1 Comment


Williams Hematology



Blood Coagulation Pathways and Protein C Anticoagulant Pathway
Hereditary Deficiencies Associated with Thrombotic Disease

Protein C Pathway Components

Protein C

Protein C Gene

Protein C Mutations

Protein S


Endothelial Protein C Receptor

Expression of Activated Protein C Activity
Inhibition of Coagulation Proteases


Tissue Factor Pathway Inhibitor

Other Protease Inhibitors
Chapter References

Normally, the blood coagulation system is active, but idling, and is poised for explosive generation of thrombin. The requirement for positive feedback activation of clotting factors (e.g., factors V, VIII, XI, and VII) imparts special threshold properties to the blood coagulation pathways, making the coagulant response nonlinearly responsive to stimuli. Analysis of blood coagulation as a threshold system suggests all-or-none responses to various levels of stimuli, depending on the ensemble of reactions that determine up-regulation and down-regulation of thrombin generation. Because of synergies between cellular and humoral anticoagulant mechanisms, the presence of multiple coagulation inhibitors with complementary modes of action prevents massive thrombin generation in the absence of a substantial procoagulant stimulus. This chapter highlights plasma mechanisms that inhibit blood coagulation, with an emphasis on mechanisms whose defects cause hereditary thrombophilias. The majority of hereditary defects involves the anticoagulant protein C pathway, which includes protein C and protein S as anticoagulant factors, thrombomodulin and endothelial protein C receptor as cofactors for activation of protein C, and factors Va and VIIIa as substrates for activated protein C. Variant factor V containing Gln506 in place of Arg506 causes activated protein C resistance by impairing the efficiency of the protein C pathway. Plasma protease inhibitors are essential to block clotting proteases. Antithrombin neutralizes all proteases of the intrinsic coagulation pathway, including thrombin and factors Xa, IXa, XIa, and XIIa, in reactions stimulated by glycosaminoglycans. Tissue factor pathway inhibitor neutralizes the extrinsic coagulation pathway factors VIIa and Xa. Other plasma protease inhibitors can also neutralize various coagulation proteases, although the clinical significance of these reactions is less apparent than the reaction of antithrombin with thrombin.

Acronyms and abbreviations that appear in this chapter include: APC, activated protein C; C4BP, C4b-binding protein; DIC, disseminated intravascular coagulation; EPCR, endothelial cell protein C receptor; EPI, extrinsic pathway inhibitor; Gla, g-carboxyglutamic acid; HDL, high-density lipoprotein; LACI, lipoprotein-associated coagulation inhibitor; SHBG, sex-hormone-binding globulin–like region; TFPI, tissue factor pathway inhibitor; previously also described as, TSR, thrombin-sensitive region.

Control of coagulation reactions is essential for normal hemostasis. As part of the tangled web of host defense systems that respond to vascular injury, the blood coagulation factors act in concert with the endothelium and platelets to generate a protective fibrin-platelet clot, forming a hemostatic plug. Pathologic thrombosis occurs when the protective clot is extended beyond its beneficial size, when a clot occurs inappropriately at sites of vascular disease, or when a clot embolizes to other sites in the circulatory bed. For normal hemostasis, both procoagulant and anticoagulant factors must interact with the vascular components and cell surfaces, including the vessel wall (see Chap. 114) and platelets (see Chap. 111 and Chap. 112). Moreover, the action of the fibrinolytic system must be integrated with coagulation reactions for timely formation and dissolution of blood clots (see Chap 116). For extensive discussion of regulation of coagulation, the reader is referred to recent books that focus only on hemostasis and thrombosis.1,2 This chapter on control of coagulation highlights the major physiologic mechanisms for down-regulation of blood coagulation reactions and the plasma proteins that inhibit blood coagulation, with an emphasis on those mechanisms whose defects are clinically significant based on insights gleaned from consideration of the hereditary thrombophilias (see Chap 127).
Although more than 40 years have elapsed since the elaboration of the cascade model for blood coagulation pathways,3,4 the basic outline of sequential conversions of protease zymogens to active serine proteases is still useful, albeit with important modifications, to represent blood coagulation reactions (Fig. 113-1). The major conceptual advances in the past two decades emphasize both positive and negative feedback reactions as depicted in Fig. 113-1.

FIGURE 113-1 Blood coagulation and protein C pathways. Thrombin can be either procoagulant (left) or anticoagulant (right) depending on cofactors and surfaces. Coagulant thrombin clots fibrinogen and activates platelets, factor V, factor VIII, factor XI, and factor XIII. Conversion of zymogen protein C to anticoagulant APC by thrombomodulin-bound thrombin is enhanced by endothelial protein C receptor (EPCR). APC with its nonenzymatic cofactor, protein S, inactivates factors Va and VIIIa by highly selective proteolysis (e.g., at Arg506 and Arg306 in factor Va), yielding inactivated (i) factors Vi and VIIIi. This anticoagulant action may be enhanced by platelets, endothelial cells, or their microparticles. HDL can also provide protein S-dependent anticoagulant APC-cofactor activity. The direct actions of protein S inhibiting generation of thrombin by directly reacting with factors Va and Xa are also indicated. Adapted from Griffin with permission.308

In positive feedback reactions, procoagulant thrombin activates platelets and factors V, VIII, and XI5,6,7,8,9 and 10 (see Chap. 112). Small amounts of thrombin can be generated by trace amounts of tissue factor via the extrinsic pathway. Subsequently, thrombin can activate factors XI, VIII, and V, thereby stimulating each of the steps in the intrinsic pathway and thus amplifying thrombin generation. In negative feedback reactions that involve the protein C pathway, binding of thrombin to thrombomodulin converts the bound thrombin to an anticoagulant enzyme that converts the protein C zymogen to the anticoagulant serine protease, activated protein C (APC) (Fig. 113-1). This surface-dependent reaction is enhanced by the endothelial cell protein C receptor (EPCR).7,11,12 In a subsequent negative feedback loop, APC with the aid of its nonenzymatic cofactor, protein S, inactivates factors Va and VIIIa by highly selective proteolysis, yielding inactive (i) cofactors, factors Vi and VIIIi. Protein S also can directly inhibit factors VIIIa, Xa, and Va.13,14,15,16,17,18 and 19 Thus, APC and protein S inhibit multiple steps in the intrinsic coagulation pathway. At each step in the coagulation pathways, each clotting protease can be inhibited by one or more protease inhibitors. Given the highly nonlinear nature of the coagulation pathways with both positive and negative feedback reactions, synergy between the protein C pathway and plasma protease inhibitors is important for regulating thrombin generation.
Plasma from all normal subjects contains circulating active enzymes, factor VIIa,20,21 and APC,22 as well as various polypeptide fragments generated by the action of clotting proteases, namely fibrinopeptides,23,24 prothrombin fragment 1+2,25 and activation peptides for factors IX and X.26,27 Thus, there is continuous activation of coagulation factors at a basal physiologic low level. The presence of multiple clotting factors that require positive feedback activation (e.g., factors V, VIII, XI, and VII) imparts special threshold properties to the blood coagulation pathways, making the coagulant response nonlinearly responsive to stimuli. Theoretical analysis of blood coagulation as a threshold system suggests there can be an all-or-none response to various levels of stimulation, depending on the ensemble of activating and inhibitory reactions that defines up-regulation and down-regulation of thrombin generation.28,29 It appears that the coagulation system is active, but idling, and is poised for extensive and explosive generation of thrombin. Because of synergy among various cellular and humoral anticoagulant mechanisms that establish a threshold system, the presence of multiple coagulation inhibitors with complementary modes of action prevents massive thrombin generation in the absence of a substantial procoagulant stimulus.
Evidence for the physiologic importance of specific factors for controlling coagulation reactions comes from clinical observations and animal model studies. Major identified genetic risk factors for venous thrombosis involve protein structural defects in factor V, protein C, protein S, and antithrombin. There are also gene regulatory defects associated with thrombotic disease such as the nt G20210A polymorphism in the prothrombin gene that causes elevated levels of prothrombin and the defects in protein C regulatory elements that decrease the expression of protein C (see Chap. 127). Deficiencies of thrombomodulin are less well established as clinically significant, but they may be associated with increased risk of arterial thrombosis (see Chap 127). Hereditary abnormalities of EPCR have not yet been definitively linked to increased risks of thrombosis.
Schematic representations of the structures of protein C, protein S, thrombomodulin, and EPCR are shown in Fig. 113-2. These proteins contain multiple domains, each of which may mediate different molecular functions. Values for the molecular weight, normal plasma concentration, chromosomal location, and gene structures of these factors are given in Table 113-1. Factors Va and VIIIa, as substrates of APC, are also participants in the reactions of the protein C pathway. Moreover, certain forms of factor V can also act as an APC cofactor,30,31 as detailed below.

FIGURE 113-2 Membrane-bound protein C, protein S, thrombomodulin, and the endothelial cell protein C receptor (EPCR). Each protein is a multidomain protein that extends above the surface of cell membranes, and different domains mediate different functions of each protein. Protein C and protein S can bind reversibly to phospholipid membranes through their NH2-terminal domains which contain 9 or 11 g-carboxyglutamic acid (Gla) residues that bind 4 to 6 Ca2+ ions. Thrombomodulin and EPCR are integral membrane proteins that are embedded in cell membranes by a single hydrophobic transmembrane sequence. Adapted from Esmon with permission.108


Protein C
In 1976, Stenflo designated a bovine plasma vitamin-K-dependent protein that eluted in the third peak (peak C) from an anion exchange column as bovine “protein C.”32 Protein C was found to be identical to a previously identified anticoagulant factor, autoprothrombin II-A.33,34 Biochemical studies showed that protein C is a zymogen that can be isolated from either bovine or human plasma and that can be converted to an anticoagulantly active serine protease by the action of thrombin.35,36
Protein C is synthesized in the liver as a polypeptide precursor of 461 residues, with a prepropeptide of 42 amino acids that contains the signal for carboxylation of Glu residues by a carboxylase that forms 9 g-carboxyglutamic acid (Gla) residues and secretion of the mature protein.37,38,39 and 40 The mature glycoprotein of Mr 62,000 contains 419 residues (Fig. 113-2) and N-linked carbohydrate, and the majority of the secreted protein C molecules are cleaved by a furin-like endoprotease that releases Lys156-Arg157 and generates a two-chain zymogen that circulates in plasma at 70 nM (4 µg/ml).41,42 and 43 The heavy and light chains of plasma protein C are covalently linked by a disulfide bond that keeps the serine protease globular domain (residues 170–419) covalently tethered to the N-terminal string of three domains, the Gla domain and the epidermal growth-factor-like domains EGF1 and EGF2 (Fig. 113-2).37,38,39 and 40,44,45 and 46
The Gla domain of protein C (residues 1–42) and of APC is important for a number of functions, including binding of these proteins to phospholipid-containing membranes, thrombomodulin, and EPCR; thus, incomplete carboxylation impairs the functional anticoagulant activity of APC.47,48,49,50,51 and 52 The EGF1 domain undergoes an unusual posttranslational modification resulting in b-hydroxyaspartic acid at residue 71, and this modification appears essential for full anticoagulant activity.53,54 The two EGF modules in the light chain may also contribute to interactions of APC with protein S and of protein C with thrombomodulin. The C-terminus of the light chain is implicated in binding of APC to its substrate, factor Va.55
The serine protease domain of protein C is homologous to other chymotrypsin-like proteases, and three-dimensional modeling56,57 and x-ray crystallographic structures46 reflect the structural similarity of APC to members of the serine protease family. The serine protease domain of APC exerts its trypsin-like anticoagulant activity by highly specific interactions with factors Va and VIIIa followed by cleavage at only two Arg-containing peptide bonds (see below). APC residues 390 to 404 and 311 to 325 appear to contribute to specific recognition of factor Va.58,59
Purified protein C concentrates have been successfully used to treat patients with thrombotic episodes.60,61,62 and 63
The protein C gene, comprising nine exons and eight introns, is located on chromosome 2q14-21 and spans 11 kb (see Fig. 112-12, Table 113-1).64,65,66,67,68 and 69 The protein C gene is homologous to the genes for factors VII, IX, and X (see Chap. 112).
Many different mutations in the protein C gene have been identified that cause protein C deficiency associated with thrombosis, and a database of more than 100 different mutations was published.70 Based on three-dimensional models of the protease domain of protein C, the structural basis for protein C defects has been rationalized.57,71,72 Most mutations that cause type I protein C deficiency, characterized by parallel reductions in activity and antigen, involve amino acid residues that form the hydrophobic cores of the two folded globulin-like domains that are characteristic of serine proteases. These mutations destabilize either the process or the product of protein folding, and they result in unstable molecules that are poorly secreted and/or exhibit a very short circulatory half-life. In contrast, most mutations that cause type II defects (reduced activity but normal antigen levels), i.e., circulating dysfunctional molecules, involve polar surface residues that do not affect polypeptide folding or thermodynamic stability; these polar residues presumably are involved in protein-protein interactions important for expression of anticoagulant activity.
A murine model of severe protein C deficiency due to homozygous knockout of the mouse protein C gene showed a similar phenotype as severe human protein C deficiency (Chap. 127), with perinatal consumptive coagulopathy in the brain and liver and either death or massive thrombosis that occurred either intrauterine or shortly after birth.73
Protein S was first purified from plasma by DiScipio and colleagues who named it protein S in honor of Seattle, the city of its discovery.74,75 It is a vitamin-K-dependent glycoprotein that is synthesized by hepatocytes, neuroblastoma cells, kidney cells, testis, megakaryocytes, and endothelial cells76,77,78,79 and 80 and is found in platelet a-granules.81 Protein S is inducible by IL-4 in T cells.82
Protein S is synthesized as a precursor protein of 676 amino acids which gives rise to a mature secreted single-chain glycoprotein of 635 residues with three N-linked carbohydrate side chains (Fig. 113-2, Table 113-1).83,84,85 and 86 Eleven Gla residues in the N-terminal region of mature protein S contribute to Ca2+-mediated binding of the protein to phospholipid membranes. The thrombin-sensitive region (TSR), residues 47 to 72, follows the Gla-domain (Fig. 113-2). Four EGF modules each contain one unusual residue of b-hydroxyaspartic acid or b-hydroxyasparagine whose function has not been established, although they likely contribute to Ca2+-binding.84,87,88 and 89
The C-terminal region of protein S, residues 270 to 635, the sex-hormone-binding globulin (SHBG)-like region contains binding sites for C4b-binding protein (C4BP) (see below)90,91 and for factor V as well as factor Va.92,93 Thus, different domains of protein S exhibit a number of different binding sites for different plasma proteins.
The protein S gene, comprising 15 exons and 14 introns, is located on chromosome 3p11.1-11.2 and spans 80 kb (see Fig. 112-13, Table 113-1).68,94,95,96,97,98,99,100 and 101 The protein S gene has limited homology with other genes for vitamin-K-dependent factors in the Gla and EGF domains (see Chap. 112) and notable homology of the region coding for residues 240 to 635 with genes of the SHBG family. Because humans contain a protein S pseudogene that contains several stop codons and is not translated, the normal active gene is designated as the protein S 1 or protein Sa gene, and the pseudogene is protein S 2 or protein Sb gene. The pseudogene is 97 percent homologous with the normal gene and is located very near the normal protein S gene on chromosome 3.
Many different mutations in the protein S gene have been identified that cause protein S deficiency associated with thrombosis, and a database of more than 100 different mutations was published.102 One protein S polymorphism present in less than 1 percent of Caucasians causes replacement of Ser460 by Pro and results in absence of N-linked carbohydrate on Asn458 in the variant, designated protein S Heerlen.103 The functional consequences of the absence of this carbohydrate or of the presence of Pro460 for protein S functions have not been established.
Thrombomodulin was discovered and named by Esmon and Owen, who demonstrated that endothelial cell surfaces possess a nonenzymatic cofactor that accelerates protein C activation by thrombin.104,105 Moreover, binding of thrombin to thrombomodulin converts thrombin from a procoagulant enzyme to an anticoagulant enzyme because thrombomodulin-bound thrombin loses its normal ability to clot fibrinogen or activate platelets.106,107 Thrombomodulin is a multidomain transmembrane protein comprising an N-terminal lectin-like domain, six EGF domains, a Ser/Thr-rich region, a single membrane-spanning sequence, and an intracellular C-terminal tail (Fig. 113-2).108,109,110,111,112 and 113 Much is known about structure-function relationships of this protein.108,114,115,116,117,118 and 119 EGF domains 4, 5, and 6 are essential for activation of protein C, with the latter two domains binding thrombin and the first domain binding protein C. The mature protein has 557 amino acid residues and variable amounts of N-linked and O-linked carbohydrates that cause variability in molecular size. Glycosaminoglycans, notably chondroitin sulfate, covalently attached to the Ser/Thr-rich region, contribute to the functional properties of thrombomodulin by enhancing either protein C activation by thrombin or by accelerating neutralization of thrombin by protease inhibitors. Modulation of the substrate specificity of thrombin by thrombomodulin involves conformational changes in thrombin caused by binding of thrombomodulin.120,121
Low levels of soluble thrombomodulin circulate in plasma, presumably as a result of limited proteolysis of the protein near its transmembrane cell surface anchor.122 The functional significance of circulating thrombomodulin is unknown, although variations in its plasma level arise in different clinical conditions.
The thrombomodulin gene, which lacks introns, is located on chromosome 20p12 and spans 3.7 kb (Fig. 112-21) (Table 113-1).111,112,123,124 Down-regulation of the thrombomodulin gene is stimulated by a variety of inflammatory agents, including endotoxin, IL-1, and TNF-a and is up-regulated by retinoic acid (see Esmon125). In general, agents that down-regulate thrombomodulin expression usually up-regulate tissue factor expression in a manner consistent with the general concept that these cellular factors exert opposing activities on the hemostatic balance.
Inherited deficiencies of thrombomodulin are not well established as clinically significant, but they may be associated with increased risk of thrombosis.126,127 and 128
An endothelial cell protein C receptor (EPCR) that binds both protein C and APC was cloned by Fukudome and Esmon in 1994.129 Subsequent studies, especially those by Esmon’s laboratory, have defined many properties of the murine, bovine, and human EPCR.52,131,132,133,134,135,136,137,138,139 and 140
The mature EPCR glycoprotein contains 221 amino acid residues and N-linked carbohydrate, giving an Mr of 46,000. EPCR is an integral membrane protein that is homologous to CD1/MHC class I molecules. The N-terminus is part of an extracellular domain which is connected to a single transmembrane sequence that is followed by a short Arg-Arg-Cys-COOH cytoplasmic tail (Fig. 113-2). The cytoplasmic tail can be palmitoylated, and this modification may localize EPCR to caveolae.140 EPCR binds protein C and APC equally well and appears to do so through their Gla domains. EPCR is mainly located on the surface of large vessels, in contrast to the predominant localization of thrombomodulin in the microcirculation. EPCR on endothelial surfaces enhances by fivefold the rate of activation of protein C by thrombin:thrombomodulin, as depicted in Fig. 113-3. Based on the homology between EPCR and CD1/MHC class I molecules, a three-dimensional model of EPCR was constructed, allowing speculation about EPCR structure-function relationships.141

FIGURE 113-3 Thrombin-dependent activation of protein C on cell surface. On an endothelial surface, protein C (PC) is activated by limited proteolysis by the thrombin:thrombomodulin complex (IIa:TM) which liberates a dodecapeptide (residues 158–169) from protein C to generate the anticoagulant serine protease, activated protein C (APC). This proteolytic activation of protein C is accelerated fivefold by the endothelial cell protein C receptor (EPCR). Based on the scheme of van de Poel with permission.256

Soluble EPCR is found in normal human plasma at 100 ng/ml; in purified reaction mixtures, soluble EPCR at relatively high levels inhibits the anticoagulant action of APC against factor Va but not the reaction of APC with protease inhibitors.131,142 Levels of soluble EPCR are increased during disseminated intravascular coagulation (DIC) and in patients with systemic lupus erythematosus, and EPCR increases are not correlated with alterations in circulating thrombomodulin levels.143 Because soluble EPCR binds protein C and APC with an affinity similar to the membrane-bound molecule, it is speculated that EPCR binds the protein C and the APC Gla domains without thermodynamically significant contributions from membrane phospholipids.
APC is thought to exert anti-inflammatory activity that is independent of its anticoagulant activity144,145 (for review, see Esmon140). It is possible that EPCR in APC:EPCR complexes modulates the biologic activity of APC by shifting its proteolytic specificity toward currently unknown substrates. Currently, the involvement of EPCR in physiologic hemostasis is unknown.
The EPCR gene, comprising four exons and three introns, is located on chromosome 20q11.2 and spans 6 kb.146
Protein C is converted to an active serine protease due to cleavage by thrombin at the Arg169-Leu170 peptide bond in a Ca2+-dependent reaction that is accelerated by orders of magnitude by thrombomodulin (see above).35,36,105
Proof that thrombin is a physiologic activator of protein C includes the demonstrations that thrombin infusions into baboons generate anticoagulant activity due to APC.147,148 Interestingly, thrombin infusion into hyperlipidemic monkeys with atherosclerosis generated less APC and caused a poorer ex vivo response to APC compared with normolipidemic control monkeys,149 showing that hyperlipidemia and vascular disease can affect protein C activation.
Proof that thrombomodulin is a physiologic antithrombotic cofactor comes from studies of mice containing a targeted point mutation in thrombomodulin that markedly impairs its ability to activate protein C. Mice with a specific Glu to Pro mutation in the loop between EGF4 and EGF5 in thrombomodulin have a hypercoagulable state, fibrin deposition, and a severely reduced ability to activate protein C.150,151 However, thrombomodulin has additional biologic functions, including a critical as yet undefined role in fetal development, because complete knockout of the thrombomodulin gene is associated with embryonic lethality before development of an intact cardiovascular system.152 Factor Va also enhances the rate of activation of protein C by thrombin, although the physiologic significance of this reaction has not been assessed.153
Proof that ischemia causes protein C activation in vivo comes from several studies. Even a brief occlusion of the left anterior descending coronary artery in pigs results in APC generation.154 During cerebral ischemia in humans undergoing routine endarterectomy, APC increases in the venous cerebral blood.155 Protein C is significantly activated during cardiopulmonary bypass, mainly during the minutes immediately after aortic unclamping in the ischemic vascular beds.156 Streptokinase therapy for acute myocardial infarction increases circulating APC.157
Circulating APC concentration in normal human subjects is highly correlated with circulating levels of protein C zymogen.158 Based on protein C infusion studies in protein-C-deficient subjects, it appears that basal activation of protein C is strongly determined by the concentration of protein C.159
Although EPCR has not yet been shown to contribute to physiologic protein C activation, in vitro studies of thrombin-dependent protein C activation using purified EPCR and thrombomodulin in reconstituted phospholipid vesicles shows that EPCR without negatively charged phospholipids can provide the surface for protein C activation by thrombin:thrombomodulin, suggesting that EPCR concentration in different vascular beds will determine the effectiveness of the thrombin:thrombomodulin complex.135 This concept for protein C activation is presented in Fig. 113-3, which schematically indicates phospholipid-independent activation of protein C. This model for protein C activation allows EPCR to substitute for negatively charged phospholipids, and it also implies that a cellular response to a specific stimulus might bring EPCR and thrombomodulin in close proximity such that protein C activation is enhanced by a specific agonist that itself does not generate thrombin or up-regulate thrombomodulin (see Fig. 113-3).
Thrombomodulin is abundantly present in the small blood vessels but less so in large vessels, whereas EPCR is more abundant in large vessels than in small vessels.125 Thrombomodulin levels vary markedly in different tissues,160 with significant consequences for a variable tendency for fibrin deposition in different organs.108,140,150,151 In contrast to an initial report that thrombomodulin is absent in brain,161 low levels are expressed in brain,162,163,164 and 165 and brain-specific activation of protein C in humans occurs during carotid occlusion.155
Proteolytic cleavage and activation of protein C can also be effected by meizothrombin, plasmin, or factor Xa.166,167,168 and 169 On the surface of cultured endothelial cells, negatively charged sulfated polysaccharides in the presence of phospholipid vesicles containing phosphatidyl ethanolamine can enhance the rate of protein C activation by factor Xa to approach the protein C activation rate of thrombin:thrombomodulin.169 No data have indicated whether protein C activation by plasmin or factor Xa is physiologically relevant.
The mechanism of APC anticoagulant activity involves factors V and VIII, the two homologous coagulation cofactors that circulate as inactive molecules and are converted to active cofactors by limited proteolysis (see Chap. 112 and Fig. 112-15 and Fig. 112-17). APC circulates at 40 pM in normal humans, and there is an inverse correlation between fibrinopeptide A and APC levels in healthy nonsmoking adults, suggesting APC is a significant regulator of basal thrombin activity.22,170 In contrast to the availability of substantial information about the modes of action of APC and protein S as anticoagulant factors, no specific molecular mechanisms for the expression of anti-inflammatory activity by APC and/or protein S have been demonstrated.
Factors V and VIII are synthesized as large single-chain precursor coagulation cofactors of Mr 330,000, consisting of three homologous A domains (A1, A2, and A3) and two homologous C domains (C1 and C2) with a very large intervening, generally nonhomologous domain, designated the B domain, that connects the A2 and A3 domains. In factors Va and VIIIa, the A domains form heterotrimeric structures like ceruloplasmin, while the C domains form head-to-tail heterodimeric structures.171,172,173 and 174 As depicted in Fig. 113-4, activation of factor V is accomplished by limited proteolysis at Arg709, Arg1018, and Arg1545 by thrombin, factor Xa, or other proteases; cleavage at Arg1545 is the key step for generating factor Va. The various forms of factor Va (Fig. 113-4) are composed of two polypeptide chains, one bearing the A1-A2 domains and the other bearing the A3-C1-C2 domains. The noncovalent interactions between the two chains are stabilized by Ca2+ ions because these chains dissociate in the absence of divalent metal ions. Although generally similar to factor V activation, factor VIII activation (Fig. 112-17) involves formation of a heterotrimer of polypeptide chains, one each containing the A1 domain, the A2 domain, and the A3-C1-C2 domains respectively. In contrast to heterodimeric factor Va, heterotrimeric factor VIIIa is intrinsically unstable due to spontaneous dissociation of the A2 domain.175

FIGURE 113-4 Proteolytic activation and inactivation of factors V and Va. Lines represent polypeptide structures of factor V, active factor Va species, and inactive factor Vi. Activation of factor V by thrombin, factor Xa, or Russell’s viper’s venom is associated with cleavages at Arg709, Arg1018, and Arg1545. Inactivation of factor Va by APC involves cleavages at Arg506, Arg306, and Arg679. If factor Va is cleaved by APC only at Arg506, designated as factor Vaa, it exhibits approximately 70 percent procoagulant activity. Cleavage at Arg306 is the most important cleavage for full inactivation of factor Va and is markedly phospholipid-dependent and enhanced approximately 20-fold by protein S. Protein S–dependent cleavage at Arg306 by APC is also enhanced by HDL. The bottom line indicates that dissociation of the A2 domain is associated with inactivation of factor Va. Based on an unpublished scheme of T. Hackeng with permission.

Irreversible proteolytic inactivation of factors Va and VIIIa by APC can be accomplished by proteolysis at Arg506 and Arg306 in factor Va and Arg562 and Arg362 in factor VIIIa8,175,176,177,178,179,180 and 181 (Fig. 112-15,Fig. 112-17,Fig. 112-15). Currently, the most common identifiable venous thrombosis risk factor involves a mutation of Arg506 to Gln in factor V that results in APC resistance (see Chap. 127). The complexities of APC-dependent inactivation of factor Va and VIIIa are compounded by the number of different molecular forms of Va and VIIIa that can be generated by limited proteolysis by a variety of proteases and by their differing susceptibilities to APC and to the different APC cofactors. While some elements of these complexities are clear, most details are not presently well understood, and APC resistance can be caused by a number of molecular defects in APC cofactors or in APC’s substrates.
APC resistance is defined as an abnormally reduced anticoagulant response of a plasma sample to APC (see Chap. 127) and can be caused by many potential abnormalities in the protein C pathway. Such abnormalities could include defective APC cofactors, defective APC substrates, or antibodies or other agents that interfere with the normal functioning of the protein C pathway.
A report of familial venous thrombosis associated with APC resistance without any identifiable defect led to an intensive search for a genetic explanation182 that was soon found to involve replacement of G by A at nucleotide 1691 in exon 10 of the factor V gene which causes the amino acid replacement of Arg506 by Gln.8,183,184 and 185 This factor V variant, which arose in a single Caucasian founder some 21,000 to 34,000 years ago,186 is known as Gln506-factor V or factor V Leiden. This mutation is currently a common, but not the only, cause of APC resistance.
The molecular mechanism for APC resistance of Gln506-factor V is based on the fact that the variant molecule is inactivated 10 times slower than normal Arg506-factor Va.185,187,188,189,190 and 191 The variant factor Va exhibits only a partial resistance to APC because cleavage at Arg306 in factor Va also occurs, causing complete loss of factor Va activity. This finding helps explain why APC resistance due to Gln506-factor V is a rather mild risk factor for venous thrombosis and why a combination of genetic risk factors or a combination of a genetic and acquired risk factors for venous thrombosis is found in a significant fraction of symptomatic patients (see Chap. 127). Another possibility to help explain the mild risk of venous thrombosis associated with Gln506-factor V is that factor Va may be inactivated in vivo by proteases other than APC that cleave at sites other than residue Arg506.
A factor V haplotype, designated R2, has also been associated with mild APC resistance,192 although it appears that the R2 haplotype may only be a risk factor when present along with a Gln506-factor V allele.193
Plasma and recombinant factor V can exist in two biochemically distinct forms, designated factor V1 and factor V2.194,195,196 and 197 Factor V1 has N-linked carbohydrate on Asn2181, near the phospholipid binding region of the C2 domain, whereas factor V2 has none. Because the N-linked carbohydrate appears to decrease the apparent affinity of factor V1 or Va1 for phospholipid, it reduces the specific clotting activity and susceptibility to APC. Normal plasma contains a mixture of factors V1 and V2. Removal of the carbohydrate attached to factor V increases the rate of inactivation of factor Va by APC, although the clinical significance of this phenomenon is unknown.198
APC resistance with no identifiable genetic or acquired abnormalities is well described in patients with venous and arterial thrombosis.199,200,201,202 and 203 Further studies are needed to identify the causes of APC resistance in these patients, and further work is needed to develop and compare various APC resistance assays that have different sensitivities to different physiologic variable or plasma components. For example, APTT-based assays are not equivalently sensitive as are dilute tissue-factor-based assays to plasma HDL levels or oral contraceptive use.204,205 Plasma variables, such as prothrombin levels, may affect the response to APC by inhibiting APC action.206
Proteolytic activation of factors Va and VIIIa can generate different forms of each active cofactor that differ in specific activity. For example, factor VIIIa generated by factor Xa has lower specific activity and longer half-life than that generated by thrombin,175,207 and factor Va generated by cleavage only at Arg709 and Arg1018 (without cleavage at Arg1545) has a lower specific activity than that generated after cleavage at Arg1545.208 Factor Va can be cleaved at Arg1765, and this could generate forms of factor Va with differing specific activities.209 Factor VIIa:tissue factor complexes can cleave factor V at novel sites to produce a form of factor V that can be destroyed by APC without the requirement for full activation of the cofactor precursor.210
APC anticoagulant activity is enhanced by a number of factors that are termed APC cofactors; these include certain Ca2+ ions, phospholipids, protein S, factor V, and HDL.
Phospholipids as APC Cofactors Certain phospholipids such as phosphatidyl serine, phosphatidyl ethanolamine, and cardiolipin enhance the anticoagulant activity of APC.211,212,213 and 214
Protein S Protein S forms a 1:1 complex with APC215 and enhances by 10- to 20-fold the rate of APC’s cleavage at Arg306 in factor Va but not the Arg506 cleavage.190 Part of the mechanism for this activity of protein S may be related to its ability to bring the active site of APC closer to the plane of the phospholipid membrane on which the APC:protein S complex is located when the complex is formed.216,217 Protein S also facilitates the action of APC against factor VIIIa.218,219 Protein S enhances APC’s action, in part at least, by ablating the ability of factor Xa to protect factor Va from APC.220 The TSR and EGF domains of protein S are implicated in binding APC for expression of anticoagulant activity by the APC:protein S complex.221,222,223,224,225 and 226 Cleavage of the TSR by thrombin abolishes normal binding of protein S to phospholipid and its APC-cofactor anticoagulant activity.227,228,229 and 230
Factor V Factor V apparently can have anticoagulant as well as procoagulant properties because it enhances the anticoagulant action of APC against factor VIIIa in a reaction in which protein S acts synergistically with factor V.30,31,231,232 and 233 Cleavage at Arg1545, which optimizes factor Va procoagulant activity, ablates the molecule’s anticoagulant cofactor activity. However, when factor V is cleaved at Arg506 by APC, its APC cofactor activity is increased 10-fold. This suggests that Gln506-factor V has two potential prothrombotic defects, namely, resistance of the variant factor Va to APC inactivation and resistance of the variant factor V to activation of its APC cofactor function.232
High-Density Lipoprotein High-density lipoprotein (HDL) enhances the anticoagulant activity of APC both in plasma and in purified reaction mixtures. This APC cofactor activity requires protein S and involves, at least in part, stimulation of APC’s cleavage at Arg306 in factor Va.204 In animal models, HDL inhibits DIC induced by endotoxin infusion in baboons and ferric chloride–induced arterial thrombosis in rats.234,235 HDL is heterogeneous in both protein and lipid composition, and the components responsible for this activity have not been identified. The clinical significance of this anticoagulant property of HDL is unknown.
APC is a normal component of circulating blood, and it likely contributes to antithrombotic surveillance mechanisms.22 Circulating APC levels are determined by the balance of mechanisms for APC generation compared with mechanisms for APC inhibition and clearance. Determinants of APC generation include: (1) protein C zymogen levels; (2) endogenous thrombin generation; and (3) thrombomodulin and EPCR availability. Clearance of APC appears to be mainly caused by inhibition of APC by protease inhibitors.236,237 The major plasma inhibitors of APC include a1-antitrypsin, protein C inhibitor, and a2-macroglobulin.236,238,239,240,241,242,243,244,245,246 and 247
Protein S has both indirect and direct anticoagulant activity because of its ability to serve as a nonenzymatic APC cofactor (see above and Fig. 113-1) and because, independent of APC, it inhibits coagulation reactions by directly binding to procoagulant factors. APC-independent activity is based on the ability of protein S to inhibit directly the activity of the prothrombinase complex by reversibly binding to factor Va and/or factor Xa (Fig. 113-5).14,15 and 16 As depicted in Fig. 113-5, protein S can bind factors Xa or Va to form inactive complexes. It is possible that ternary complexes of protein S:fVa:fXa might be formed.248 The TSR and the EGF3 domains of protein S are thought to bind factor Xa, contributing to APC-independent anticoagulant activity.222,249 Protein S can also bind factor VIIIa and inhibit activation of factor X by factor IXa:VIIIa complexes.18,250

FIGURE 113-5 Protein S direct inhibition of prothrombin activation. Protein S inhibits directly the activity of the prothrombinase complex by reversibly binding to factor Va and/or factor Xa. On the left, protein S is depicted in solution and bound to a phospholipid (PL) membrane surface. On the right, inactive complexes of factors Va:PS and Xa:PS are depicted.14,15 and 16 It is possible that ternary complexes of protein S:fVa:fXa also can exist.248 Protein S can also bind factor VIIIa and inhibit activation of factor X by factor IXa:VIIIa complexes (not shown).18 Based on an unpublished scheme of S. Yegneswaran with permission.

C4b-binding protein is a plasma protein that enhances inactivation of the complement cascade by binding to C4b and promoting proteolytic inactivation of C4b by factor I. C4b-binding protein reversibly binds protein S with high affinity,251,252 and 253 and formation of this complex affects some of the anticoagulant activities of protein S (see review by Dahlbäck12). When factor Va is the targeted substrate, the APC-cofactor activity of protein S is neutralized by its binding to C4b-binding protein.254,255 However, the association of C4b-binding protein with protein S does not ablate its ability to serve as an APC cofactor when the substrate is factor VIIIa256 or its ability to inhibit the prothrombinase complex. This latter observation is explained by the ability of C4b-binding protein to block binding of protein S to factor Va but not to factor Xa. C4b-binding protein in plasma is a heteropolymer containing two different kinds of disulfide-linked polypeptides, six or seven a chains and a single b chain, and the latter chain is responsible for binding protein S.257,258 and 259 Residues 30 to 45 of the b chain bind to the SHBG domain of protein S.90,260,261 Because the affinity of protein S for C4b-binding protein is so high, the amount of free protein S in plasma is determined by the absolute concentrations of the two proteins, such that normally there is approximately 240 nM protein S: C4b-binding protein complexes and 120 nM free protein S.253 During an acute phase reaction, the level of the C4b-binding protein a chain but not the b chain is increased, so that the change in total C4b-binding protein does not alter the level of free and bound protein S.262
Antithrombin, initially designated antithrombin III, is the clinically most important inhibitor of clotting factor proteases (see Chap 127). Antithrombin can neutralize all proteases of the intrinsic coagulation pathway, including thrombin and factors Xa, IXa, XIa, and XIIa, in reactions that are enhanced by heparin and related glycosaminoglycans (see Chap 112). Tissue factor pathway inhibitor (TFPI), previously also described as lipoprotein-associated coagulation inhibitor (LACI), can neutralize factors VIIa and Xa, proteases of the extrinsic coagulation pathway. In addition, other plasma protease inhibitors can neutralize various coagulation proteases, although the clinical significance of these reactions is less well defined than the reaction of antithrombin with thrombin.
Antithrombin is synthesized in the liver and is present in plasma at 150 µg/ml. Antithrombin is a typical member of the serine protease inhibitor (serpin) superfamily.263 Heparin and endogenous endothelial heparan sulfates, which are heterogeneous but structurally similar to heparin, accelerate antithrombin’s inhibitory actions (see Chap 133).
The neutralization of proteases by antithrombin is due to a stable enzyme:antithrombin complex that is formed by a molecular mechanism characteristic of inhibitory serpins.263,264,265,266,267,268 and 269 Following binding of a protease to a “reactive site” loop in a serpin, a single peptide bond in the serpin is cleaved with formation of an acyl-enzyme intermediate via the active site Ser residue. This metastable enzyme:serpin complex can either break apart or form a more stable covalent enzyme:serpin complex. To break apart the enzyme:serpin covalent complex, deacylation liberates the cleaved product and regenerates the active site Ser residue of the protease. However, serpins have a remarkable ability to undergo major conformational changes following cleavage at the reactive site residue that can lock the enzyme in the protease:serpin complex.263,267,268 and 269 The dominant structural feature of native serpins is a large five-stranded b-sheet that defines the structure of an ellipsoidal protein. Following cleavage at the reactive center residue in the reactive center loop by a protease, this extended loop is able to partially or completely insert itself into the five-stranded b-sheet, forming a very stable six-stranded b-sheet. If this insertion reaction proceeds before deacylation occurs, then the protease remains covalently attached to the reactive center P1 residue through the protease’s active site Ser residue, and a stable covalent protease:inhibitor complex is formed.
Heparin enhancement of the rate of reaction between antithrombin and thrombin is caused by two distinct effects of heparin, one involving conformational effects on antithrombin and the other involving “approximation” effects on both thrombin and antithrombin.270,271,272,273,274 and 275 For the first effect, a particular pentasaccharide within heparin is most potent at causing a conformational change that converts antithrombin from its native state of moderate reactivity to a conformation with relatively high reactivity. This pentasaccharide contains a specific sulfated sequence of glucosamine and iduronic acid residues,270,274,276,277 and it accelerates the reaction of antithrombin with not only thrombin but essentially any target protease. On the other hand, the approximation effect mainly affects its reaction with thrombin and is due to the fact that both thrombin and antithrombin have high affinity for heparin. When both thrombin and antithrombin are simultaneously bound to heparin, they encounter each other much more frequently than when they are free in solution, thus increasing the reaction rate. Heparan sulfates also act in this manner. This approximation mechanism is not very significant for proteases other than thrombin (e.g., factor Xa) unless the protease has a very high affinity for heparin.
After cleavage of a propeptide from a 464-residue precursor polypeptide, mature antithrombin contains 432 amino acid residues.278 It has four sites for N-linked carbohydrate attachment, one of which (Asn135) is variably glycosylated, giving rise to a b-isoform that has higher affinity for heparin.279,280 Heparin binding to antithrombin is mediated by a number of positively charged Arg and Lys residues that are located in the N-terminal sequence of the protein within residues 41 to 49 and 107 to 156, whereas the reactive center loop containing the scissile peptide bond at Arg393-Ser394 is near the C-terminus.
The antithrombin gene comprising seven exons and six introns spans 13.4 kb and is located on chromosome 1q23-25.281,282,283 and 284
Hereditary deficiencies of antithrombin are well recognized as risk factors for venous thrombosis (see Chap 127). Over a hundred different mutations have been reported to be associated with thrombosis, and a complete database of mutations is published and is available on the world wide web through the courtesy of Lane and colleagues285 at http://www.med.ic.ac.uk/dd/ddhc. Mutations that cause antithrombin deficiency are scattered throughout the molecule. Defects can be classified as type I, characterized by parallel decreases in antigen and activity, or type II, characterized by circulating dysfunctional molecules such that plasma has decreased activity but normal or near-normal antigen levels. Type II defects are further classified based on whether the dysfunction involves only reactive center defects (tested in the absence of heparin), only heparin-binding defects, or both of these properties (pleiotropic effects). Reactive center defects carry the largest risk of thrombosis, while heparin-binding defects are associated with less risk of venous thrombosis (see Chap 127).
The mature TFPI protein, previously known as lipoprotein-associated coagulation inhibitor (LACI) or extrinsic pathway inhibitor (EPI), has an Mr of 34,000 and contains an acidic N-terminal sequence, three homologous but distinct Kunitz-type protease inhibitor domains, and a C-terminal positively charged basic amino acid sequence.9,286 Although present at only 100 ng/ml in normal plasma, TFPI is a significant inhibitor of the extrinsic coagulation pathway that functions synergistically with the protein C pathway and antithrombin to suppress thrombin generation. TFPI is synthesized by endothelial cells and smooth muscle cells.9,287 Approximately half of TFPI in plasma is associated with lipoproteins, mainly LDL, and a substantial amount of TFPI is released when heparin is infused.288,289
Studies from the laboratories of Broze and others support the reaction scheme for neutralization of factors Xa and VIIa by TFPI depicted in Fig. 113-6.9,99,290,291,292 and 293 Initially, the second Kunitz domain of TFPI reacts with and inhibits the active site of factor Xa. Subsequently, this binary complex reacts with factor VIIa in the TF:VIIa complex to form a quaternary protein complex on a membrane. TFPI can react with factor VIIa in the absence of factor Xa, but at a much slower rate. Interestingly, TFPI can neutralize factor Xa when the enzyme is bound in a prothrombinase complex, i.e., in a Xa:Va:phospholipid complex. Because TFPI requires factor Xa for kinetically favorable reactions with factor VIIa, TFPI does not shut off the initiation of the extrinsic pathway by tissue factor until some significant amount of factor Xa is generated. Then TFPI provides negative feedback inhibition of the generation of factor Xa by the VIIa:tissue factor complex. For hemostasis, amplification of an initiating signal generated by tissue factor then requires extensive thrombin generation by the intrinsic pathway that is caused by the positive feedback action of thrombin (see Fig. 113-1 and Chap. 112).

FIGURE 113-6 Feedback inhibition of factor VIIa by TFPI in factor Xa:TFPI complexes. Surface-bound factor VIIa:tissue factor (TF) complexes generate factors IXa and Xa (upper left). Free TFPI is a multivalent protease inhibitor containing three Kunitz-type protease inhibitor domains. After factor Xa complexes with and is inhibited by the Kunitz-2 domain of TFPI, the Xa:TFPI complex (right side) can bind to and inhibit a TF:factor VIIa complex, forming a quaternary complex (lower center). Alternatively, TFPI might combine with a surface-bound ternary complex of TF:VIIa:Xa (lower left) to form a final quaternary complex (lower center). Adapted from Broze with permission.9

Proof that TFPI functions physiologically as an inhibitor of coagulation comes from animal model studies showing that depletion of TFPI predisposes animals to endotoxin-induced DIC and the generalized Schwarzman reaction and that treatment of animals with TFPI reduces mortality from E. coli septic shock.294,295 and 296 Mice carrying complete deficiency of TFPI in gene knockout studies do not survive beyond the neonatal period and die of hemorrhage with signs of fibrin formation, suggestive of consumptive coagulopathy.297
The sequence of TFPI was established from cloning of its cDNA and the TFPI gene that contains 9 exons, spans 85 kb, and is located on chromosome 2q31-32.1.286,298,299
Hereditary abnormalities of TFPI have not yet been definitively associated with an increased risk of thrombosis, although one recent report has suggested a linkage to venous thrombosis.300
Thrombin in plasma can be inhibited not only by antithrombin but also by a2-macroglobulin, an acute phase reactant. Heparin cofactor II, a serpin whose inhibitory activity is greatly enhanced by dermatan sulfate, also inhibits thrombin in vivo and in vitro by an approximation mechanism.301,302 In purified reaction mixtures, protein C inhibitor also efficiently neutralizes thrombin in the presence of thrombomodulin.303,304 Hereditary defects of protease inhibitors other than antithrombin have not yet been linked to an increased risk of thrombosis, although several reports linking heparin cofactor II deficiency to venous thrombosis have appeared.305,306 and 307

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Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology



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