The assembly of the factor X-activating complex on activated human platelets

Transcription

1 Journal of Thrombosis and Haemostasis, 1: REVIEW The assembly of the factor X-activating complex on activated human platelets S. S. AHMAD,* F. S. LONDON* and P. N. WALSH*y *The Sol Sherry Thrombosis Research Center and the Department of Biochemistry; and ydepartment of Medicine, Temple University School of Medicine, Philadelphia, USA Summary. Platelet membranes provide procoagulant surfaces for the assembly and expression of the factor X-activating complex and promote the proteolytic activation and assembly of the prothrombinase complex resulting in normal hemostasis. Recent studies from our laboratory and others indicate that platelets possess specific, high-affinity, saturable, receptors for factors XI, XIa, IX, IXa, X, VIII, VIIIa, V, Va and Xa, prothrombin, and thrombin. Studies described in this review support the hypothesis that the factor X-activating complex on the platelet surface consists of three receptors (for the enzyme, factor IXa; the substrate, factor X; and the cofactor, factor VIIIa), the colocalization of which results in a 24 million-fold acceleration of the rate of factor X activation. Whether the procoagulant surface of platelets is defined exclusively by procoagulant phospholipids, or whether specific protein receptors exist for the coagulant factors and proteases, is currently unresolved. The interaction between coagulation proteins and platelets is critical to the maintenance of normal hemostasis and is pathogenetically important in human disease. Keywords: agonists, factor IXa, factor VIIIa, factor X activation, factor X, platelet membrane receptors, platelets, prothrombin Introduction The coagulation of blood consists of an intricate system of limited, proteolytic events that ultimately lead to the formation of an insoluble fibrin clot. Classically, the sequence of coagulation reactions referred to as the coagulation cascade [1] or waterfall [2] has been understood as two alternative or convergent pathways activated either by the contact system proteases [3], e.g. factor (F)XII, prekallikrein, and high molecular weight kininogen, or alternatively, by the activation of FVII and FX, when tissue factor is exposed at the site of vascular injury. A more current understanding of the sequence of reactions occurring during the initiation and the consolidation of blood Correspondence: Dr P. N. Walsh, The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, USA. Tel.: þ ; fax: þ ; pnw@temple.edu Received 11 March 2002, accepted 30 April 2002 coagulation is shown in Fig. 1. The major points to be inferred from this scheme are the following: (i) virtually all important interactions of coagulation proteins involved in hemostasis occur on platelets where catalytic complexes of enzymes, cofactors, and substrates are assembled, generating thrombin in sufficient concentrations to convert fibrinogen to fibrin [4]; (ii) the initiation of blood coagulation occurs when tissue factor exposed on cell membranes at sites of vascular injury results in the generation of small quantities of FXa that are required to facilitate the inactivation of FVIIa and FXa by tissue factor pathway inhibitor [5 11]; (iii) very small quantities of thrombin, insufficient for hemostasis, are generated, resulting in (a) the activation of FXI on activated platelet membranes; (b) the conversion of FIX to FIXa; and (c) the subsequent activation of FX leading to the generation of sufficient amounts of thrombin to effect hemostasis [12,13]. In this review, some of the plateletreceptor-mediated coagulation protein interactions are discussed with special focus on platelet-receptor-mediated FX activation. Platelet-coagulation protein interactions The interaction between coagulation proteins and platelets is an aspect of hemostasis that is both critical to the maintenance of normal hemostasis and pathogenically important in human disease [14,15]. In 1972, we initially presented evidence that human platelets provide an activated membrane surface for the assembly of FX-activating complex [16,17]. In the intervening years, a large body of evidence has been accumulated both from our laboratory [18 36] and from those of other investigators (for review see reference [37]). Both the membranes of activated platelets and other cells, as well as phospholipid membranes of defined composition containing aminophospholipids, provide binding sites for the assembly of FIXa, FVIIIa and FX and for the efficient activation of FX, which in solution is an extremely inefficient process [30,38]. The study of platelet coagulant protein interactions has resulted in the view that platelets have plasma membrane receptors for coagulation proteins including thrombin [15], fibrinogen [15], FXa [39 44], FVa [43,44], FXI [45], FXIa [46], FIXa and FIX [23], FX and FII [32] and for FVIII and FVIIIa [25,47]. It has been demonstrated that human FXa binds specifically and reversibly in the presence of calcium ions to high-affinity receptors on platelets and that FXa binding is closely correlated with enhanced rates of prothrombin

2 FX-activating complex assembly on activated human platelets 49 been reviewed [4,37]. In this paper, we will focus our attention [48] on the assembly of FX-activating complex on platelet membranes. Biochemistry of the components of the factor X-activating complex FIX biochemistry Fig. 1. A model of the sequence of reactions occurring during the initiation and the consolidation of blood coagulation (see text for explanation). The crescentic forms represent the cell membranes that localize various coagulation reactions, with the tissue factor/fviia complex assembled on tissue factor bearing cells (gray-filled membrane) and the remainder of the complexes assembled on the activated platelet membrane (empty crescent). The Roman numerals represent coagulation proteins in the zymogen or cofactor form with the a representing the active enzyme or cofactor. The circles represent zymogens; the circles with segmental excisions represent enzymes; the ellipses represent cofactors; and, the rectangles represent Kunitz-type inhibitors with the solid block representing the block imposed by the inhibitor. The designation pxi represents platelet FXI. The arrows represent conversions from zymogens to enzymes. Other abbreviations include: HK, high molecular weight kininogen; TFPI, tissue factor pathway inhibitor; PNII, protease nexin II; II, prothrombin; TF, tissue factor. activation [39 44]. The functional consequence of the assembly of the prothrombinase complex on platelets is a fold acceleration of the rate of prothrombin activation by FXa. The reversible, saturable, high-affinity binding of FXa to platelets depends on the presence of FVa, which has been suggested as the binding site for FXa [39,40,43,44], and coordinate binding studies of FXa and FVa to platelets indicate that a stoichiometric complex is bound [43]. The details of the interactions of platelets with thrombin, FX, FXa, fibrinogen, and fibrin, and the significance of these interactions for platelet activation and aggregation are beyond the scope of this review. However, some of these platelet-coagulation protein interactions have recently Human FIX is a single chain glycoprotein (M r ) containing 18% carbohydrate and consisting of 415 amino acids [48 50]. After synthesis in the liver it circulates in plasma as a zymogen that is activated by limited proteolysis either by FXIa (intrinsic pathway) or by the FVIIa-tissue factor complex (extrinsic pathway) (Fig. 1) [51 53]. FIXa (M r ) results from cleavage of two peptide bonds and the formation of an activation peptide (M r ) and two chain FIXa, which consists of disulfide-linked, heavy (M r ) and light (M r ) chains [52]. The FIX gene (34 kb) contains seven introns and eight exons coding for distinct structural domains that are highly conserved among the other homologous vitamin K-dependent plasma coagulation proteins (FX, FVII, prothrombin, protein C and protein S) [54]. Exon I encodes the signal peptide and exons II III the propeptide (which is cleaved from the mature protein prior to secretion through the Golgi apparatus) and the g-carboxyglutamic acid (Gla) domain (comprising residues 1 46, including 12 glutamic acid residues that are post-translationally modified to the dicarboxylic Gla form by a vitamin K-dependent carboxylase) [54]. Exons IVand Vencode two similar epidermal growth factor (EGF)-like domains (residues ), each of which contains six cysteines; the first epidermal growth factor (EGF1) domain of FIX contains one high-affinity Ca 2þ binding site [55 57]. Exon VI encodes an activation peptide (residues , containing two carbohydrate-binding sites), and exons VII-VIII (and the 3 0 portion of exon VI) code for residues , which comprise the trypsin-like heavy-chain domain of FIXa [54 58]. FIXa activates FX by cleaving a specific peptide bond (R 52 I 53 ) in the N- terminal region of the FX heavy chain [59,60]. It recognizes FX as its normal macromolecular substrate and can also cleave and activate FVIII but less effectively than thrombin or FXa [61,62]. Recent studies by Perera et al. [63] provide three-dimensional structures of human FIX and FIXa in ionic aqueous medium by computational methodology and provide correlations between: (i) actual FIX database mutations [64]; (ii) partial X-ray crystal structure of inhibited FIXa [65,66]; and (iii) FIXa protein protein interactions [67]. FX biochemistry FX is a vitamin K-dependent glycoprotein whose sequence is similar to that of FIX (and prothrombin and FVII) in the region of the active site [51,68]. Like FIX, FX also contains an N-terminal Gla region followed by two EGF domains, an activation peptide and a trypsin-like serine protease domain [68]. The zymogen (M r ) circulates in plasma at a

3 50 S. S. Ahmad et al concentration of 5 10 mg ml 1 and consists of a M r light chain and a M r catalytic domain held together by disulfide bonds [68,69]. FX contains 15% carbohydrate, most of which is released on activation by FIXa or by FVIIa-tissue factor, both of which activate FX by hydrolysis of an internal Arg Ile bond in the heavy chain [70]. The Gla domain is required for the Ca 2þ -dependent interaction with phospholipid surfaces and is necessary for biological activity [53,71]. The EGF1 domain contains one aspartic acid residue post-translationally modified to b-hydroxyaspartic acid (Hya). FX is essentially 100% hydroxylated at this site [72]. Recent studies report determination of structure of human FXa [73] and bovine prothrombin fragment 1 [74]. FVIII von Willebrand factor (VWF) complex FVIII participates nonenzymatically as a regulatory protein or cofactor in the activation of FX by FIXa in the presence of calcium ions and phospholipids [75]. FVIII may be stabilized by its association with VWF, is activated by thrombin [76 78] or by FXa in the presence of lipids [79], and can undergo proteolysis and inactivation by plasmin [80] and by activated protein C [79]. A detailed review of the biochemistry of the FVIII molecular complex is beyond the scope of this discussion. However, the gene for FVIII has been isolated and the cdna sequence has been determined, revealing that the gene codes for a protein of M r [81 83]. The bp gene has 26 exons ranging in size from 69 to 3106 bp comprising 9000 bp of coding sequence. FVIII is synthesized as a single polypeptide chain containing 2351 amino acids. This sequence indicates a domain structure with an arrangement of A1 A2 B A3 C1 C2, where A represents a repeating domain that shares sequence homology with ceruloplasmin and FV, B represents a domain unique to FVIII and C represents a repeating domain that shares homology with FV and discoidin I, a lectin [71,81 84]. During proteolytic activation of FVIII, the B domain is excised, leading to a non-covalent association of three polypeptide chains that participate as the active cofactor in complex formation with FIXa, FX and phospholipids [71,84 86]. Activation of FVIII is initiated by release from VWF by a cleavage in the A3 domain resulting in the removal of a peptide segment and is completed by a cleavage between the A1 and A2 domains [71,84]. FVIIIa consists of a heterotrimeric molecule consisting of the A1 and A2 domains of the heavy chain and the A3, C1 and C2 of the light chain [85,86]. The activated form of FVIIIa is transient due to rapid dissociation of the A2 domain from the remainder of the molecule [85 88]. Assembly of factor X-activating complex on platelet membrane According to the revised theory of blood coagulation (Fig. 1), although activated endothelial cells and monocytes have been shown to support assembly of FX-activating and prothrombinase complexes [89 94], virtually all of the enzymatic reactions occur on the surface of platelet membrane receptors that colocalize the enzymes, the cofactors, and the substrates in a kinetically favorable complex, the assembly of which requires platelet activation. Therefore, we will first discuss the mechanisms of platelet activation that are necessary and sufficient for the binding of FIXa, activated FVIII (FVIIIa) and FX to platelet receptors and to the assembly of FX-activating complex. Mechanisms of platelet activation Circulating resting platelets do not bind components of the FX activation complex [37]. Activation of platelets with ADP, while leading to many other functional outcomes, does not result, even at 100 mmol L 1, in exposure of binding sites for FX [32] or FVIIIa [25] or in assembly of a functional FX-activating complex [15,17,35,37]. However, activation of platelets with strong agonists such as thrombin or collagen, or the proteaseactivated receptor (PAR) 1-activating peptide SFLLRN, results in the expression of binding sites for all the constituents of the FIXa-catalyzed FX-activating complex, namely the enzyme FIXa, the cofactor FVIIIa, and the substrate FX [25,32,35]. It has been established that a combination of thrombin and collagen promote assembly of the prothrombinase complex in a synergistic manner [15,37,95 100], which is closely correlated with the exposure on platelet membranes of negatively charged aminophospholipids, whereas the combination of thrombin (1 nmol L 1 ) and collagen (10 mgml 1 ) has no greater effect on the binding of FVIIIa than of thrombin (1 nmol L 1 ) alone [25]. Signal-transduction pathways for these two effective agonists originate with their cellular receptors, which are PAR 1 and PAR 4 for thrombin [ ], and glycoprotein VI and possibly integrin a1b2 for collagen [ ]. PAR 1, the best-characterized thrombin receptor, is known to transduce the ligand signal via heterotrimeric G proteins [ ]. Candidate G proteins involved in the process include Gq (which activates the phospholipase C-driven rise in internal calcium and appearance of diacylglycerol, which activates protein kinase C), possibly G12/13, and possibly Gi, which is known to be activated following thrombin stimulation (and signals both through the Gia subunit downregulation of adenylate cyclase and through activation of tyrosine kinases leading to phosphatidylinositol-3 kinase activation). Recent evidence indicates, however, that activation through Gi may require ADP occupancy of its Gi-linked receptor P2Y12 [119] and thus does not constitute a proximal step in thrombin signal transduction. Such feedback signaling is known to be important for platelet responses such as secretion and aggregation in response to weak agonists. Specifically, aspirin (0.5 mmol L 1 ) and indomethacin (20 mmol L 1 ), which inhibit arachadonic acid metabolism, prostaglandin E1 (2 mmol L 1 ) plus RA233 (200 mmol L 1 ), and p-chloromercuriphenylsulfonate (1 mmol L 1 ) have no effect on FX activation while completely inhibiting secretion [35]. In addition, stirring of platelets leading to cross-linking of platelets through fibrinogen binding to its receptor integrin a2bb3 is not required for platelet-supported FX activation although it is required for aggregation [15,37,95 100]. The

4 FX-activating complex assembly on activated human platelets 51 release reaction, although providing FVa from a-granules for optimal assembly of the prothrombinase complex, appears to be unnecessary for FX activation. Indeed, inhibition of protein kinase C with the compound RO blocks release and therefore aggregation in the absence of added fibrinogen; however, FX activation is uninhibited and is actually increased slightly [120] at RO concentrations that are just effective at totally inhibiting secretion (8 16 mmol L 1 ). Another wellknown consequence of Gqa signaling, the rise in cytoplasmic calcium, also appears to be unnecessary for FX activation although total chelation of cytoplasmic calcium with 100 mmol L 1 BAPTA-AM does completely block secretion [121]. One step known to be required for platelet-supported FX activation is the exposure of aminophospholipids. Not only can artificial vesicles containing aminophospholipids above a threshold concentration promote FX activation with similar kinetics as activated platelets [27,30,122], but the calciumdependent aminophospholipid binding protein Annexin V inhibits FX activation [27]. Scott syndrome platelets with a genetic defect in aminophospholipid exposure following platelet activation show defective FVIIIa binding and FX activation [19]. However, annexin V shows different inhibition kinetics on activated platelets and on phospholipid vesicles containing aminophospholipids [27], thus raising the question whether platelet proteins expressed on the surface of activated platelets may play a role in efficient and effective assembly of the FXactivating complex. Indeed, the affinity of FIXa for phospholipid vesicles, in the absence of FVIIIa, is much less than for activated platelets [27], and phospholipid vesicle-supported FX activation appears to be much more sensitive to variations in salt concentration than are the kinetics of platelet-supported FX activation [28]. A great deal of information has accumulated in recent years about the physiological agonists, receptors and signal transduction mechanisms involved in the activation of platelets leading to shape change, aggregation and secretion [ ]. In contrast, in spite of significant advances in our understanding of the biochemical mechanisms leading to phospholipid asymmetry and translocation [95 100, ] as well as platelet microvesiculation, the signaling events by which thrombin or collagen lead to platelet procoagulant activities are poorly understood [37]. Factor X activation on activated platelet membranes The activation of FX is a cell membrane mediated event that can occur either when tissue factor and FVIIa are exposed on the cell surface [131] or when cell membrane binding sites are exposed for the components of the intrinsic FX-activating complex on platelets, endothelial cells or monocytes [15]. The weight of evidence points to the platelet membrane as the normal physiological site of coagulation enzyme cofactor substrate assembly involved in the intrinsic pathway [15,35,71,84,132]. A large amount of work has focused on the biochemical mechanisms of FX activation on phospholipid model systems. Our laboratory provided the first evidence that platelets have an essential role in the activation of FX by the intrinsic pathway and none whatsoever in the extrinsic pathway of FX activation [35,132], although more recent evidence suggests that platelets can acquire tissue factor from microparticles circulating in blood [133,134]. As discussed in the above section, platelet activation by a variety of agonists including collagen and thrombin but not ADP is required to promote the activation of FX on the platelet surface [35,98,135]. Subsequently, we examined the mechanisms by which platelets can promote FX activation by FIXa [19,22 24,30]. Our studies indicate the presence of a discrete number ( binding sites per platelet) of high-affinity, saturable receptors for FIXa that are expressed on the surface of thrombin-activated platelets in the presence of calcium ions [23]. A detailed and rigorous comparison between FIX and FIXa binding to platelets demonstrated that in the absence of FVIII and FX, FIXa can occupy 550 binding sites per platelet of which 300 can also be occupied by FIX, the zymogen. Thus, FIXa can displace all the bound FIX, but FIX can displace only half the FIXa. Although in the absence of FVIII and FX, the affinity of binding of FIX and FIXa are similar (K d 2.5 nmol L 1 ), in the presence of FVIII and FX the affinity of FIXa binding is increased five-fold (K d 0.5 nmol L 1 ), whereas the binding of FIX is unaffected [23]. Nesheim et al. [47] have demonstrated the presence on thrombin-activated platelets of 450 binding sites per platelet for FVIII with a K d 3.0 nmol L 1 (i.e. with stoichiometry and affinity similar to those that describe FIXa binding). Our recent studies [25] on the platelet receptor-mediated FVIII binding indicate that both procofactor (FVIII) and active cofactor (FVIIIa) binding were specific, saturable, and reversible. FVIII binds to specific, high-affinity receptors on the platelet membrane (n ¼ 484; K d ¼ 3.7 nmol L 1 ), and FVIIIa interacts with an additional sites per platelet with enhanced affinity (K d ¼ 1.5 nmol L 1 ). FVIIIa binding to activated platelets in the presence of FIXa and FX is closely coupled with the rates of FX activation. The presence of EGR-FIXa (i.e. active site-inhibited FIXa) and FX increases both the number and the affinity of binding sites on activated platelets for both FVIII and FVIIIa, emphasizing the validity of a three-receptor model in the assembly of the FX-activating complex on the platelet surface. Platelet-receptor occupancy by FIXa is closely correlated with rates of FX activation [24]. In addition, kinetic studies from our laboratory demonstrated that although unactivated platelets have no significant effect on the kinetics of FX activation by FIXa either in the presence or absence of thrombin-activated FVIII, thrombin-activated platelets decrease the K m by more than 200-fold and permit FVIIIa to increase the k cat more than fold [30]. Thus, the combined effects of thrombinactivated platelets and FVIIIa result in an overall increase in catalytic efficiency (k cat /K m ) of more than fold. Our recent studies of the mechanism of platelet-mediated FX activation have demonstrated the presence of a shared FX/prothrombin binding site ( sites per platelet, K d 320 nmol L 1 ) on the surface of activated platelets [32]. Binding of

5 52 S. S. Ahmad et al either FX or prothrombin to these sites also requires the presence of Ca 2þ ions and the activation of platelets by a physiologic activator (thrombin or thrombin-receptor peptide, SFLLRN). These studies demonstrate that FX bound to the surface of activated platelets is preferentially activated by platelet-bound FIXa [33]. Finally, the binding of FVIIIa to activated platelets generates a high-affinity, low-capacity binding site that is absolutely specific for FX and does not recognize prothrombin [136,137]. Taken together, these observations support the hypothesis that activated platelets expose multiple high-affinity, specific receptors, occupancy of which is essential for the enormous acceleration (> fold increase in k cat / K m ) of FX activation on the platelet surface [30]. Following the generation of FXa, similar interactions occur on the activated platelet surface resulting in the assembly of the prothrombin activating complex consisting of the enzyme (FXa), the cofactor (FVa), and the substrate (prothrombin). Over the past 20 years, a great deal has been learned about the assembly of the prothrombinase complex on both human [40 42] and bovine platelets [39,43,44,138,139]. Although there are differences in some of the particular results of these studies, it is evident that FVa binds directly to platelets [43,44,138] and provides a high-affinity binding site for FXa [40,42,43]. In the presence of excess human FVa there are between 200 [42] and 5000 [139] sites for FXa on human platelets with a K d between 30 [42] and 200 pmol L 1 [139]. The binding of human FVa to human platelets is not saturated [138,139] at concentrations up to 12 nmol L 1 where, in the presence of FXa and prothrombin, 3000 molecules are bound per cell [138]. Nonetheless, the kinetic sequel of FVa binding, in terms of its ability to enhance the catalytic efficiency of FXa towards prothrombin, saturates either in the picamolar [138,139] or the nanomolar [140] range. Structural and functional characterization of the factor X-activating complex This section focuses on the characterization of the structural domains within enzyme (FIXa), cofactor (FVIIIa), and substrate (FX) molecules that interact within the FX-activating complex and defines the molecular domains and specific amino acid residues that mediate these important interactions. A major determinant of FIX/FIXa binding to human platelet receptors resides within its Gla domain [31] whereas the EGF1 domain of FIXa appears not to be involved in FIXa binding to platelets [21]. Our studies with both the Gla-modified and Gla-domainless FIXa molecules have shown that FIXa binding to platelets is mediated in part, but not exclusively, by residues in the Gla domain of FIX [31]. We then used nine different recombinant FIX molecules to show that the high-affinity, specific binding of FIXa to activated platelets in the presence or absence of FVIIIa and FX is mediated at least in part by amino acids exposed in the N-terminal region of the FIX Gla domain within position 3 11, possibly by residues 4, 5, 9, 10, and 11 [20]. Finally, we constructed a computer-generated molecular model of the Gla domain of FIX based upon the known structure of bovine prothrombin fragment 1 in order to identify amino acid residues exposed on the surface of the protein that might comprise a binding site for interaction with a platelet receptor [26]. Based upon this model, we prepared rationally designed, conformationally constrained synthetic peptides and used them to probe the functional role of the Gla domain in the interaction of FIX/ FIXa with platelets [26]. Taken together, these studies with chimeric and mutant FIX/FIXa molecules and with isolated protein domains and conformationally constrained synthetic peptides have confirmed the conclusion that the Gla domain of FIX/FIXa (as well as other vitamin K-dependent proteins), contains a sequence of amino acids (G 4 Q 11 ), referred to as the omega loop, that mediates its specific interaction with phospholipid membranes and with the activated platelet surface [18,20,21,26,31,36, ]. The evidence for this conclusion is that Gla-deficient and Gla-domainless FIX/IXa [31], chimeric FIX/IXa molecules with FVII or FX residues inserted into the G 4 Q 11 sequence of FIX [20], and FIX/IXa molecules with mutations at specific residues within the G 4 Q 11 sequence [20] bind to a reduced number of sites (n 250 per platelet, compared with 500 per platelet for normal plasma-derived or wild-type FIXa) with decreased affinity (K d nmol L 1, either in the presence or absence of FVIIIa, compared with 2.5 nmol L 1 for normal plasma-derived or wild-type FIXa in the absence of FVIIIa and 0.5 nmol L 1 in its presence) and promote FX activation at significantly decreased rates, a defect that is entirely explained by the decreased binding affinity. These observations are confirmed by the capacity of a conformationally constrained synthetic peptide (G 4 Q 11 ) to compete with FIX/IXa binding to the shared platelet binding site (n 250 per platelet) with a K i (3.0 nmol L 1 ) equal to the K d (3.0 nmol L 1 ) for FIX/IXa binding to the shared platelet binding site [26]. However, since this same peptide is required at fold higher concentration (K i 165 mmol L 1 )to inhibit rates of platelet-mediated FIXa-catalyzed FX activation, we have postulated the presence of an additional plateletbinding site within FIXa that mediates its assembly into the FX-activating complex [26,36]. These observations have given rise to the quite unexpected and interesting suggestion that although the Gla domain of FIX/ FIXa is important, as indicated by studies from a number of different laboratories, including our own, in mediating the interaction of this vitamin K-dependent protein with phospholipid membranes, the portion of the Gla domain (G 4 Q 11 ) implicated in phospholipid binding does not by itself mediate the interaction of FIXa with the site on activated platelets involved in the assembly of the functional FX-activating complex. In order to define the FIXa domain that is involved in the functional assembly of the FX-activating complex, we examined the second epidermal growth factor (EGF2) domain. Our initial studies with a chimeric FIXa molecule in which EGF2 was replaced with that of FX suggested that the EGF2 domain may be important for specific, high-affinity FIXa binding to platelets in the presence of FVIIIa and FX [18,36, ]. To further localize important residues, we have prepared several mutant FIXa molecules using the homologous sequences from

6 FX-activating complex assembly on activated human platelets 53 FVII: FVII-EGF2 (FIXD88-124,rFVII91-127),* loop 1 (FIXD88-99,rFVII91-102), loop 2 (FIXD95-109,rFVII98-112), and loop 3 (FIXD ,rFVII ). The mutants were tested for their ability to bind to platelets and activate FX. Direct binding studies with SFLLRN-amide (25 mmol L 1 ) activated platelets in the presence of FVIIIa (2 nmol L 1 ) and FX (1.5 mmol L 1 ) showed that loop 1 (FIX FVII ) and loop 2 (FIX FVII ) molecules were bound to reduced numbers of sites (380 and 290 sites per platelet, respectively) with decreased affinity (K d ¼ 19 nmol L 1 and 35 nmol L 1, respectively) compared with FIXa N (585 sites per platelet; K d ¼ 0.55 nmol L 1 ) or FIXa wt (640 sites per platelet; K d ¼ 0.8 nmol L 1 ). In contrast, the loop 3 (FIX FVII ) chimera interacted with platelets with normal affinity and stoichiometry. Our data clearly show that residues of FIXa are important for platelet binding and assembly of the FX activating complex [142,143]. To further examine the role of the EGF2 domain in FIXa binding we have prepared a rationally designed, conformationally constrained synthetic FIX EGF2 domain peptide and screened it for its capacity to inhibit FIXa binding to platelets and platelet-mediated FX activation. This peptide contains 45 residues (L 84 V 128 ) including six cysteines with three disulfide bridges, formed by selective protection and deprotection strategies [144] as in the native protein. In competition experiments using thrombin (0.1 U ml 1 ) activated platelets in the presence of both FVIIIa (2 nmol L 1 ) and FX (1.5 mmol L 1 ) the EGF2 peptide inhibited 125 I-FIXa binding to 300 sites per platelet with a K i 700 nmol L 1 compared with native FIXa (K i 2.5 nmol L 1 ). This inhibitory activity was lost upon reduction and alkylation of the peptide [142,143]. In future studies, it will be important to utilize this EGF2 peptide (L 84 V 128 )in addition to the Gla peptide (G 4 Q 11 ) to further examine the relative contributions of the EGF2 domain and the Gla domain in mediating FIXa interaction with platelet receptors important for the assembly of the FX-activating complex. Little is known about the molecular domain within the substrate (FX) that binds to platelets and promotes assembly of FX-activating complex. Our studies indicate that FX bound to the surface of activated platelets via the Gla domain [32] is preferentially activated by platelet bound FIXa [33]. It appears that the shared FX/prothrombin binding sites are quite distinct from those of FIX/FIXa and FVIIIa. Neither FX nor prothrombin (up to 1 mmol L 1 ) is able to displace a significant fraction of bound FIX or FIXa from the platelet surface [32]. In fact, when FVIIIa is present or absent, FIX brings about an increase in the affinity of the interaction between FIXa and activated platelets [29]. Further, the number of sites ( ) for enzyme (FIXa) and cofactor (FVIII/FVIIIa) is significantly lower than the shared FX/prothrombin binding sites. Similarly, in the prothrombinase complex, the presence of prothrombin *In this notation, ~ designates the deletion of the indicated amino acid sequence, whereas 5 designates the insertion of corresponding residues from FVII. has not been found to decrease the binding of FV, FVa [40,43,138] or FXa to unactivated platelets, and FX has been shown not to affect FXa binding to activated platelets [41,42]. In future studies, it will be important to identify the molecular domains within FX and prothrombin that mediate binding to the shared FX/prothrombin binding site on activated platelets and promote assembly of FX-activating complex, and to define the mechanisms by which high-affinity, low-capacity, specific sites are generated for FX (by FVIIIa) and for prothrombin (possibly by FVa). In an attempt to define the molecular domains within the cofactor FVIIIa that bind to platelets and promote assembly of the FX-activating complex, we have recently demonstrated that both procofactor (FVIII) and active cofactor (FVIIIa) bind to platelets with enhanced affinity in the presence of the enzyme (EGR-FIXa) and the substrate (FX), thus emphasizing the validity of a three-receptor complex on the platelet surface [25,145]. Recent studies indicate that it is the spontaneous dissociation of the A2 domain from the metal-linked A1/A3- C1-C2 dimer that causes the instability of FVIIIa activity [146,147], and that the presence of the A2 domain increases both the affinity and the stoichiometry of FVIIIa binding to activated platelets [25]. Earlier studies by Fay et al. [ ] indicated that the A2 subunit of FVIIIa markedly increases the catalytic activity (i.e. k cat of FIXa-catalyzed FX activation) by enhancing the reaction rate 100-fold. Furthermore, reconstitution of heterotrimeric FVIIIa from the isolated A2 subunit and A1/A3-C1-C2 dimer is also enhanced several fold in the presence of FIXa and phospholipid [152]. Based on these observations, a primary role of the A2 domain has been suggested in modulating the active site of FIXa in the FXactivating complex. Our results [25] confirm previous evidence [151,152] that the presence of both the A2 subunit and the A1/A3-C1-C2 dimer stabilizes the FX-activating complex, and clearly indicate that the presence of FVIII A2 subunit alone (but not the A1 subunit) increases the affinity of active cofactor (FVIIIa) binding in the presence of FIXa and FX. Addition of both A1 and A2 domains further enhances the affinity of FVIIIa binding to activated platelets in the presence of EGR-FIXa and FX [25], emphasizing that FX activation is a plateletreceptor-mediated process tightly coupled to receptor occupancy by FIXa, FVIIIa, and FX, and dependent upon multiple protein-protein contacts. More recent studies using functional assays [153] further indicated that the A1 and A2 subunits of FVIIIa synergistically stimulate FIXa catalytic activity yielding an overall increase in k cat of over 1000-fold, compared to FIXa alone. Taken together, our results from equilibrium binding studies and those of Fay et al. [151,153] from FX activation studies are consistent with the hypothesis that the primary mechanism for decay of the FX-activating complex under physiological conditions is the dissociation of the A2 subunit [ ]. Moreover, the addition of supplemental A1 domain (250 nmol L 1 ) enhances the affinity of FX binding (K d 2 nmol L 1 mol L 1 from 10 nmol L 1 ) while also increasing the number of FX binding sites (1800 from 1200 sites per platelet) [157].

7 54 S. S. Ahmad et al We have also focused our attention on defining the specific domains of FVIIIa that are responsible for platelet-receptormediated interactions [ ]. The anti-c2-domain monoclonal antibody, ESH4, which recognizes residues and inhibits phospholipid binding, inhibited 125 I-FVIIIa interaction with activated platelets (IC 50 ¼ 110 nmol L 1 ) in equilibrium binding assays, both in the presence and absence of EGR- FIXa and FX. In contrast, ESH8, which recognizes residues in the C2 domain of FVIII, did not inhibit FVIIIa platelet interaction but interestingly abolished both the enhanced stoichiometry and affinity of FVIIIa binding to platelets observed in the presence of EGR-FIXa and FX (K d ¼ nmol L 1 ; sites per platelet both in the absence and presence of EGR FIXa and FX). By using 125 I-FVIII (LC) (i.e. 72 kda A3-C1-C2 domain) and the radiolabeled LC fragments (obtained by protease digestion), we were able to show saturable and reversible equilibrium binding of LC to platelets (K d ¼ nmol L 1 ; sites per platelets). The presence of ESH4 (200 nmol L 1 ) in binding assays partially inhibited (60%) FVIII (LC) binding (K d ¼65 21 nmol L 1 ; sites per platelets). These data provide strong evidence for the presence of [1] a major platelet-receptor-mediated binding site (within residues ) in the C2 domain; and [2], an additional binding site (within residues ) in the C2 domain that increases the stoichiometry and affinity of FVIIIa binding to activated platelets observed in the presence of FIXa and FX. More recently we have examined the contribution of putative FVIII C2 domain (residues ) platelet binding sites to the binding of the FVIII/FVIIIa to activated platelets [159]. The rc2-domain interacts with the same number of binding sites (700 sites per platelets) with impaired affinity (K d 14 nmol L 1 ) compared with FVIIIa or FVIIIa (LC) (700 sites per platelet; K d 2 nmol L 1 ). This observation is further supported by competition studies with synthetic peptides corresponding to FVIII residues (comprising the epitope for the FVIII MoAb ESH4), which also showed a marked inhibition of FVIIIa binding (60%) compared with competition by native FVIIIa or FVIIIa (LC). In contrast a C2-domain peptide corresponding to residues (comprising the epitope for the FVIII MoAb ESH8) failed to inhibit FVIIIa binding to activated platelets [158,159]. These studies support the hypothesis that the C2-domain alone does not provide the totality of the binding energy for FVIIIa interaction with the platelet membrane and suggests that the A3 and/or C1 domain of FVIII (LC) may also contribute to the interaction of FVIIIa with activated platelets [159]. Three-receptor model for platelet-mediated factor IXa-catalyzed factor X activation The specific hypotheses resulting from our recent work and the work of many other laboratories are presented in schematic form in Fig. 2. Both FIX and FIXa bind specifically and with high affinity (K d 3.0 nmol L 1 ) to a discrete number of sites (n 250 per platelet) exposed on the activated platelet surface Fig. 2. The assembly of the FX activating complex on platelet membranes. The molecular domains of FIXa, FVIIIa and FX are shown with number of binding sites designated as N and values of dissociation constants as K d. EGF refers to epidermal growth factor-like domain; Gla represents g-carboxyglutamic acid domain. The elliptical structure inserted in the membrane represents a binding site of unknown composition that interacts with the EGF2 domain of FIXa. see text for explanation and details. [23]. Moreover, FIXa binds to an additional number (n 250 per platelet) of high-affinity (K d 3.0 nmol L 1 ), specific sites [23], which respond to the presence of FVIIIa with enhanced binding affinity (K d 0.5 nmol L 1 ). FX also binds to a highcapacity (n per platelet), low-affinity (K d 320 nmol L 1 ) platelet-binding site that is shared with prothrombin [32], occupancy of which is closely coupled to rates of FX activation in the absence of FVIII [33]. We have confirmed the demonstration by Nesheim et al. [47] of a specific, high-affinity (K d 3.7 nmol L 1 ) FVIII binding site (n 480 per platelet) and have demonstrated that the active cofactor, FVIIIa, binds to an additional sites with enhanced affinity (K d 1.5 nmol L 1 ), occupancy of which is closely coupled to rates of FX activation [25]. FVIIIa bound to the activated platelet surface generates a high-affinity (K d 30 nmol L 1 ), low-capacity (n 1200 per platelet) binding site for the substrate, FX, which, in contrast to the shared high-capacity (n per platelet), low-affinity (K d 320 nmol L 1 ) FX/FII plateletbinding site, is absolutely specific for FX [157]. The zymogen (FIX) potentiates FIXa-catalyzed FX activation by specifically and selectively increasing the affinity (four-fold) of FIXa binding to the activated platelet surface [29]. Coordinate binding studies [160] of FIXa and FVIIIa demonstrate that in the presence of saturating concentrations of FX, FIXa and procofactor FVIII form an equimolar complex, whereas 3 4 molecules of the active cofactor FVIIIa are bound (K d 0.8 nmol L 1 ) per molecule of FIXa (K d 0.5 nmol L 1 ) in the presence of saturating concentrations of FIX. Coordinate binding studies [161] of FX and FVIIIa demonstrate that in the presence of saturating concentrations of EGR-FIXa and prothrombin (to block the shared FX/FII binding site), FX binds to three-fold more platelet binding sites than in their absence (n 1400 per platelet) with relatively low affinity (K d 250 nmol L 1 ),

8 FX-activating complex assembly on activated human platelets 55 whereas in the presence of FVIIIa, FX binds with 25- to 50- fold enhanced affinity (K d 5 9 nmol L 1 ) in an equimolar stoichiometric complex, suggesting that the active cofactor (FVIIIa) binds with high affinity to the activated platelet surface and presents the substrate (FX) to the enzyme (FIXa). An inference to be drawn from these studies is that each of the components of the FX-activating complex binds to a separate and distinct receptor site on the activated platelet membrane and that the presence of the other two components significantly enhances the affinity and specificity of binding of any one component. Another inference to be drawn from our hypothesis is that the platelet FX-activating complex may function in part by providing a large target area for substrate binding and for funneling the bound substrate from a high-capacity, lowaffinity binding site to the low-capacity, high-affinity, highly specific enzyme cofactor complex. It should be emphasized that confirmation of this hypothesis, which can only come from careful correlations of equilibrium binding measurements with kinetic studies of FX activation on the platelet surface, has the potential to fundamentally alter our concepts of the assembly of the FX-activating complex derived from studies with model systems using phospholipid vesicles, since for the first time our data support the view that three separate, unique and specific receptorsoccupiedindependentlybythree-dimensional diffusion, mediate the contiguous localization of the enzyme, the cofactor, and the substrate into a complex by lateral, two-dimensional diffusion. It should also be emphasized that many of the equilibrium dissociation constants denoted may reflect the combined binding energy of both protein receptor and protein protein interactions. In many instances, the equilibrium dissociation constants of protein protein interactions are known, as shown, for example, in Fig. 2 for the binding of FX to FVIIIa [162,163] and the binding of FIXa to FVIIIa [67,151,164,165]. Shown in Fig. 3 is a global, sequential, reaction scheme for FIXa-mediated FX activation presenting our currently available equilibrium dissociation constants and stoichiometries for ligand receptor interactions and the kinetic constants (K m and k cat ) for FX activation in solution and on the activated platelet surface, either in the presence or in the absence of FVIIIa. As complex as this three-dimensional scheme may at first inspection appear, careful analysis of the equilibrium dissociation constants, stoichiometries and kinetic constants will reveal that thermodynamic equilibrium is maintained throughout, and in many instances equimolar stoichiometries of enzyme, cofactor and substrate are maintained. In conclusion, we believe that the elucidation of mechanisms by which the FX-activating complex is assembled on the platelet surface will provide information essential for understanding the central role of platelets in promoting normal hemostasis. Finally, we also suggest that the FX-activating complex as a product of platelet receptor interaction is a potentially important point of attack for the development of specific antithrombotic agents that might have the capacity to prevent thrombus formation without inducing a hemorrhagic state [145]. 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