- Email: [email protected]
Blood coagulation
Best Practice & Research Clinical Obstetrics & Gynaecology Vol. 17, No. 3, pp. 369 –383, 2003 doi:10.1053/S1521-6934(03)00014-2, www.elsevier.com/locate/jnlabr/ybeog
1 Blood coagulation Lucy A. Norris*
BA Mod. (Biochem) , MSc Mol Med , PhD
Research Biochemist Coagulation Research Laboratory, Department of Obstetrics and Gynaecology, Trinity College Centre for Health Sciences, St James’s Hospital, Dublin 8, Ireland
Blood coagulation can be initiated by two pathways: the extrinsic pathway, which is triggered by release of tissue factor from the site of injury, and the intrinsic system, which is stimulated by contact with a negatively charged surface. Following initial triggering, a series of serine proteases are sequentially activated, culminating in the formation of thrombin, the enzyme responsible for the conversion of soluble fibrinogen to the insoluble fibrin clot. Activation of coagulation is tightly regulated. Initiation by tissue factor is inhibited by tissue factor pathway inhibitor. Antithrombin can inactivate many of the serine proteases, including thrombin, by forming stable complexes which are rapidly cleared from the circulation. Protein C and protein S combine to inactivate coagulation factors V and VIII. The deposition of excess fibrin is prevented by the fibrinolytic system which can lyse fibrin into fibrin degradation products. Both genetic and environmental factors can influence the activation of coagulation and may predispose affected individuals to thrombosis. Key words: coagulation; thrombin; fibrin; thrombosis.
In a healthy individual, blood circulates as a liquid; however, the occurrence of vascular injury requires blood to gel rapidly to form a clot in order to prevent haemorrhage. When the endothelium becomes damaged, platelets adhere to the subendothelium and activation of the coagulation cascade causes the production of fibrin, which forms a mesh over the platelet plug, sealing the site of injury. In addition, chemokines facilitate the attraction of leukocytes to the area, which prevent infection and assist in wound healing. Although an almost instantaneous and explosive activation of the haemostatic system is required to prevent blood loss, the ability of blood to clot must be carefully regulated because inappropriate clot formation can lead to thrombotic complications. To achieve this, the normal healthy endothelium acts as an antithrombotic surface. Activation of the coagulation cascade is triggered by the release of tissue factor from the site of injury and, in the presence of pro-coagulant cell surfaces, production of fibrin can be achieved in seconds. The explosive activation of the haemostatic system is possible because of the so-called ‘cascade’ system of coagulation in which inactive zymogens and cofactors are sequentially activated by proteolytic cleavage. Almost immediately, the fibrinolytic system is stimulated—limiting fibrin deposition to the site of injury—and feedback in a system of naturally occurring anticoagulants feeds back and blocks further activation of the coagulation cascade. * Tel.: þ 353-1-6082116; Fax: þ353-1-4531614. E-mail address: [email protected] (L. A. Norris). 1521-6934/03/$ - see front matter Q 2003 Elsevier Science Ltd. All rights reserved.
370 Lucy A. Norris
Although thrombosis is a multifactorial disease, dysregulation of the haemostatic pathways is a major contributor to thrombus formation. A number of genetic and environmental factors can cause excessive upregulation of coagulation enzymes. This upregulation may overwhelm the anticoagulant response—particularly in those individuals in whom one or more of the key anticoagulant pathways is impaired.
INITIATION OF BLOOD COAGULATION Traditionally, two main pathways have been described for blood coagulation: the intrinsic or contact pathway and the extrinsic or tissue factor pathway (Figure 1). Although originally these pathways were thought to be mutually exclusive and of equal importance, recent evidence disputes this. The intrinsic pathway can be activated when blood comes into contact with a negatively charged surface resulting in the activation of factor XII to XIIa. However, deficiencies in factor XII are not associated with bleeding and may even be associated with thrombosis.1 In addition, the activation of factor IX can be achieved by both the extrinsic and intrinsic pathways. The main function of the intrinsic pathway may be to amplify the coagulation activation triggered by the tissue factor pathway. Extrinsic/tissue factor pathway The tissue factor pathway is triggered when the membrane-bound protein tissue factor (TF) comes into contact with plasma containing factor VII or VIIa. Unlike other members of the coagulation cascade, tissue factor is always present as an active cofactor and is thought to be essential for life.2 Tissue factor is not normally expressed in cells that come into contact with plasma; however, upon vascular injury, cells expressing membrane-bound tissue factor are exposed to plasma and can bind factor VII. In addition, monocytes and smooth muscle cells can be stimulated to produce tissue factor by cytokines and other inflammatory mediators.3 The binding of factor VII to tissue factor leads to the formation of TF-VIIa which, when attached to the cell membrane, becomes the most potent activator of the coagulation cascade known. Factor VII is a typical vitamin-K-dependent plasma protein produced in the liver. Although almost 99% of factor VII is present in its inactivated state, 1% is present as an active serine protease, factor VIIa.4 However, when factor VIIa is free and not part of the TF-VIIa complex, it is a relatively weak enzyme. Increased factor VII has been reported as a risk factor for thrombotic disease5,6; however, this relationship is dependent on the method of measuring factor VII levels.7 Factor VII deficiency is a rare event and individuals with severe deficiency present with major life-threatening bleeding episodes.8 Factor VII is also influenced by polymorphisms in the factor VII gene; mutations in the promoter region and a GLN/ARG substitution in the protease domain have been associated with lower levels of factor VII, giving rise to the suggestion that factor VII genotype may influence thrombotic risk9—but this has not been shown conclusively.10 Once formed, the TF-VIIa complex on the cell surface has two potential substrates: factor IX, which is converted to IXa, and factor X, which is converted to Xa. In vitro,
INTRINSIC PATHWAY EXTRINSIC PATHWAY TF
Negatively charged surface XII
XI
X
IX
HMWK
XIIa
VII
TF-VIIa
HMWKa XIa
TFPI
TFPI
CI-inh IXa Ca PL
Tenase complex
Prothrombin VIIIa
AT
AT Xa Va
VIII Prothrombinase
PL V
Thrombin
Fibrinogen
Fibrin
Figure 1. The coagulation cascade showing both intrinsic and extrinsic activation, inhibitors and feedback activation (dashed lines). HMWK ¼ high molecular-weight kininogen. C1-inh ¼ C1-inhibitor. TF ¼ tissue factor. TFPI ¼ tissue factor pathway inhibitor. PL ¼ phospholipids. Ca ¼ calcium. AT ¼ antithrombin.
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AT
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factor X is the preferred substrate of TF-VIIa; however, the predominant pathway in vivo is unknown and may depend on local factors.4 Intrinsic system The intrinsic or contact pathway can also activate factor IX. The binding of factor XII (Hageman factor) to an artificial or negatively charged surface leads to a local increase in concentration and results in the autoactivation of factor XII to XIIa. Once assembled on a negatively charged surface, XIIa causes the conversion of prekallikrein to kallikrein and factor XI to XIa.11 Cleavage of high-molecular-weight kininogen (HMWK) (a protein with diverse biological properties) also occurs. These changes culminate in the formation of factor IXa.12 Although in vitro defects in the intrinsic pathway manifest as a prolongation of partial thromboplastin time, in vivo, deficiencies in these factors do not cause bleeding. The exception to this is factor XI, which causes mild bleeding following trauma or injury.13 The observation that thrombin can also activate factor XI suggests that the main role of factor XI is not in the initiation of coagulation but is in the secondary production of thrombin which is essential for haemostasis.14 The Leiden thrombophilia study showed that increased levels of factor XI is a risk factor for thrombosis: it suggested that the role of factor XI was twofold— secondary generation of thrombin and downregulation of fibrinolysis via thrombinactivatable fibrinolysis inhibitor (TAFI).15 Although the proteins of the intrinsic pathway are often considered biologically irrelevant, more recent studies have suggested that these proteins have diverse biological roles throughout the vascular system. Prekallikrein and HMWK are involved in the regulation of blood pressure and participate in fibrinolysis. Factor XII can activate neutrophils and upregulate the release of cytokines from monocytes and macrophages.16
FORMATION OF THE ‘TENASE’ COMPLEX When factor IX is activated to IXa by either the intrinsic or extrinsic pathway, it forms a complex with factor VIIIa, calcium and phospholipids which can activate factor X to Xa. This complex is sometimes known as the ‘tenase’ complex and is crucial to haemostasis. Absence of factor VIII or factor IX produce the same haemorrhagic disease known as haemophilia, the severity of which is related to the degree of deficiency of factor VIII or IX. Factor VIII exists in plasma mostly as a non-covalent complex with von Willebrand factor (vWF) which protects it from proteolytic activation to VIIIa. vWF binding to platelets attached to the surface of damaged endothelium facilitates activation of factor VIII to VIIIa and dissociation of vWF.17 Thrombin can also feedback and activate factor VIII, a process that is not prevented by vWF.18
CONVERSION OF PROTHROMBIN TO THROMBIN The final step in producing the active thrombin enzyme is the formation of the prothrombinase complex. This consists of factor Xa bound to factor Va, its
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non-enzymatic cofactor, calcium and a phospholipid membrane surface. In the presence of anionic phospholipids, inactive factor V is converted to Va. Thrombin may also activate factor V, as can the tissue factor-VIIa complex.19 Factor V mutations are the commonest inherited risk factors for thrombosis; many of these mutations—including the common factor V Leiden polymorphism—affect the inactivation of factor V by the inhibitor, protein C.20 Factor X is a vitamin-K-dependent coagulation factor which can be converted to the active serine protease, factor Xa, by the tissue factor-VIIa complex, or by the tenase complex. Because the direct activation of factor X by TF-VIIa is rapidly downregulated by the tissue factor pathway inhibitor (TFPI), the tenase complex is the most important activator of factor X, which explains the clinical severity of the haemophilias. Although factor Xa alone can catalyse the conversion of prothrombin to thrombin, the reaction is very slow and is greatly accelerated by the addition of factor Va and the binding of the complex to the phospholipid surface of either activated platelets or monocytes.21 The tenase complex converts inactive prothrombin to a-thrombin, a serine protease. Cleavage of prothrombin to thrombin occurs via a number of steps. Initial cleavage occurs at Arg320 to give rise to the intermediate product meizothrombin. Cleavage at Arg271 produces the active enzyme a-thrombin and a by-product, prothrombin fragment 1.2.22 As circulating thrombin concentration is difficult to measure, the more stable prothrombin fragment 1.2 is frequently used as a marker of prothrombin-tothrombin conversion and is an indicator of a hypercoagulable state.23 A mutation in the 30 untranslated region of the prothrombin gene (G20120-A) results in elevated plasma prothrombin levels and has been reported as a risk factor for thrombosis.24 Thrombin has many functions within haemostasis. It can convert fibrinogen to fibrin and is also capable of activating platelets, coagulation factors V, VIII and IX, and the inhibitors protein C and thrombin-activatable fibrinolysis inhibitor (TAFI).25 This facility is possible because of a number of distinct binding sites on the thrombin molecule and because of the ability of the enzyme to bind to cofactors which can render it procoagulant or anticoagulant.26
FIBRIN FORMATION The formation of fibrin is the final stage of the coagulation process. Soluble fibrinogen is converted into an insoluble fibrin polymer, which seals the site of injury and protects damaged tissue during wound healing. This tightly controlled process occurs in several stages. First, the dimeric fibrinogen molecule is cleaved by thrombin to produce soluble fibrin monomers, fibrinopeptide A and fibrinopeptide B. Fibrinopeptide A is frequently used as an early marker of fibrinogen-to-fibrin conversion.23 Non-covalent interactions result in the formation of fibres or strands which then aggregate to form a mesh. Finally, the newly formed fibrin is stabilized by cross-linking catalysed by thrombin-activated coagulation factor XIIIa.27 Although fibrinogen plays a key role in coagulation, it is also important in other cellular processes. Soluble fibrinogen acts as a signalling molecule and is important in adhesive processes required for the trafficking of immune cells required during wound healing.28 In addition, both fibrinogen and fibrin play a role in tumour genesis by promoting angiogenesis essential for tumour growth.29
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REGULATION OF BLOOD COAGULATION The regulation of blood coagulation is essential to avoid a generalized activation of the system and massive fibrin deposition. To be effective, the system must be active only at a local site of vascular injury and must remain active only for a sufficient period of time to produce enough fibrin to seal the wound. To achieve this, a number of regulatory mechanisms are in place. As described above, each coagulation protein is sequentially activated, so that only a small proportion of an active serine protease is available at any given time. Second, coagulation activation and formation of the complexes above can proceed only on the surface of activated cells and platelets—where negatively charged phospholipids are exposed and the appropriate receptors are available for binding. Similarly, the essential trigger for the cascade, tissue factor, is available only on the surface of activated monocytes and cells which are exposed to plasma only as a result of vascular injury. The most important regulatory pathways, however, are a series of anticoagulant proteins and cofactors which bind to activated coagulation factors and limit their period of activity. Finally, in the presence of fibrin, fibrinolysis is stimulated which dissolves fibrin into fibrin degradation products. Tissue factor pathway inhibitor (TFPI) The initial trigger for coagulation activation is the TF-VIIa complex which is a potent activator of both factor IX and X. For this reason, the activity of this complex must be quickly inactivated. This is achieved by a specific inhibitor of the complex, tissue factor pathway inhibitor (TFPI), released constitutively from endothelial cells. TFPI is a multidomain inhibitor. Initially it forms a complex with Xa through one of its domains and the complex can then bind to the TF-VIIa complex with another binding domain to form a quaternary complex.30 The effect is rapid inhibition of extrinsic activation of the cascade. In addition TFPI also promotes the internalization and subsequent degradation of the inhibited complex.31 In some model systems, pharmacological agents which block tissue factor have been shown to be effective in reducing thrombosis without the bleeding complications associated with other antithrombotic drugs. This suggests that the TF-VIIa may be a useful target for the design of antithrombotic drugs. Recent preliminary studies have shown that TFPI may be an effective agent against thrombosis and may reduce mortality in sepsis patients.4 Antithrombin Antithrombin is a serine protease inhibitor that can inhibit many of the activated coagulation enzymes. Key components of the cascade, such as factors IXa, Xa, TF-VIIa complex and thrombin, are rapidly bound by antithrombin and neutralized. The ability of antithrombin to inhibit these factors is greatly accelerated by heparin sulphate proteoglycans and this is the basis for the anticoagulant action of the pharmaceutical heparins. Antithrombin inhibits free thrombin and Xa more efficiently than thrombin and Xa bound to activation complexes; this has the effect of removing these proteases from the general circulation and confining their activity to the site of clot formation. Thrombin bound to antithrombin forms the stable thrombin –antithrombin (TAT) complex, which is rapidly cleared from the circulation.32 TAT complex levels are also used as a marker of hypercoagulability because they reflect thrombin production.33
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Because antithrombin plays an essential role in controlling coagulation activity, even modest deficiencies in the levels of activity can cause a clinical problem. An antithrombin concentration below 60 – 70% is associated with thrombosis.34,35 Approximately 5% of patients with venous thrombosis are found to be antithrombin deficient, and both inherited and acquired deficiencies have been described.36 The protein C anticoagulant pathway The protein C anticoagulant pathway is thought to be the major mechanism by which thrombosis in the microcirculation is prevented. Protein C is a vitamin-K-dependent plasma protein which circulates as an inactive zymogen.37 To become activated (Figure 2), it must first bind via its GLA domain to a transmembrane receptor expressed on the endothelium called the endothelial protein C receptor (EPCR).38 The activation of protein C also requires thrombin. Free thrombin must first bind to another transmembrane protein, thrombomodulin, which is expressed constitutively by endothelial cells.39 This protein tethers thrombin to the endothelium where EPCR presents protein C for activation. Thrombin complexed to thrombomodulin activates protein C, possibly through a conformational change in the thrombin enzyme.40 Activated protein C (APC) is inactive as an inhibitor while it remains bound to the EPCR.41 To become active, APC must dissociate from EPCR and bind to its cofactor protein S. Protein S is also a vitamin-K-dependent plasma protein; however, unlike protein C, it has no enzymatic activity of its own; the main function of protein S is to act as a cofactor for APC.42 The protein S/APC complex can inactivate both factor Va and factor VIIIa, effectively shutting down prothrombinase and tenase activity respectively.43 Thus thrombin, by activating the protein C pathway, shuts down its own production. APC/protein S binds preferentially to factors Va and VIIIa rather than their inactive precursors.44,45 In the case of VIIIa, inactivation by APC can occur as a result of cleavage of Arg336 and Arg562.46 The inactivation of factor Va involves cleavage at Arg506 and Arg306 and is a two-phase process. The initial rapid phase of factor Va inactivation is due to cleavage at Arg506. Cleavage of factor V results in a 50-fold decrease in affinity for Xa and a drastic reduction in prothrombinase activity. A second, slower phase corresponds to cleavage at Arg306.47 In patients with the factor V Leiden mutation, the arginine amino acid residue at position 506 is replaced by glutamine.48 The effect of this mutation is to produce factor Va which cannot be completely inactivated by APC, hence the term ‘APC resistance’ to describe the phenotype of this mutation.48 APC resistance is the most common inherited risk factor for venous thrombosis currently known. In the general population, the mutation appears to be limited to Caucasians and occurs in 2 –15% of Europeans.49 Inherited deficiencies have also been described for protein C and protein S. Over 160 mutations have been described for the protein C gene, most of which lead to absent or defective protein C.50 However, in affected individuals, protein C mutations are rarely categorized and patients are assumed to be heterozygous if they produce 40 –60% of normal activity from the unaffected allele. Homozygous protein C deficiency is a serious condition leading to life-threatening thrombotic complications frequently occurring immediately after birth.51 Mutations in the protein S gene also lead to a deficiency in protein S; these deficiencies account for between 2 and 5% of patients with a history of thrombosis.52 Protein S circulates in two forms: free unbound protein S and protein S bound to C4bbinding protein.53 Based on plasma measurements, three different types of protein S deficiency of clinical importance have been defined. Type 1 deficiency is defined as
376 Lucy A. Norris
PC
T
T
TM Cell membrane
E P C R
E P TM PC C R
APC
E P C R
PS PS
Vi
VIIIi
Va PS APC
VIIIa PS APC
Figure 2. Schematic representation of protein C activation. T ¼ thrombin. TM ¼ thrombomodulin. PC ¼ protein C. EPCR ¼ endothelial protein C receptor. PC ¼ protein C. PS ¼ protein S. Vi ¼ inactivated factor Va. VIIIi ¼ inactivated factor VIIIa. APC ¼ activated protein C.
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a reduction in free and total protein S. Patients with type II deficiency have normal protein S antigen levels but reduced functional activity, suggesting that an abnormal protein is being produced. The third type shows normal total protein S levels with a reduction in free unbound protein S only.54 However, assays for protein S are influenced by a number of factors so that differences in phenotype may not always be explained by differences in genotype. 55 Recent studies have suggested that polymorphisms in the thrombomodulin gene may influence the risk of coronary heart disease in African Americans; however, larger studies are required to confirm this.39 Similarly, a 23-base-pair insertion in the EPCR gene has been identified; however, the significance of this mutation remains to be determined.56
Inhibitors of the intrinsic system The major inhibitor of the intrinsic system is C1-inhibitor, which inhibits factor XIIa. Binding of C1 inhibitor irreversibly inactivates at least 90% of XIIa; however, when XIIa is bound to a kaolin surface it is protected from C1-inhibitor activation.57 a1Antitrypsin can inhibit factor XIa and acts in a similar manner to antithrombin, forming a stable complex with its target serine protease.58 a2-Macroglobulin is a broad-spectrum proteinase inhibitor which acts as a secondary inhibitor of many proteinases involved in the coagulation cascade, including kallikrein and thrombin and the fibrinolytic enzyme plasmin.11 Deficiencies in these inhibitors do not cause thrombosis, partly because of the secondary nature of the intrinsic system and partly because antithrombin can inhibit almost all of the serine proteases involved in the coagulation cascade. As with other components of the intrinsic system, these inhibitors have functions outside the haemostatic system—for example, a2-macroglobulin is thought to be involved in the regulation of the immune system59, and deficiencies in C1-inhibitor are associated with hereditary angiodema.60
FIBRINOLYSIS Fibrinolysis is the final control mechanism that limits clot formation. The fibrinolytic system is a series of enzymes which, when activated, cleave fibrin into fibrin fragments known as fibrin degradation products (Figure 3). The control of this mechanism is crucial to maintaining haemostatic balance.
Plasminogen and plasmin The enzyme responsible for lysis of fibrin to fibrin degradation products is plasmin. This enzyme circulates as an inactive zymogen, plasminogen.61 Plasminogen can bind to fibrin via lysine binding sites on the heavy-chain portion of the plasminogen molecule; the subsequent binding of a plasminogen activator leads to cleavage of the plasminogen molecule at the Arg561 – Val bond and the formation of the active protease plasmin.62 The presence of plasminogen activators and their inhibitors is essential in controlling fibrinolysis.
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PLASMINOGEN PAI-I tPA
Fibrin
uPA
HMWK XIIa Xla PLASMIN
Antiplasmin
α1 - antitrypsin α2 - macroglobulin Antithrombin FDPs
FIBRIN TAFI
Figure 3. The fibrinolytic system. tPA ¼ tissue plasminogen activator. uPA ¼ urokinase plasminogen activator. PAI-1 ¼ plasminogen activator inhibitor 1. HMWK ¼ high-molecular-weight kininogen. TAFI ¼ thrombin activatable fibrinolysis inhibitor. FDPs ¼ fibrin degradation products.
Plasminogen activators There are several pathways of plasminogen activation. Factor XIIa, XIa and kallikrein are all capable of converting plasminogen to plasmin.63,64 The contribution which these enzymes makes to the activation of plasminogen in vivo is uncertain, however, because deficiencies in these proteins do not appear to lead to pathological states that could be explained by impaired fibrinolysis.65,66 The major activator of plasminogen in vivo is tissue plasminogen activator (tPA), a serine protease produced by endothelial cells. In the absence of fibrin, it is an inefficient activator of plasminogen but once bound to fibrin, activation is greatly accelerated.67 Local release of tPA from the vascular endothelium in the vicinity of an injury may be stimulated by fibrin itself, by thrombin attached to the formed clot, or by the effects of venous occlusion.68 – 70 Urokinase or urinary type plasminogen activator (uPA) can also activate plasminogen. The importance of uPA is in tissues where it plays a role in the degradation of the extracellular matrix. This facilitates the migration of cells which is important in wound healing and in tumour invasion and metastasis.71,72
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Inhibitors of fibrinolysis The primary inhibitor of active plasmin is a2-antiplasmin. In plasma, a2-antiplasmin binds rapidly to plasmin to form an irreversible stable complex, plasmin –antiplasmin complex.73 This complex can be measured in plasma and is sometimes used as a marker for in vivo plasmin production.74 Free plasmin is much more rapidly bound to a2-antiplasmin than plasmin attached to fibrin because both utilize lysine binding sites.73 a2-Macroglobulin plays a limited role as a plasmin inhibitor, becoming important only when the concentration of plasmin exceeds the capacity of a2-antiplasmin. Similarly, antithrombin, a1-antitrypsin and C1-inactivator have been shown to inhibit plasmin in vitro but have a minimal physiological effect in blood.75 Inhibitors of plasminogen activator play an important role in regulating fibrinolysis. Currently, four distinct types have been described: PAI-1, PAI-2, PAI-3 and protease nexin. Of these, PAI-1 is the most important in inhibiting tPA in plasma. Synthesis of PAI1 occurs primarily in endothelial cells and is one of the most regulated proteins in humans. Stimulation of PAI-1 release is caused by a large variety of compounds, including thrombin and endotoxin.76,77 PAI-1 is present in molar excess over tPA, hence the majority of tPA circulates bound to PAI-1; the effect of this is to prevent premature lysis of fibrin in the forming clot and to inhibit systemic fibrinolysis.78 PAI-2 is found only in the plasma of pregnant women and is produced in the placenta and also in some tumour cells. The main function of PAI-2 is unclear; however, it may play a role in the regulation of limited proteolysis in some tissues.79 The most recently described fibrinolysis inhibitor is thrombin-activatable fibrinolysis inhibitor (TAFI), also known as plasma carboxypeptidase B.80 Thrombin, when bound to thrombomodulin, can activate TAFI—which can inhibit fibrinolysis by several mechanisms. First, activated TAFI cleaves the COOH terminal end of fibrin which reduces the ability of fibrin to facilitate plasminogen activation via tPA. TAFI can also directly inhibit plasmin activity.81 TAFI can also be cross-linked to fibrin and incorporated into a fibrin clot, which prevents premature lysis. Inhibitors such as APC, which prevent prothrombin-to-thrombin conversion, inhibit the activation of TAFI; this explains the apparent pro-fibrinolytic activity of APC.82
SUMMARY AND CONCLUSIONS The maintenance of haemostasis is essential to avoid bleeding and thrombosis. This is achieved by the sequential and short-lived activation of the coagulation cascade series of enzymes, ultimately resulting in the production of an insoluble fibrin clot. Both genetic and environmental factors can cause alterations in the level of activation of these enzymes, and for this reason a number of fail-safe inhibitory mechanisms are required if thrombosis is to be prevented. These pathways remove any excess procoagulant enzymes and prevent systemic fibrin production. Fibrinolysis inhibitors are also essential to prevent inappropriate breakdown of fibrin and fibrinogen. Both thrombin and fibrin orchestrate their own demise, thrombin by triggering the activation of protein C and fibrin by stimulating plasmin production and fibrinolysis. An increasing knowledge of the genetic basis of many of the inherited disorders of the haemostatic system has enhanced our understanding of the mechanisms by which coagulation is regulated and controlled. Within the last 10 years, the major cause of venous thrombosis in the microcirculation, the factor V Leiden mutation, was discovered; the next decade may bring further discoveries of this nature. Although
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Practice points † factors such as pregnancy, use of hormones and surgery cause coagulation activation which may predispose an individual to thrombosis † factor V Leiden is the leading cause of thrombosis in the microcirculation † an antithrombin level of 60 –70% or less of normal lines is associated with an increased risk of thrombosis
Research agenda † all hormonal preparations cause changes in coagulation activation and inhibition in healthy women; however, only a small proportion develop thrombosis. Factor(s) as yet undiscovered may help to identify which women are likely to develop thrombosis while taking hormone preparations † many of the coagulation genes are polymorphic; however, the significance of these polymorphisms in relation to thrombosis risk in many cases is unknown. Similarly, combinations of polymorphisms may be predictive for thrombosis under certain environmental conditions † thrombin and fibrinogen not only function in the coagulation system but are also signalling molecules. Study of the interaction between the immune system and coagulation serine proteases may increase understanding of the mechanisms of coagulation activation and the pathogenesis of thrombosis a large number of thrombotic events can be explained by inherited or acquired thrombophilia, the pathogenesis of this disease in a significant number of patients remains unexplained. The interaction between environmental factors and polymorphisms of coagulation activators and inhibitors may provide further clues to the mechanisms by which thrombosis occurs.
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In Colman RW et al (eds) Haemostasis and Thrombosis, Basic Principles and Clinical Practice, 4th edn, pp 157 –169, New York: Lippincott/Williams and Wilkins, 2001. * 20. Bertina RM, Koeleman BPC, Koster T et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64 –67. 21. Neisheim ME, Taswell JB & Mann KG. The contribution of bovine factor V and factor Va to the activity of prothrombinase. Journal of Biological Chemistry 1979; 254: 10 952–10 962. 22. Doyle MF & Haley PE. Meizothrombin: active intermediate formed during prothrombinase-catalysed activation of prothrombin. Methods in Enzymology 1993; 222: 299. 23. Bauer KA. Activation markers of coagulation. Baillie`re’s Clinical Haematology 1999; 12: 387 –406. 24. Poort SR, Rosendaal FR, Reitsma PH & Bertina RM. A common genetic variation in the 30 untranslated region of the prothrombin gene is associated with elevated prothrombin levels and an increase in venous thrombosis. Blood 1996; 388: 3698–3703. 25. Brummel KE, Paradis SG, Butenas S & Mann KG. Thrombin functions during tissue factor induced blood coagulation. Blood 2002; 100: 148–152. * 26. Tulinsky A. Molecular interactions of thrombin. Seminars in Thrombosis and Haemostasis 1996; 22: 117 –124. 27. Weisel J. Fibrin assembly. Lateral aggregation and the role of two pairs of fibrinopeptides. Journal of Biophysics 1986; 50: 1079. 28. Clark RAF. Wound repair. In Clark RAF (ed.) The Molecular and Cellular Biology of Wound Repair, p 617, New York: Plenum Press 1996. 29. Wojtukiewicz MZ, Sierko E, Klement P & Rak J. The haemostatic system and angiogenesis in malignancy. Neoplasia 2001; 3: 371–384. * 30. Broze GJ Jr. Tissue factor pathway inhibitor and the current concept of blood coagulation. Blood Coagulation and Fibrinolysis 1995; 6(supplement 1): S7 –S13. 31. Hamik A, Setiadi H, Bu GJ et al. Down regulation of monocyte tissue factor mediated by tissue factor pathway inhibitor and low density lipoprotein receptor-related protein. Journal of Biological Chemistry 1999; 74: 4962–4969. 32. Fuchs H, Shiffman M & Pizzo S. In vivo catabolism of alpha1-proteinase inhibitor-trypsin, antithrombinthrombin and alpha2macroglobulin-methylamine. Biochimica et Biophysica Acta 1982; 716: 151–157. * 33. Lopez Y, Paloma MJ, Rifon J et al. Measurement of prethrombotic markers in the assessment of acquired hypercoagulable states. Thrombosis Research 1999; 93: 71–78. 34. Kauffman R, Veltkamp J, van Tilburg N & van Es L. Acquired antithrombin III deficiency and thrombosis in the nephritic syndrome. American Journal of Medicine 1978; 65: 607–613. 35. Francis C, Pellegrini V, Harris C et al. Prophylaxis of venous thrombosis following total hip and total knee replacement using antithrombin III and heparin. Seminars in Hematology 1991; 28: 39–45. 36. Harper P, Luddington R, Daly M et al. The incidence of dysfunctional antithrombin variants: four cases in 210 patients with thromboembolic disease. British Journal of Haematology 1991; 77: 360– 364. 37. Kisiel W. Human plasma protein C: isolation, characterisation and mechanisms of activation by athrombin. Journal of Clinical Investigation 1979; 64: 761–769. 38. Esmon CT. The endothelial cell protein C receptor. Thrombosis and Haemostasis 2000; 83: 639–643.
382 Lucy A. Norris 39. Wu KK & Matijevic-Aleksic N. Thrombomodulin: a linker of coagulation and fibrinolysis and predictor of risk of arterial thrombosis. Annals of Medicine 2000; 32(supplement 1): 73–77. 40. Ye J, Esmon NL, Esmon CT & Johnson AE. The active site of thrombin is altered upon binding to thrombomodulin: two distinct structural changes are detected by fluorescence but only one correlates with protein C activation. Journal of Biological Chemistry 1991; 266: 23 016–23 021. * 41. Liaw PCY, Neuenschwander PF, Smirnov MD & Esmon CT. Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function. Journal of Biological Chemistry 2000; 275: 5447–5452. 42. Walker FJ. Regulation of activated protein C by a new protein: a role for bovine protein X. Journal of Biological Chemistry 1980; 255: 5521–5524. 43. Walker FJ & Fay PJ. Regulation of blood coagulation by protein C system. FASEB Journal 1992; 6: 2561–2567. 44. Vehar GA & Davie EW. Preparation and properties of bovine factor VIII. Biochemistry 1980; 19: 401–410. 45. Walker FJ, Sexton PW & Esmon CT. Inhibition of blood coagulation by activated protein C through selective inactivation of activated factor V. Biochimica et Biophysica Acta 1979; 571: 333 –342. 46. Amano K, Michnick DA, Moussalli M & Kaufman RJ. Mutation at either Arg336 or Arg562 in factor VIII is insufficient for complete resistance to activated protein C(APC)-mediated inactivation: implications for the APC resistance test. Thrombosis and Haemostasis 1998; 79: 557–563. 47. Nicolaes GAF, Tans G, Thomassen MC et al. Peptide bind cleavages and loss of functional activity during inactivation of factor Va and factorVaR506Q by activated protein C. Journal of Biological Chemistry 1995; 270: 21 158–21 166. * 48. Dahlba¨ck B. Resistance to activated protein C as a risk factor for thrombosis: molecular mechanisms, laboratory investigation, and clinical management. Seminars in Haematology 1997; 34: 217 –234. 49. Zivelin A, Griffin JH, Xu X et al. A single genetic origin for a common Caucasian risk factor for venous thrombosis. Blood 1997; 89: 397– 402. 50. Reitsma PH, Bernardi F, Doig RG et al. Protein C deficiency: a database of mutations, 1995 update. On behalf of the subcommittee on plasma coagulation inhibitors of the scientific and standardisation committee of the ISTH. Thrombosis and Haemostasis 1995; 73: 876–889. 51. Sehigsohn U, Berger A, Abend M et al. Homozygous protein C deficiency manifested by massive venous thrombosis in the newborn. New England Journal of Medicine 1984; 310: 559 –562. 52. Middlethorp S, Buller HR, Prins MH & Hirsh J. Approach to the thrombophilic patient. In Colman RW, et al (eds) Haemostasis and Thrombosis, Basic Principles and Clinical Practice, 4th edn, pp 1085– 1100, New York: Lippincott/Williams & Wilkins, 2001. 53. Da¨lhlback B & Stenflo J. High molecular weight complex in human plasma between Vitamin K-dependent protein S and complement component C4b-binding protein. Proceedings of the National Academy of Sciences of the USA 1981; 78: 2512– 2516. 54. Comp PC. Laboratory evaluation of protein S status. Seminars in Thrombosis and Haemostasis 1990; 16: 177–181. 55. Simmonds RE, Zoller B, Ireland H et al. Genetic and phenotypic analysis of a large (122 member), protein S deficient kindred provides an explanation for the familial coexistence of type I and type III plasma phenotypes. Blood 1997; 89: 4364–4370. 56. Biguzzi E, Merati G, Liaw PC et al. A 23bp insertion in the endothelial protein C receptor (EPCR) gene in patients with myocardial infarction and deep vein thrombosis. Thrombosis and Haemostasis 2001; 86: 945–948. 57. Pixley RA, Schapira M & Colman RW. The regulation of human factor XIIa by plasma proteinase inhibitors. Journal of Biological Chemistry 1985; 260: 1723–1729. 58. Scott CF, Schapira M, James HL et al. Inactivation of XIa by plasma protease inhibitors: predominant role of a-1-protease inhibitor and protective effect of high molecular weight kininogen. Journal of Clinical Investigation 1982; 69: 844– 852. 59. James K. Alpha2-macroglobulin and its possible importance in the immune system. Trends in Biochemical Science 1980; 5: 43. 60. Donaldson VH & Evans RR. Biochemical abnormality in hereditary angioneurotic edema. American Journal Medicine 1963; 35: 37. 61. Castellino FJ. Biochemistry of human plasminogen. Seminars in Thrombosis and Haemostasis 1984; 10: 18–23. 62. Robbins KC, Summaria L, Hseih B & Shah R. The peptide chains of human plasmin: mechanism of activation of human plasminogen to plasmin. Journal of Biological Chemistry 1967; 242: 2333. 63. Mandle RJ Jr & Kaplan AP. Hageman factor dependent fibrinolysis. Generation of fibrinolytic activity by the interaction of human factor XI and plasminogen. Blood 1979; 54: 850. 64. Colman RW. Activation of plasminogen or plasma kallikrein. Biochemical and Biophysical Research Communications 1979; 351: 273.
Blood coagulation 383 65. Colman RW, Bugdascorian A, Talamo RC et al. Williams trait: human kininogen deficiency with diminished levels of plasminogen proactivator and prekallikrein associated with abnormalities of the Hageman factor dependent pathways. Journal of Clinical Investigation 1975; 56: 1650. 66. Saito H, Ratnoff O, Waldmann A & Abraham JP. Fitzgerald trait: deficiency of a hitherto unrecognised agent Fitzgerald factor, participating in surface mediated reactions of clotting, fibrinolysis, generation of kinins and the property of diluted plasma enhancing vascular permeability (PF/dil). Journal of Clinical Investigation 1975; 55: 1082. 67. Camiolo SM, Thorsen S & Astrup T. Fibrinogenolysis and fibrinolysis with tissue plasminogen activator, urokinase, streptokinase-activated human globulin, and plasmin. Proceedings of the Society of Experimental Biology and Medicine 1971; 138: 277 –280. 68. Kaplan KL, Mather T, DeMarco L & Solomon S. Effect of fibrin on endothelial cell production of prostacyclin and tissue plasminogen activator. Arteriosclerosis 1989; 9: 43. 69. Levin EG, Marzec U, Anderson J & Harker LA. Thrombin stimulates tissue plasminogen activator and plasminogen activator inhibitor by cultured human endothelial cells. Journal of Clinical Investigation 1984; 74: 1988. 70. Amery A, Vermylen J, Maes H & Verstraete M. Enhancing the fibrinolytic activity of human blood by occlusion of blood vessels. Thrombosis Diathesis Haemorrhagica 1962; 7: 70. 71. Stahl A & Mueller BM. Binding of urokinase to its receptor promotes migration and invasion of human melanoma cells in vitro. Cancer Research 1994; 54: 3066–3071. 72. Dano K, Andreason PA, Grondahl-Hansen J et al. Plasminogen activators, tissue degradation and cancer. Advances in Cancer Research 1985; 44: 139–266. 73. Wiman B & Collen D. On the kinetics of the reaction between human antiplasmin and plasmin. European Journal of Biochemistry 1978; 84: 573. 74. Meijer P & Kluft C. The potency of the fibrinolytic system detected by a new assay for a2antiplasminplasmin complex determination in human plasma. Fibrinolysis 1992; 6(supplement 3): 94–96. * 75. Aoki N & Harpel PC. Inhibitors of the fibrinolytic enzyme system. Seminars in Thrombosis and Haemostasis 1984; 10: 24. 76. Chandler WL, Trimble SL, Loo SC et al. Effect of PAI-1 levels on the molar concentrations of active tissue plasminogen activator (tPA) and t-PA/PAI-1 complex in plasma. Blood 1990; 76: 930–937. 77. Delehrter TD & Sznycer-Laszuk R. Thrombin induction of plasminogen activator-inhibitor in cultured human endothelial cells. Journal of Clinical Investigation 1986; 77: 165. 78. Emeis JJ & Kooistra T. Interleukin-1 and lipopolysacharide induce an inhibitor of tissue type plasminogen activator in vivo and in cultured endothelial cells. Journal of Experimental Medicine 1986; 163: 1260. 79. Kruithof EK, Baker MS & Bunn CL. Biological and clinical aspects of plasminogen activator inhibitor 2. Blood 1985; 86: 4007–4024. 80. Bajzar L, Morzer J & Nesheim M. Purification and characterisation of TAFI, thrombin activatable fibrinolysis inhibitor. Journal of Biological Chemistry 1995; 270: 14 477. * 81. Wang P, Boffa MB, Bajzar L et al. A study of the mechanism of inhibition of fibrinolysis by activated thrombin activatable fibrinolysis inhibitor. Journal of Biological Chemistry 1998; 273: 27 176. 82. Bajzar L, Nesheim ME & Tracy PB. The profibrinolytic effect of activated protein C in clots formed from plasma is TAFI-dependent. Blood 1996; 88: 2093.
1 Blood coagulation Lucy A. Norris*
BA Mod. (Biochem) , MSc Mol Med , PhD
Research Biochemist Coagulation Research Laboratory, Department of Obstetrics and Gynaecology, Trinity College Centre for Health Sciences, St James’s Hospital, Dublin 8, Ireland
Blood coagulation can be initiated by two pathways: the extrinsic pathway, which is triggered by release of tissue factor from the site of injury, and the intrinsic system, which is stimulated by contact with a negatively charged surface. Following initial triggering, a series of serine proteases are sequentially activated, culminating in the formation of thrombin, the enzyme responsible for the conversion of soluble fibrinogen to the insoluble fibrin clot. Activation of coagulation is tightly regulated. Initiation by tissue factor is inhibited by tissue factor pathway inhibitor. Antithrombin can inactivate many of the serine proteases, including thrombin, by forming stable complexes which are rapidly cleared from the circulation. Protein C and protein S combine to inactivate coagulation factors V and VIII. The deposition of excess fibrin is prevented by the fibrinolytic system which can lyse fibrin into fibrin degradation products. Both genetic and environmental factors can influence the activation of coagulation and may predispose affected individuals to thrombosis. Key words: coagulation; thrombin; fibrin; thrombosis.
In a healthy individual, blood circulates as a liquid; however, the occurrence of vascular injury requires blood to gel rapidly to form a clot in order to prevent haemorrhage. When the endothelium becomes damaged, platelets adhere to the subendothelium and activation of the coagulation cascade causes the production of fibrin, which forms a mesh over the platelet plug, sealing the site of injury. In addition, chemokines facilitate the attraction of leukocytes to the area, which prevent infection and assist in wound healing. Although an almost instantaneous and explosive activation of the haemostatic system is required to prevent blood loss, the ability of blood to clot must be carefully regulated because inappropriate clot formation can lead to thrombotic complications. To achieve this, the normal healthy endothelium acts as an antithrombotic surface. Activation of the coagulation cascade is triggered by the release of tissue factor from the site of injury and, in the presence of pro-coagulant cell surfaces, production of fibrin can be achieved in seconds. The explosive activation of the haemostatic system is possible because of the so-called ‘cascade’ system of coagulation in which inactive zymogens and cofactors are sequentially activated by proteolytic cleavage. Almost immediately, the fibrinolytic system is stimulated—limiting fibrin deposition to the site of injury—and feedback in a system of naturally occurring anticoagulants feeds back and blocks further activation of the coagulation cascade. * Tel.: þ 353-1-6082116; Fax: þ353-1-4531614. E-mail address: [email protected] (L. A. Norris). 1521-6934/03/$ - see front matter Q 2003 Elsevier Science Ltd. All rights reserved.
370 Lucy A. Norris
Although thrombosis is a multifactorial disease, dysregulation of the haemostatic pathways is a major contributor to thrombus formation. A number of genetic and environmental factors can cause excessive upregulation of coagulation enzymes. This upregulation may overwhelm the anticoagulant response—particularly in those individuals in whom one or more of the key anticoagulant pathways is impaired.
INITIATION OF BLOOD COAGULATION Traditionally, two main pathways have been described for blood coagulation: the intrinsic or contact pathway and the extrinsic or tissue factor pathway (Figure 1). Although originally these pathways were thought to be mutually exclusive and of equal importance, recent evidence disputes this. The intrinsic pathway can be activated when blood comes into contact with a negatively charged surface resulting in the activation of factor XII to XIIa. However, deficiencies in factor XII are not associated with bleeding and may even be associated with thrombosis.1 In addition, the activation of factor IX can be achieved by both the extrinsic and intrinsic pathways. The main function of the intrinsic pathway may be to amplify the coagulation activation triggered by the tissue factor pathway. Extrinsic/tissue factor pathway The tissue factor pathway is triggered when the membrane-bound protein tissue factor (TF) comes into contact with plasma containing factor VII or VIIa. Unlike other members of the coagulation cascade, tissue factor is always present as an active cofactor and is thought to be essential for life.2 Tissue factor is not normally expressed in cells that come into contact with plasma; however, upon vascular injury, cells expressing membrane-bound tissue factor are exposed to plasma and can bind factor VII. In addition, monocytes and smooth muscle cells can be stimulated to produce tissue factor by cytokines and other inflammatory mediators.3 The binding of factor VII to tissue factor leads to the formation of TF-VIIa which, when attached to the cell membrane, becomes the most potent activator of the coagulation cascade known. Factor VII is a typical vitamin-K-dependent plasma protein produced in the liver. Although almost 99% of factor VII is present in its inactivated state, 1% is present as an active serine protease, factor VIIa.4 However, when factor VIIa is free and not part of the TF-VIIa complex, it is a relatively weak enzyme. Increased factor VII has been reported as a risk factor for thrombotic disease5,6; however, this relationship is dependent on the method of measuring factor VII levels.7 Factor VII deficiency is a rare event and individuals with severe deficiency present with major life-threatening bleeding episodes.8 Factor VII is also influenced by polymorphisms in the factor VII gene; mutations in the promoter region and a GLN/ARG substitution in the protease domain have been associated with lower levels of factor VII, giving rise to the suggestion that factor VII genotype may influence thrombotic risk9—but this has not been shown conclusively.10 Once formed, the TF-VIIa complex on the cell surface has two potential substrates: factor IX, which is converted to IXa, and factor X, which is converted to Xa. In vitro,
INTRINSIC PATHWAY EXTRINSIC PATHWAY TF
Negatively charged surface XII
XI
X
IX
HMWK
XIIa
VII
TF-VIIa
HMWKa XIa
TFPI
TFPI
CI-inh IXa Ca PL
Tenase complex
Prothrombin VIIIa
AT
AT Xa Va
VIII Prothrombinase
PL V
Thrombin
Fibrinogen
Fibrin
Figure 1. The coagulation cascade showing both intrinsic and extrinsic activation, inhibitors and feedback activation (dashed lines). HMWK ¼ high molecular-weight kininogen. C1-inh ¼ C1-inhibitor. TF ¼ tissue factor. TFPI ¼ tissue factor pathway inhibitor. PL ¼ phospholipids. Ca ¼ calcium. AT ¼ antithrombin.
Blood coagulation 371
AT
372 Lucy A. Norris
factor X is the preferred substrate of TF-VIIa; however, the predominant pathway in vivo is unknown and may depend on local factors.4 Intrinsic system The intrinsic or contact pathway can also activate factor IX. The binding of factor XII (Hageman factor) to an artificial or negatively charged surface leads to a local increase in concentration and results in the autoactivation of factor XII to XIIa. Once assembled on a negatively charged surface, XIIa causes the conversion of prekallikrein to kallikrein and factor XI to XIa.11 Cleavage of high-molecular-weight kininogen (HMWK) (a protein with diverse biological properties) also occurs. These changes culminate in the formation of factor IXa.12 Although in vitro defects in the intrinsic pathway manifest as a prolongation of partial thromboplastin time, in vivo, deficiencies in these factors do not cause bleeding. The exception to this is factor XI, which causes mild bleeding following trauma or injury.13 The observation that thrombin can also activate factor XI suggests that the main role of factor XI is not in the initiation of coagulation but is in the secondary production of thrombin which is essential for haemostasis.14 The Leiden thrombophilia study showed that increased levels of factor XI is a risk factor for thrombosis: it suggested that the role of factor XI was twofold— secondary generation of thrombin and downregulation of fibrinolysis via thrombinactivatable fibrinolysis inhibitor (TAFI).15 Although the proteins of the intrinsic pathway are often considered biologically irrelevant, more recent studies have suggested that these proteins have diverse biological roles throughout the vascular system. Prekallikrein and HMWK are involved in the regulation of blood pressure and participate in fibrinolysis. Factor XII can activate neutrophils and upregulate the release of cytokines from monocytes and macrophages.16
FORMATION OF THE ‘TENASE’ COMPLEX When factor IX is activated to IXa by either the intrinsic or extrinsic pathway, it forms a complex with factor VIIIa, calcium and phospholipids which can activate factor X to Xa. This complex is sometimes known as the ‘tenase’ complex and is crucial to haemostasis. Absence of factor VIII or factor IX produce the same haemorrhagic disease known as haemophilia, the severity of which is related to the degree of deficiency of factor VIII or IX. Factor VIII exists in plasma mostly as a non-covalent complex with von Willebrand factor (vWF) which protects it from proteolytic activation to VIIIa. vWF binding to platelets attached to the surface of damaged endothelium facilitates activation of factor VIII to VIIIa and dissociation of vWF.17 Thrombin can also feedback and activate factor VIII, a process that is not prevented by vWF.18
CONVERSION OF PROTHROMBIN TO THROMBIN The final step in producing the active thrombin enzyme is the formation of the prothrombinase complex. This consists of factor Xa bound to factor Va, its
Blood coagulation 373
non-enzymatic cofactor, calcium and a phospholipid membrane surface. In the presence of anionic phospholipids, inactive factor V is converted to Va. Thrombin may also activate factor V, as can the tissue factor-VIIa complex.19 Factor V mutations are the commonest inherited risk factors for thrombosis; many of these mutations—including the common factor V Leiden polymorphism—affect the inactivation of factor V by the inhibitor, protein C.20 Factor X is a vitamin-K-dependent coagulation factor which can be converted to the active serine protease, factor Xa, by the tissue factor-VIIa complex, or by the tenase complex. Because the direct activation of factor X by TF-VIIa is rapidly downregulated by the tissue factor pathway inhibitor (TFPI), the tenase complex is the most important activator of factor X, which explains the clinical severity of the haemophilias. Although factor Xa alone can catalyse the conversion of prothrombin to thrombin, the reaction is very slow and is greatly accelerated by the addition of factor Va and the binding of the complex to the phospholipid surface of either activated platelets or monocytes.21 The tenase complex converts inactive prothrombin to a-thrombin, a serine protease. Cleavage of prothrombin to thrombin occurs via a number of steps. Initial cleavage occurs at Arg320 to give rise to the intermediate product meizothrombin. Cleavage at Arg271 produces the active enzyme a-thrombin and a by-product, prothrombin fragment 1.2.22 As circulating thrombin concentration is difficult to measure, the more stable prothrombin fragment 1.2 is frequently used as a marker of prothrombin-tothrombin conversion and is an indicator of a hypercoagulable state.23 A mutation in the 30 untranslated region of the prothrombin gene (G20120-A) results in elevated plasma prothrombin levels and has been reported as a risk factor for thrombosis.24 Thrombin has many functions within haemostasis. It can convert fibrinogen to fibrin and is also capable of activating platelets, coagulation factors V, VIII and IX, and the inhibitors protein C and thrombin-activatable fibrinolysis inhibitor (TAFI).25 This facility is possible because of a number of distinct binding sites on the thrombin molecule and because of the ability of the enzyme to bind to cofactors which can render it procoagulant or anticoagulant.26
FIBRIN FORMATION The formation of fibrin is the final stage of the coagulation process. Soluble fibrinogen is converted into an insoluble fibrin polymer, which seals the site of injury and protects damaged tissue during wound healing. This tightly controlled process occurs in several stages. First, the dimeric fibrinogen molecule is cleaved by thrombin to produce soluble fibrin monomers, fibrinopeptide A and fibrinopeptide B. Fibrinopeptide A is frequently used as an early marker of fibrinogen-to-fibrin conversion.23 Non-covalent interactions result in the formation of fibres or strands which then aggregate to form a mesh. Finally, the newly formed fibrin is stabilized by cross-linking catalysed by thrombin-activated coagulation factor XIIIa.27 Although fibrinogen plays a key role in coagulation, it is also important in other cellular processes. Soluble fibrinogen acts as a signalling molecule and is important in adhesive processes required for the trafficking of immune cells required during wound healing.28 In addition, both fibrinogen and fibrin play a role in tumour genesis by promoting angiogenesis essential for tumour growth.29
374 Lucy A. Norris
REGULATION OF BLOOD COAGULATION The regulation of blood coagulation is essential to avoid a generalized activation of the system and massive fibrin deposition. To be effective, the system must be active only at a local site of vascular injury and must remain active only for a sufficient period of time to produce enough fibrin to seal the wound. To achieve this, a number of regulatory mechanisms are in place. As described above, each coagulation protein is sequentially activated, so that only a small proportion of an active serine protease is available at any given time. Second, coagulation activation and formation of the complexes above can proceed only on the surface of activated cells and platelets—where negatively charged phospholipids are exposed and the appropriate receptors are available for binding. Similarly, the essential trigger for the cascade, tissue factor, is available only on the surface of activated monocytes and cells which are exposed to plasma only as a result of vascular injury. The most important regulatory pathways, however, are a series of anticoagulant proteins and cofactors which bind to activated coagulation factors and limit their period of activity. Finally, in the presence of fibrin, fibrinolysis is stimulated which dissolves fibrin into fibrin degradation products. Tissue factor pathway inhibitor (TFPI) The initial trigger for coagulation activation is the TF-VIIa complex which is a potent activator of both factor IX and X. For this reason, the activity of this complex must be quickly inactivated. This is achieved by a specific inhibitor of the complex, tissue factor pathway inhibitor (TFPI), released constitutively from endothelial cells. TFPI is a multidomain inhibitor. Initially it forms a complex with Xa through one of its domains and the complex can then bind to the TF-VIIa complex with another binding domain to form a quaternary complex.30 The effect is rapid inhibition of extrinsic activation of the cascade. In addition TFPI also promotes the internalization and subsequent degradation of the inhibited complex.31 In some model systems, pharmacological agents which block tissue factor have been shown to be effective in reducing thrombosis without the bleeding complications associated with other antithrombotic drugs. This suggests that the TF-VIIa may be a useful target for the design of antithrombotic drugs. Recent preliminary studies have shown that TFPI may be an effective agent against thrombosis and may reduce mortality in sepsis patients.4 Antithrombin Antithrombin is a serine protease inhibitor that can inhibit many of the activated coagulation enzymes. Key components of the cascade, such as factors IXa, Xa, TF-VIIa complex and thrombin, are rapidly bound by antithrombin and neutralized. The ability of antithrombin to inhibit these factors is greatly accelerated by heparin sulphate proteoglycans and this is the basis for the anticoagulant action of the pharmaceutical heparins. Antithrombin inhibits free thrombin and Xa more efficiently than thrombin and Xa bound to activation complexes; this has the effect of removing these proteases from the general circulation and confining their activity to the site of clot formation. Thrombin bound to antithrombin forms the stable thrombin –antithrombin (TAT) complex, which is rapidly cleared from the circulation.32 TAT complex levels are also used as a marker of hypercoagulability because they reflect thrombin production.33
Blood coagulation 375
Because antithrombin plays an essential role in controlling coagulation activity, even modest deficiencies in the levels of activity can cause a clinical problem. An antithrombin concentration below 60 – 70% is associated with thrombosis.34,35 Approximately 5% of patients with venous thrombosis are found to be antithrombin deficient, and both inherited and acquired deficiencies have been described.36 The protein C anticoagulant pathway The protein C anticoagulant pathway is thought to be the major mechanism by which thrombosis in the microcirculation is prevented. Protein C is a vitamin-K-dependent plasma protein which circulates as an inactive zymogen.37 To become activated (Figure 2), it must first bind via its GLA domain to a transmembrane receptor expressed on the endothelium called the endothelial protein C receptor (EPCR).38 The activation of protein C also requires thrombin. Free thrombin must first bind to another transmembrane protein, thrombomodulin, which is expressed constitutively by endothelial cells.39 This protein tethers thrombin to the endothelium where EPCR presents protein C for activation. Thrombin complexed to thrombomodulin activates protein C, possibly through a conformational change in the thrombin enzyme.40 Activated protein C (APC) is inactive as an inhibitor while it remains bound to the EPCR.41 To become active, APC must dissociate from EPCR and bind to its cofactor protein S. Protein S is also a vitamin-K-dependent plasma protein; however, unlike protein C, it has no enzymatic activity of its own; the main function of protein S is to act as a cofactor for APC.42 The protein S/APC complex can inactivate both factor Va and factor VIIIa, effectively shutting down prothrombinase and tenase activity respectively.43 Thus thrombin, by activating the protein C pathway, shuts down its own production. APC/protein S binds preferentially to factors Va and VIIIa rather than their inactive precursors.44,45 In the case of VIIIa, inactivation by APC can occur as a result of cleavage of Arg336 and Arg562.46 The inactivation of factor Va involves cleavage at Arg506 and Arg306 and is a two-phase process. The initial rapid phase of factor Va inactivation is due to cleavage at Arg506. Cleavage of factor V results in a 50-fold decrease in affinity for Xa and a drastic reduction in prothrombinase activity. A second, slower phase corresponds to cleavage at Arg306.47 In patients with the factor V Leiden mutation, the arginine amino acid residue at position 506 is replaced by glutamine.48 The effect of this mutation is to produce factor Va which cannot be completely inactivated by APC, hence the term ‘APC resistance’ to describe the phenotype of this mutation.48 APC resistance is the most common inherited risk factor for venous thrombosis currently known. In the general population, the mutation appears to be limited to Caucasians and occurs in 2 –15% of Europeans.49 Inherited deficiencies have also been described for protein C and protein S. Over 160 mutations have been described for the protein C gene, most of which lead to absent or defective protein C.50 However, in affected individuals, protein C mutations are rarely categorized and patients are assumed to be heterozygous if they produce 40 –60% of normal activity from the unaffected allele. Homozygous protein C deficiency is a serious condition leading to life-threatening thrombotic complications frequently occurring immediately after birth.51 Mutations in the protein S gene also lead to a deficiency in protein S; these deficiencies account for between 2 and 5% of patients with a history of thrombosis.52 Protein S circulates in two forms: free unbound protein S and protein S bound to C4bbinding protein.53 Based on plasma measurements, three different types of protein S deficiency of clinical importance have been defined. Type 1 deficiency is defined as
376 Lucy A. Norris
PC
T
T
TM Cell membrane
E P C R
E P TM PC C R
APC
E P C R
PS PS
Vi
VIIIi
Va PS APC
VIIIa PS APC
Figure 2. Schematic representation of protein C activation. T ¼ thrombin. TM ¼ thrombomodulin. PC ¼ protein C. EPCR ¼ endothelial protein C receptor. PC ¼ protein C. PS ¼ protein S. Vi ¼ inactivated factor Va. VIIIi ¼ inactivated factor VIIIa. APC ¼ activated protein C.
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a reduction in free and total protein S. Patients with type II deficiency have normal protein S antigen levels but reduced functional activity, suggesting that an abnormal protein is being produced. The third type shows normal total protein S levels with a reduction in free unbound protein S only.54 However, assays for protein S are influenced by a number of factors so that differences in phenotype may not always be explained by differences in genotype. 55 Recent studies have suggested that polymorphisms in the thrombomodulin gene may influence the risk of coronary heart disease in African Americans; however, larger studies are required to confirm this.39 Similarly, a 23-base-pair insertion in the EPCR gene has been identified; however, the significance of this mutation remains to be determined.56
Inhibitors of the intrinsic system The major inhibitor of the intrinsic system is C1-inhibitor, which inhibits factor XIIa. Binding of C1 inhibitor irreversibly inactivates at least 90% of XIIa; however, when XIIa is bound to a kaolin surface it is protected from C1-inhibitor activation.57 a1Antitrypsin can inhibit factor XIa and acts in a similar manner to antithrombin, forming a stable complex with its target serine protease.58 a2-Macroglobulin is a broad-spectrum proteinase inhibitor which acts as a secondary inhibitor of many proteinases involved in the coagulation cascade, including kallikrein and thrombin and the fibrinolytic enzyme plasmin.11 Deficiencies in these inhibitors do not cause thrombosis, partly because of the secondary nature of the intrinsic system and partly because antithrombin can inhibit almost all of the serine proteases involved in the coagulation cascade. As with other components of the intrinsic system, these inhibitors have functions outside the haemostatic system—for example, a2-macroglobulin is thought to be involved in the regulation of the immune system59, and deficiencies in C1-inhibitor are associated with hereditary angiodema.60
FIBRINOLYSIS Fibrinolysis is the final control mechanism that limits clot formation. The fibrinolytic system is a series of enzymes which, when activated, cleave fibrin into fibrin fragments known as fibrin degradation products (Figure 3). The control of this mechanism is crucial to maintaining haemostatic balance.
Plasminogen and plasmin The enzyme responsible for lysis of fibrin to fibrin degradation products is plasmin. This enzyme circulates as an inactive zymogen, plasminogen.61 Plasminogen can bind to fibrin via lysine binding sites on the heavy-chain portion of the plasminogen molecule; the subsequent binding of a plasminogen activator leads to cleavage of the plasminogen molecule at the Arg561 – Val bond and the formation of the active protease plasmin.62 The presence of plasminogen activators and their inhibitors is essential in controlling fibrinolysis.
378 Lucy A. Norris
PLASMINOGEN PAI-I tPA
Fibrin
uPA
HMWK XIIa Xla PLASMIN
Antiplasmin
α1 - antitrypsin α2 - macroglobulin Antithrombin FDPs
FIBRIN TAFI
Figure 3. The fibrinolytic system. tPA ¼ tissue plasminogen activator. uPA ¼ urokinase plasminogen activator. PAI-1 ¼ plasminogen activator inhibitor 1. HMWK ¼ high-molecular-weight kininogen. TAFI ¼ thrombin activatable fibrinolysis inhibitor. FDPs ¼ fibrin degradation products.
Plasminogen activators There are several pathways of plasminogen activation. Factor XIIa, XIa and kallikrein are all capable of converting plasminogen to plasmin.63,64 The contribution which these enzymes makes to the activation of plasminogen in vivo is uncertain, however, because deficiencies in these proteins do not appear to lead to pathological states that could be explained by impaired fibrinolysis.65,66 The major activator of plasminogen in vivo is tissue plasminogen activator (tPA), a serine protease produced by endothelial cells. In the absence of fibrin, it is an inefficient activator of plasminogen but once bound to fibrin, activation is greatly accelerated.67 Local release of tPA from the vascular endothelium in the vicinity of an injury may be stimulated by fibrin itself, by thrombin attached to the formed clot, or by the effects of venous occlusion.68 – 70 Urokinase or urinary type plasminogen activator (uPA) can also activate plasminogen. The importance of uPA is in tissues where it plays a role in the degradation of the extracellular matrix. This facilitates the migration of cells which is important in wound healing and in tumour invasion and metastasis.71,72
Blood coagulation 379
Inhibitors of fibrinolysis The primary inhibitor of active plasmin is a2-antiplasmin. In plasma, a2-antiplasmin binds rapidly to plasmin to form an irreversible stable complex, plasmin –antiplasmin complex.73 This complex can be measured in plasma and is sometimes used as a marker for in vivo plasmin production.74 Free plasmin is much more rapidly bound to a2-antiplasmin than plasmin attached to fibrin because both utilize lysine binding sites.73 a2-Macroglobulin plays a limited role as a plasmin inhibitor, becoming important only when the concentration of plasmin exceeds the capacity of a2-antiplasmin. Similarly, antithrombin, a1-antitrypsin and C1-inactivator have been shown to inhibit plasmin in vitro but have a minimal physiological effect in blood.75 Inhibitors of plasminogen activator play an important role in regulating fibrinolysis. Currently, four distinct types have been described: PAI-1, PAI-2, PAI-3 and protease nexin. Of these, PAI-1 is the most important in inhibiting tPA in plasma. Synthesis of PAI1 occurs primarily in endothelial cells and is one of the most regulated proteins in humans. Stimulation of PAI-1 release is caused by a large variety of compounds, including thrombin and endotoxin.76,77 PAI-1 is present in molar excess over tPA, hence the majority of tPA circulates bound to PAI-1; the effect of this is to prevent premature lysis of fibrin in the forming clot and to inhibit systemic fibrinolysis.78 PAI-2 is found only in the plasma of pregnant women and is produced in the placenta and also in some tumour cells. The main function of PAI-2 is unclear; however, it may play a role in the regulation of limited proteolysis in some tissues.79 The most recently described fibrinolysis inhibitor is thrombin-activatable fibrinolysis inhibitor (TAFI), also known as plasma carboxypeptidase B.80 Thrombin, when bound to thrombomodulin, can activate TAFI—which can inhibit fibrinolysis by several mechanisms. First, activated TAFI cleaves the COOH terminal end of fibrin which reduces the ability of fibrin to facilitate plasminogen activation via tPA. TAFI can also directly inhibit plasmin activity.81 TAFI can also be cross-linked to fibrin and incorporated into a fibrin clot, which prevents premature lysis. Inhibitors such as APC, which prevent prothrombin-to-thrombin conversion, inhibit the activation of TAFI; this explains the apparent pro-fibrinolytic activity of APC.82
SUMMARY AND CONCLUSIONS The maintenance of haemostasis is essential to avoid bleeding and thrombosis. This is achieved by the sequential and short-lived activation of the coagulation cascade series of enzymes, ultimately resulting in the production of an insoluble fibrin clot. Both genetic and environmental factors can cause alterations in the level of activation of these enzymes, and for this reason a number of fail-safe inhibitory mechanisms are required if thrombosis is to be prevented. These pathways remove any excess procoagulant enzymes and prevent systemic fibrin production. Fibrinolysis inhibitors are also essential to prevent inappropriate breakdown of fibrin and fibrinogen. Both thrombin and fibrin orchestrate their own demise, thrombin by triggering the activation of protein C and fibrin by stimulating plasmin production and fibrinolysis. An increasing knowledge of the genetic basis of many of the inherited disorders of the haemostatic system has enhanced our understanding of the mechanisms by which coagulation is regulated and controlled. Within the last 10 years, the major cause of venous thrombosis in the microcirculation, the factor V Leiden mutation, was discovered; the next decade may bring further discoveries of this nature. Although
380 Lucy A. Norris
Practice points † factors such as pregnancy, use of hormones and surgery cause coagulation activation which may predispose an individual to thrombosis † factor V Leiden is the leading cause of thrombosis in the microcirculation † an antithrombin level of 60 –70% or less of normal lines is associated with an increased risk of thrombosis
Research agenda † all hormonal preparations cause changes in coagulation activation and inhibition in healthy women; however, only a small proportion develop thrombosis. Factor(s) as yet undiscovered may help to identify which women are likely to develop thrombosis while taking hormone preparations † many of the coagulation genes are polymorphic; however, the significance of these polymorphisms in relation to thrombosis risk in many cases is unknown. Similarly, combinations of polymorphisms may be predictive for thrombosis under certain environmental conditions † thrombin and fibrinogen not only function in the coagulation system but are also signalling molecules. Study of the interaction between the immune system and coagulation serine proteases may increase understanding of the mechanisms of coagulation activation and the pathogenesis of thrombosis a large number of thrombotic events can be explained by inherited or acquired thrombophilia, the pathogenesis of this disease in a significant number of patients remains unexplained. The interaction between environmental factors and polymorphisms of coagulation activators and inhibitors may provide further clues to the mechanisms by which thrombosis occurs.
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