application of dabigatran etexilate should be considered as an alternative for the dose-adjusted administration of vitamin K antagonists:
- High-dosed (2 × 150 mg of dabigatran per day) at a HAS-BLED score of 0–2 due to the increased efficacy in the prevention of embolism in conjunction with decreased rates of intracranial hemorrhage and similar incidence of major bleeding compared to warfarin
- Low-dosed (2 × 110 mg of dabigatran per day) at a HAS-BLED score ≥ 3 due to similar efficacy in the prevention of embolism in conjunction with a comparable rate of intracranial hemorrhage and major bleeding compared to warfarin .
- High (above 7% per year)
- Moderate (4–7% per year)
- Low (below 4% per year).
In order to assist in the decision-making process as to the intensity and duration of anticoagulation, a concept of individualized anticoagulation monitored through D-dimer antigen determination has been discussed aiming at increased reliability in the prevention of bleeding complications and recurring thromboembolic events . According to this concept, increased D-dimer antigen can be interpreted as a possible risk indicator for VTE if other causes (e.g., hematoma, effusion, wound healing, pregnancy or liver cirrhosis, have been excluded or are unlikely).
Continued low D-dimer concentration following the reduction or termination of anticoagulation is thought to indicate a low risk of recurrence of a thromboembolic event.
However, an increase in D-dimer level within a few days detected presumably indicates a relatively high risk of recurrent thromboembolism and, thus, seems to warrant continued anticoagulation.
The rate of recurrence within the first months following a thromboembolic event is higher than after a longer period. Different indications for therapeutic anticoagulation lead to correspondingly different assessments of the risk of thromboembolic events. Therefore, an indication- and risk-oriented approach is recommended to determine the duration of anticoagulation . In addition, it is advisable to regularly monitor the individual bleeding risk under therapeutic anticoagulation.
The decision about individual risk-adapted heparin bridging against the background of an indicated perioperative interruption of anticoagulation or in manifest bleeding can only be made based on sound clinical evidence . The risk of bleeding during surgical intervention is increased by patient-related factors (hemostatic disorders, also including the intake of platelet function inhibitors; previous perioperative bleeding) as well as various surgery-related factors, such as larger-scale trauma, urgency of surgical intervention, major surgery, limited practical knowledge of the surgeon, few possibilities of bleeding control. The interruption of oral anticoagulation is imperative in patients at high risk for perioperative bleeding. Perioperative bridging should be considered at high and moderate risk of thromboembolism ().
Anticoagulation monitoring is an important aspect in the prevention of therapeutic complications for numerous anticoagulants. Both bleeding and recurrent thromboembolism can only be prevented under optimal anticoagulant adjustment. By contrast, prophylactic anticoagulation usually does not require any monitoring. The efficacy of new oral anticoagulants needs not be monitored by coagulation tests in clinical routine even under therapeutic use if the limitations of indication (e.g., in patients with renal insufficiency) are taken into account. Nevertheless, when using these substances, it is important to know the possibilities and limitations of monitoring in suspected subtherapeutic or supra therapeutic levels for assessment of the clinical situation.
Depending on the site of action, anti thrombotic drugs are classified into:
- Platelet function inhibitors
- Fibrinolytic agents.
In regions affected by atherosclerotic alterations, the adhesion, activation and aggregation of platelets may lead to vascular occlusion. In order to prevent this development, substances from the group of platelet function inhibitors induce platelet dysfunction by various mechanisms. An overview of the function and clinical significance of the individual platelet function inhibitors as well as information on diagnostic monitoring is provided in .
Sites of action and function of platelet function inhibitors
Collagen and von Willebrand factor (VWF) are presented at the site of the endothelial defect. Glycoproteins (GP) Ib/V/IX (a receptor for VWF; CD42b) and GP Ia/IIa (α2-/β1-integrin; a receptor for collagen) of the platelet membrane induce the zipper-like adhesion of platelets to the subendothelial matrix, thus triggering platelet activation ().
Membrane flip-flop trans locates pro coagulant phospholipids of the platelets from inside to the outside, numerous mediators and Ca2+ are released and GP IIb/IIIa is to an increasing extent expressed on the membrane surface (inside-out signaling). The negatively charged phospholipids bind Ca2+. Coagulation factors can now also bind to the platelet surface (see ). Thus, a small number of thrombin molecules can locally trigger plasma coagulation on the surface of activated platelets. The released mediators recruit more platelets and trigger a positive feedback mechanism because released adenosine diphosphate binds to the receptor P2Y12 and/or thromboxane A2 (TXA2) binds to the thromboxane receptor (TXR), thus enhancing platelet activation.
The increased expression of GP IIb/IIIa (alpha2b/beta3 integrin; CD41a) leads to platelet aggregation because fibrinogen and VWF act as bridging molecules linking two GP IIb/IIIa on neighboring platelets.
During platelet activation, cyclooxygenase (COX)-1 is responsible for the synthesis of prostaglandin from arachidonic acid. Subsequently, TXA2 is formed by thromboxane synthase. Acetylsalicylic acid irreversibly inactivates COX-1, thereby indirectly inhibiting platelet aggregation. The reduction of activation of the intrinsic pathway by ADP receptor antagonists also has an indirect inhibiting effect on platelet aggregation. GP IIb/IIIa receptor antagonists directly inactivate aggregation by efficiently inhibiting cross-bridging between adjacent platelets.
Therapy as well as primary and secondary prevention of cardiovascular events such as myocardial infarction, stroke and peripheral arterial occlusive disease.
Monotherapy: acetylsalicylic acid (ASA) elevates the risk of a hemorrhagic complication during surgery by 50% but does not increase operative mortality. ASA that is taken for primary prevention can be interrupted for surgery. The European Society of Cardiology (ESC) recommends in general that ASA for secondary prevention should not be discontinued perioperatively. Nonetheless, for intraocular, intraspinal and intracranial procedures, even small hemorrhages can cause significant morbidity, so that temporarily discontinuing ASA would seem to be necessary.
Dual therapy: the simultaneous intake of ASA and P2Y12 inhibitors (e.g., ticlopidine, clopidogrel) usually by some patients who have received a coronary stent can cause major problems in the perioperative period. Patients with a coronary stent must take ASA for life and a P2Y12 inhibitor either for at least six weeks (bare metal stents) or for at least twelve months (drug eluting stents). Early termination of dual therapy is associated with a 90-fold increased risk of stent thrombosis. Elective surgery should be postponed until ASA mono therapy has been established. If an operation cannot be postponed and must be performed during the critical period, it is recommended that dual inhibition of platelet aggregation be continued perioperatively. If the dual inhibition of platelet aggregation has been interrupted for surgery, P2Y12 inhibitors must be restarted as soon as possible after the operation.
Monitoring of platelet function in patients undergoing therapy with platelet aggregation inhibitors is increasingly recommended. It is crucial to avoid activation of the platelets, for example due to long transport times.
Anticoagulants are classified into directly and indirectly acting ones. Phenprocoumon and warfarin indirectly inhibit coagulation by decreasing the γ-carboxylation of the prothrombin complex (factors II, VII, IX and X). Other anticoagulants act by inhibiting F Xa and/or thrombin (F IIa).
Sites of anticoagulant action
The effect of the individual anticoagulants is as follows:
- Antithrombin (AT) inhibits coagulation by the proteolytic degradation of thrombin and inactivation of serine proteases. Binding to AT is only possible in the presence of a specific pentasaccharide sequence of heparin. The binding leads to an altered AT conformation, whereby inactivation of substrates by AT is 100–1,000-fold faster. Heparin is immediately released again and free to activate more AT molecules. An intermolecular cross linking mechanism is needed for the inactivation of thrombin by AT; this inactivation process is indirectly enhanced by heparin concurrently binding to the secondary binding site of thrombin (exosite 2). This tertiary complex needs a heparin chain length of at least 17 monosaccharide units (molecular weight > 5400 kDa). Consequently, unfractionated heparins usually have similar anti-F IIa and anti-F Xa activities. The anti-F Xa activity of low molecular weight heparins is higher than their anti-F IIa activity. By its highly specific binding to AT, the synthetic pentasaccharide fondaparinux indirectly and selectively enhances the AT-mediated inhibition of F Xa approximately 300-fold. Fondaparinux has no anti-F IIa activity.
- Direct F Xa inhibitors bind directly to F Xa, thus preventing thrombin formation during the clotting intensification phase
- Direct thrombin inhibitors (DTI) bind to the active center of thrombin (F IIa; fibrin-bound or free) and indirectly block their interaction with substrates. F IIa has the active binding site as well as two secondary bindings sites, exosite 1 (fibrinogen) and exosite 2 (heparin). The naturally occurring, bifunctional DTI hirudin irreversibly binds to exosite 1 and the active binding site. By contrast, the bifunctional binding of the synthetic DTI bivalirudin and the monofunctional binding of the synthetic DTIs argatroban and dabigatran to the active binding site are reversible.
The vitamin K antagonists phenprocoumon and warfarin inhibit the hepatic synthesis of vitamin K dependent coagulation factors.
Thromboprophylaxsis following deep vein thrombosis, pulmonary embolism, atrial fibrillation and cardiac valve replacement.
Unfractionated heparin (UFH) and fractionated (low molecular weight) heparin (FH) are distinguished. UFH inhibits the activity of thrombin. The anti-F Xa activity of FHs is higher than their anti-F IIa activity. The effect of UFH and FH is mediated by AT ().
Unfractionated heparin: thromboprophylaxis in patients with mechanical heart valves, during cardiopulmonary bypass and in intensive care.
Fractionated heparin: thromboprophylaxis.
The dose must be adapted at eGFR below 30 [mL × min–1 × (1.73 m2)–1] which is often seen in older patients. In these cases, the effect of heparin must be monitored by determining the anti-F Xa activity ().
Because of the long half-life of fondaparinux (approximately 17 hours), a single subcutaneous injection per day suffices. In patients with an eGFR below 50 [mL × min–1 × (1.73 m2)–1] the dose must be lowered to 1.5 mg daily subcutaneously in prophylactic use. Routine monitoring of the anti-F Xa activity is only required in decreased GFR. Fondaparinux has almost no effect in the coagulation screening tests (). If the anti-F Xa activity needs to be determined, the test must be calibrated with fondaparinux.
The NOACs , also called non-vitamin-K oral anticoagulants are anticoagulants acting on specific factors within the coagulation cascade.
The NOACs include:
- The thrombin inhibitor dabigatran given as prodrug dabigatran etexilate (Pradaxa), which is hydrolyzed in the body to become a direct thrombin inhibitor
- Direct acting oral anticoagulants (DOACs). These drugs include the F Xa inhibitors apixiban (Eliquis), edoxaban (Lixiana), and rivaroxaban (Xarelto).
Clinical trials have shown NOACs therapeutic equivalent, or even superior, to vitamin K antagonists (VKA), with lower rates of bleeding.
Approximately 12% of hospitalization is caused by the interactions of phenprocoumon or warfarin with other drugs. Renal and hepatic (dys)function, hemorrhagic risk, age, and interactions with other drugs must be considered when NOACs are prescribed . The accompanying hemorrhagic risk of NOACs is low (slight bleeding 0.8% and heavy bleeding 1.4–2.1%).
- Stroke prevention in non-valvular atrial fibrillation
- Prevention of venous thromboembolism (total knee or total hip replacement)
- Treatment of deep vein thrombosis (DVT) and pulmonary embolism (PE)
- Prevention of recurrent DVT and PE.
Apixiban (Eliquis), edoxaban (Lixiana), and rivaroxaban (Xarelto)
- Prophylaxis of venous thromboembolism (total knee or total hip replacement)
- Prophylaxis of venous thromboembolism following total knee or total hip replacement
- Treatment of deep vein thrombosis (DVT) and pulmonary embolism (PE)
- Prophylaxis of deep vein thrombosis (DVT) and pulmonary embolism (PE)
- Prophylaxis of stroke and systemic embolism in non-valvular atrial fibrillation
- Prophylaxis of atherotrombotic events in acute coronary syndrome (with aspirin alone or aspirin and clopidrogel.
NOACs are small molecules given at fixed doses. Monitoring is only required in special cases, as pharmacodynamic and pharmacokinetic responses are reliably predicted in patients with adequate renal function who are not taking other interacting drugs . Several trials have evaluated the efficacy and safety of the new oral anticoagulants (NOACs) for venous thromboembolism (VTE) prevention in patients undergoing total hip arthroplasty (THA). Twenty eight to 39 days of pharmacological prophylaxis with an NOAC compared with enoxaparin reduced the relative odds of symptomatic VTE by about 60% in patients undergoing THA with no excess of major bleeding . A three-year prospective study of the presentation and clinical outcomes of major bleeding episodes associated with oral anticoagulant use in the UK (ORANGE study) showed the following results: bleeding sites were intracranial (44%), gastrointestinal (33%), and other (24%). The in-hospital mortality was 21% overall, and 33% for patients with intracranial hemorrhage. Compared to warfarin-treated patients, patients treated with direct NOACs were older and had lower odds of subdural/epidural, subarachnoid and intracerebral bleeding. The mortality rate due to major bleeding was not different between patients being treated with warfarin or direct NOACs.
- Potential indications for coagulation testing include emergency situations (i.e., trauma) urgent surgery, urgent invasive procedures, major bleeding, overdose/attempted suicide, acute thrombosis, renal failure, adherence verification, and potential drug-drug interactions
- Routine clotting test are not reliable for monitoring patients
- Before starting therapy renal function monitoring prevents adverse events
- Each of the NOACs, except dabigatran, is metabolized by the liver. As such, routine liver function monitoring (every 6–12 months) should be considered in patients with, or at risk for, hepatic dysfunction
- Because of insufficient studies in pregnancy and during childbed, women with VTE should not be treated with NOACs.
- Because of insufficient studies, tumor patients with VTE should not be treated with NOACs.
Plasma concentrations can be measured for all four NOACs using liquid chromatography-tandem mass spectrometry. Information on the clinical utility of coagulation assays largely comes from ex vivo studies using plasma from dabigatran treated patients or healthy volunteers and in vitro studies with dabigatran added to plasma samples .
Determination of the anticoagulant effect of heparin.
Whole blood is added to a tube containing a surface activator. Coagulation is activated via the intrinsic pathway and through in-vitro activation of F XII. The appearance of the first clot is timed. The type of activator affects the clotting time (celite, 100–170 sec.; glass, 110–190 sec.; kaolin, 90–150 sec.). In a non anticoagulated patient, the ACT is about 107 ± 13 seconds. For monitoring the heparin effect during interventions (e.g., cardiopulmonary bypass), heparin is titrated to maintain an ACT > 400–600 sec. to prevent clot formation (> 250 sec. in heparin-coated systems). During extra corporeal membrane oxygenation, an ACT in the region of 200 sec. is considered sufficient. The ACT is influenced by numerous factors and is prolonged, for example, in thrombocytopenia below (30–50) × 109/L and factor deficiency including hypo fibrinogenemia.
Determination of the anticoagulant effect of direct thrombin inhibitors (lepirudin, desirudin, bivalirudin, argatroban, dabigatran).
Prothrombin is activated by ecarin, a snake venom from Echis carinatus.
In the ECT, the time elapsing until the appearance of a clot is measured. The ECT depends on the prothrombin and fibrinogen levels in the patient plasma, contrary to the concentration-independent ECA where prothrombin is added to the reaction and cleavage of a chromogenic substrate by meizothrombin is measured spectrophotometrically.
The activity of meizothrombin and meizothrombin-desF1 is inhibited in a concentration-dependent matter by direct thrombin inhibitors. Heparin does not influence the ECA and ECT assays. There is a linear correlation between the ECT and/or ECA response time and the concentration of the direct thrombin inhibitor allowing:
- The definition of substance-specific drug levels for therapy
- The allocation to regions with increased rates of complications, regarding both the occurrence of thromboembolism in anticoagulant under dose and bleeding in anticoagulant overdose.
Determination of the anticoagulant effect of unfractionated heparin.
Heparin activates antithrombin (AT) forming a heparin/AT complex (AT inhibits coagulation by activating serine proteases and proteolytic degradation of thrombin which very rapidly inhibits thrombin. A defined excess of thrombin added to the sample leads to the inactivation of a proportion of thrombin proportional to the heparin concentration. The residual, non-inactivated thrombin cleaves p-nitroaniline (p-NA) from a chromogenic substrate . The heparin concentration is calculated based on the increase in absorption determined spectrophotometrically at 405 nm and expressed in IU/mL.
Evaluation of the anticoagulant effect of coagulation therapy with anticoagulant drugs acting on F Xa within the coagulation cascade e.g., fractionated heparin, and some NOACs like apixaban, endoxaban, and rivaroxaban.
A defined amount of F Xa is added to the test sample; part of it binds to AT and is inactivated, a reaction catalyzed by heparin (). The residual, non-inactivated F Xa cleaves p-NA from a chromogenic substrate. Heparin levels below 0.1 IU/mL are no longer detectable because platelet factor 4 (PF4) binds to heparin. Therefore, dextran sulfate is added to the sample to release heparin from PF4. The heparin concentration is calculated based on the increase in absorption determined spectrophotometrically at 405 nm and expressed in IU/mL. Calibration must be performed according to the WHO standard for low molecular weight heparin. If used to determine the anti-F Xa activity of danaparoid or fondaparinux calibration is achieved with these anticoagulants.
Anti-F Xa activity is determined in risk patients (renal insufficiency, over- or underweight, children) and during pregnancy . Optimal anti-F Xa activities (if collected 4 h after subcutaneous administration) are reported as 0.4–1.0 IU/mL in plasma in therapeutic application and as 0.2–0.5 IU/mL in plasma in prophylactic application. The lower detection limit for heparins of the anti-F Xa assay is 0.01–0.05 IU/mL .
Suspected high anticoagulant activity of direct thrombin inhibitors (hirudin, argatroban, dabigatran).
The hemoclot thrombin inhibitor assay is based on the inhibition of a defined quantity of thrombin by thrombin inhibitors . The plasma to be analyzed is diluted and pooled normal human plasma is added. Coagulation is triggered using human α-thrombin. The time elapsing until the formation of a clot is directly proportional to the concentration of the thrombin inhibitor in the analyzed plasma. The reference interval for dabigatran 2 h after intake is 50–300 μg/L.
Some anticoagulants must be subjected to continuous laboratory diagnostic monitoring, while others need only to be monitored in specific patients or scenarios.
Patients undergoing therapy with phenprocoumon and warfarin must be monitored because of the risk of bleeding and/or recurrent thromboembolism based on inter individual differences in adjustment and dosage. This is achieved by determining the PT expressed as INR. The normal INR value is 1.0, the target value is usually 2–3 under therapy and 2.5–3.5 after mechanical heart valve replacement. Refer to .
Surgery associated with a low risk of bleeding (dento- surgical interventions, cardiac catheter examinations) is performed under therapeutic INR.
Major surgery requires bridging with coagulants with a short-term effect. Bridging implies a 5-fold increased risk of bleeding. As a sub therapeutic INR is reached after 4–7 days, vitamin K antagonist therapy should be temporarily discontinued, if possible. The decision about bridging with low molecular weight heparin, (e.g., in atrial fibrillation) depends on the CHA2DS2-VASc score. Perioperative bridging with unfractionated heparin is required in patients under intensive care and patients with mechanical heart valves. Anticoagulation using argatroban (intravenously) or danaparoid (subcutaneously and/or intravenously) is an alternative in patients at risk of heparin-induced thrombocytopenia type II.
The anti-F IIa (antithrombin) activity assay is used to monitor unfractionated heparin therapy (). As a rule, the anticoagulant effect during low molecular weight heparin therapy needs not be monitored by the anti-F Xa assay, but be determined in older patients with an eGFR < 30 [mL × min–1 × (1.73 m2)–1] for dose adjustment. However, the selected drug must be calibrated with the corresponding heparin.
In contrast to vitamin K antagonists and heparin, no routine coagulation. However, monitoring is required in patients taking NOACs. Laboratory diagnostic monitoring of the anticoagulant effect of NOACs is recommended in the following scenarios .
- Hemorrhagic risk
- Before surgery or an invasive procedure when the patient has taken a NOACs during the previous 24 h and his GFR is < 30 [mL × min–1 × (1.73 m2)–1]
- Identification of sub therapeutic or supra therapeutic levels in patients taking other drugs that are known to significantly affect the NOAC pharmacokinetics
- Identification of sub therapeutic or supra therapeutic levels in patients at the extremes of body weight
- Patients with deteriorating renal function
- Peri operative management
- Reversal of anticoagulation
- Suspicion of overdose
- Assessment of compliance in patients suffering thrombotic events while on NOAC treatment.
- Coagulation tests that are readily available in most laboratories. Their result is semi quantitative and should be considered as indicating supra therapeutic, therapeutic or sub therapeutic anticoagulation in emergency or other clinical scenarios
- Tests such as HPLC tandem mass spectrometry. NOACs are determined quantitative and specific.
Thrombolytic therapy may be indicated in special situations in connection with thromboembolic events . Contra indications of fibrinolytic therapy must be excluded and the latest technical information applied. In general, a distinction is made between systemic fibrinolytic therapy and local (catheter-controlled) fibrinolysis frequently performed during radiological interventions. Available fibrinolytic agents include streptokinase, urokinase, rt-PA (alteplase) and tenectplase. Due to the increased risk of bleeding, fibrinolytic therapy is not recommended in deep vein thrombosis. However, fibrinolysis may still be employed in individual cases because of its superiority in clot recanalization. By contrast, systemic fibrinolytic therapy is the therapy of choice in hemodynamically unstable patients with pulmonary embolism . Early fibrinolysis with rt-PA (initial bolus 10 mg/1–2 min.; maintenance dose 90 mg/2 h; not more than 100 mg in total) is usually the standard therapy.
In acute coronary syndrome and/or acute myocardial infarction, fibrinolytic therapy using, for example, rt-PA (e.g., within 6 h after the onset of symptoms; initial bolus 15 mg; maintenance dose 50 mg/30 min., then 35 mg/60 min.; not more than 100 mg in total; dose adjustment required in patients with a body weight below 65 kg) should always be considered if standard percutaneous coronary intervention cannot be performed within 90 min. .
In acute ischemic stroke, systemic fibrinolysis using rt-PA (0.9 mg per kg of body weight; not more than 90 mg; 10% of the total dose as initial bolus, the rest distributed over 60 min.; not more than 90 mg in total) must be started within 3 h (to not more than 4.5 h) after the onset of symptoms; the risk of bleeding increases thereafter /, /. Local intraarterial, catheter-based thrombolytic therapy in acute ischemic stroke is only performed on selected patients in specialized centers. Intraarterial thrombolytic therapy can also be considered in acute ischemia of the extremities, whereas systemic thrombolysis is objected in this case .
Special diagnostic monitoring of fibrinolytic therapy beyond the determination of global coagulation parameters (aPTT) is not necessary. In pulmonary embolism, rt-PA treatment should be accompanied by intravenous heparinization as soon as the aPTT values are below 2 times the upper reference interval value. The aPTT target interval is between 50 and 70 sec. (1.5–2.5-fold the reference interval).
In patients with acute coronary syndrome, platelet function inhibition therapy using acetylsalicylic acid can be started concurrently with rt-PA fibrinolysis. By contrast, the administration of acetylsalicylic acid or intravenous heparin is contraindicated in patients receiving rt-PA for treatment of ischemic stroke within the first 24 h after start of therapy.
The recommended duration of therapeutic anticoagulation depends on the indication. Aspects to be considered include the evidence level 1/2, strong/low recommendation and quality of studies:
- A, excellent quality of study (e.g., two studies with evidence level I; randomized case-control studies)
- B, good quality of study (e.g., 1 study with evidence level I)
- C, moderate quality of study (e.g., studies with evidence level II; cohort or outcome studies).
Therapeutic anticoagulation is accomplished in VTE with vitamin K antagonists (VKA) and/or for cancer with low-molecular-weight heparin (LMWH) /, /. Initial therapy of pulmonary embolism is to employ the concept of overlapping treatment with LMWH, unfractionated heparin (UFH) or fondaparinux in therapeutic doses until an INR > 2 is attained (recommendation 1A). Venous catheder application (VCA) therapy starts on the first day of treatment (1A). The target INR using VCA in venous thrombosis or embolism is in the range of 2.5 (2.0–3.0). For the possible use of dabigatran etexilate, see .
A risk/benefit analysis should be performed on a regular basis in patients on unlimited-duration anticoagulant therapy (1C). Immediate fibrinolytic therapy is indicated in massive, hemodynamically relevant pulmonary embolism (1B). In most cases, the presence of laboratory-detected thrombophilia does not influence the duration of anticoagulation if the guidelines are adhered to.
In patients with valvular or structural heart disease receiving VCA therapy, the target INR is in the range of 2.5 (2.0–3.0); exception: mechanical heart valve target INR 3.0 (2.5–3.5) . Evidence of the indication for anti thrombocytic therapy is to be provided, as necessary. Dabigatran etexilate is not permitted in valvular atrial fibrillation.
For recommended duration of therapeutic anticoagulation refer to:
As expected with all antithrombotic agents, there is an associated increased risk of bleeding complications. Prevalence of total mortality and severe hemorrhage associated with oral anticoagulants was lower in patients treated with DOCA in comparison to VCA therapy .
For the majority of severe bleeding events, the widespread approach is to withdraw the DOAC, then provide supportive measures and watchful waiting with the expectation that the bleeding event will resolve with time. However urgent reversal of anticoagulation may be advatageous in patients with serious or life-threatening bleeding. Idaruczimab has been approved for reversal of anticoagulation in dabigatran-treated patients and adexanet alfa for factor Xa inhibitor treated patients .
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* Data expressed in mg/L (nmol/L)
** Molecular weight of the smallest subunit
* Data expressed in mg/L (nmol/L)
* Data expressed in mg/L (nmol/L); PAI, plasminogen activator inhibitor
* Ristocetin is an antibiotic inducing platelet aggregation in the presence of the von Willebrand factor. It is used for testing an important partial function of the vWF.
Degree of severity
F VIII or F IX activity
Minimal values prior to the next
von Willebrand syndrome
* CNS, central nervous system
HA, hemophilia A; DIC, disseminated intravascular coagulation; vWS, von Willebrand synfrome; Dc, dilutional coagulopathy
t-PA, tissue-type plasminogen activator
* Relative contra indications; CNS, central nervous system
INR; international normalized ratio
Values expressed as mean and 2.5th and 97.5 percentiles. PT, prothrombin time; aPTT, activated partial prothrombin time; Ag, antigen determination; F, factor; among the factors, the coagulation activity is expressed in %. FW, fetal week; TFPI, tissue factor pathway inhibitor.
Values expressed as mean and 2.5th and 97.5 percentiles. Factor concentration in U/mL
Clinical and laboratory findings
Abbreviations: N, normal; L, low; P, prolonged
Clinical and laboratory findings