Factors IXa and Xa play distinct roles in tissue factor-dependent initiation of coagulation

Tissue factor is the major initiator of coagulation. Both factor IX and factor X are activated by the complex of factor VIIa and tissue factor (VIIa/TF). The goal of this study was to determine the specific roles of factors IXa and Xa in initiating coagulation. We used a model system of in vitro coagulation initiated by VIIa/TF and that included unactivated platelets and plasma concentrations of factors II, V, VIII, IX, and X, tissue factor pathway inhibitor, and antithrombin III. In some cases, factor IX and/or factor X were activated by tissue factor- bearing monocytes, but in some experiments, picomolar concentrations of preactivated factor IX or factor X were used to initiate the reactions. Timed samples were assayed for both platelet activation and thrombin activity. Factor Xa was 10 times more potent than factor IXa in initiating platelet activation, but factor IXa was much more effective in promoting thrombin generation than was factor Xa. In the presence of VIIa/TF, factor X was required for both platelet activation and thrombin generation, while factor IX was only required for thrombin generation. We conclude that VIIa/TF-activated factors IXa and Xa have distinct physiologic roles. The main role of factor Xa that is initially activated by VIIa/TF is to activate platelets by generating an initial, small amount of thrombin in the vicinity of platelets. Factor IXa, on the other hand, enhances thrombin generation by providing factor Xa on the platelet surface, leading to prothrombinase formation. Only tiny amounts of factors IX and X need to be activated by VIIa/TF to perform these distinct functions. Our experiments show that initiation of coagulation is highly dependent on activation of small amounts of factors IXa and Xa in proximity to platelet surfaces and that these factors play distinct roles in subsequent events, leading to an explosion of thrombin generation. Furthermore, the specific roles of factors IXa and Xa generated by VIIa/TF are not necessarily reflected by the kinetics of factor IXa and Xa generation.     

Monocyte and Haemostasis

Human monocytes may play a central role in haemostasis since they are the only circulating blood cells capable to express tissue factor (TF) and therefore trigger the extrinsic pathway of coagulation. The aim of this review is to illustrate that monocytes also participate in haemostasis independently of TF expression. Indeed, the exposure of anionic phospholipids provides a procoagulant surface. Activation of factor X involves specific membrane receptor EPR-1 (Effector cell Protease Receptor-1). Mac-1 and membrane factor Va also bind factor X/Xa. In addition, monocytic proteases have been reported to modulate the coagulation cascade. Monocyte adhesion and cell—cell interactions represent important mechanisms implicated in TF expression. Furthermore, Tissue factor pathway inhibitor (TFPI), the natural inhibitor of TF activity, is expressed by monocytes. This suggests that monocytes could participate to the local regulation of coagulation initiation. The importance of monocytes in the development of vascular diseases remains to be clarified. In thrombotic disorders, it may be worthwhile not to limit the investigations to the expression of TF.     

Enhancement by human umbilical vein endothelial cells of factor Xa- catalyzed activation of factor VII

In blood coagulation on endothelium, an unperturbed vascular endothelial cell surface apparently provides activity equivalent to the phospholipid needed for generation of factor Xa and thrombin in soluble systems. Phospholipid in soluble systems also markedly enhances the ability of factor Xa to activate factor VII; therefore we investigated the influence of an unperturbed monolayer of human umbilical vein endothelial cells (HUVEC) upon factor VII activation. HUVEC were found to augment factor Xa-catalyzed activation of factor VII. This appeared to result from the binding of trace amounts of factor Xa to the cells. Adding active site-inhibited factor Xa to reaction mixtures, but not factor X, abolished the enhanced activation. Adding either anti-factor V antibodies or exogenous factor Va had no effect upon reaction rates. Thus factor Va does not function as a cofactor for the reaction. In further experiments the effect upon activation of factor VII and prothrombin was studied by varying the order of addition of factor Xa and factor Va to supernatants of HUVEC monolayers. Evidence was obtained that HUVEC, unlike platelets, possess a functional factor Xa binding site that is independent of factor Va. Since phospholipid is the only known cofactor for factor Xa/Ca2+-induced activation of factor VII, the demonstration of enhanced activation of factor VII in the presence of unperturbed cultured HUVEC supports a hypothesis that the functional equivalent of procoagulant phospholipid is available on the luminal surface of vascular endothelium in vivo.     

Factor Xa binding to annexin 2 mediates signal transduction via protease-activated receptor 1

The serine protease zymogen factor X is converted to its catalytically active form factor Xa by the binary complex of factor VIIa bound to its cell surface receptor tissue factor (TF) or by the intrinsic Xase complex, which consists of active factors VIII (VIIIa), IX (IXa), factor X, and Ca2+. Factor Xa has procoagulant activity by conversion of prothrombin to thrombin and also induces signal transduction, either alone or in the ternary TF:VIIa:factor Xa coagulation initiation complex. Factor Xa cleaves and activates protease activated receptor (PAR)1 or -2, but factor Xa signaling efficiency varies among cell types. We show here that annexin 2 acts as a receptor for factor Xa on the surface of human umbilical vein endothelial cells and that annexin 2 facilitates factor Xa activation of PAR-1 but does not enhance coagulant function of factor Xa. Overexpression of TF abolishes annexin 2 dependence on factor Xa signaling and diminishes binding to cell surface annexin 2, whereas selectively abolishing TF promotes the annexin 2/factor Xa interaction. We propose that annexin 2 serves to regulate factor Xa signaling specifically in the absence of cell surface TF and may thus play physiological or pathological roles when factor Xa is generated in a TF-depleted environment.     

Platelet procoagulant complex assembly in a tissue factor-initiated system

Summary. The aim of this study was to examine the assembly of the factor IXa/VIIIa (Xase) and factor Xa/Va (IIase) complexes on the platelet surface in a system designed to mimic tissue factor-initiated coagulation. The experimental system contained tissue factor-bearing monocytes, unactivated platelets, and plasma concentrations of factors V, VIII, IX, X, prothrombin, tissue factor pathway inhibitor (TFPI), antithrombin III (ATIII), and small amounts of factor VIIa. The time courses of platelet activation, coagulation factor binding and thrombin generation were compared. In this system, thrombin generation by the combination of monocytes and platelets was synergistic compared to each cell type alone. Platelet activation and thrombin generation were minimal in the absence of prothrombin or factor X. After a lag period, platelet activation began, followed by progressive binding of factors Va and VIIIa. This was followed by factor IXa and Xa binding and the onset of thrombin generation. Unexpectedly, a transient early increase in platelet-associated factor IX and X was also seen, that was due to release from platelets. The amount of factor IX bound to isolated activated platelets was increased by addition of factor VIIIa, or by activation of factor IX to IXa. In contrast, factor VIIIa binding was not altered by the presence of factor IX or IXa. We conclude that in a tissue factor-initiated system, assembly of the procoagulant complexes on the platelet surface begins after platelet activation occurs. Platelet activation requires thrombin generation in the vicinity of the tissue factor bearing cells. The cofactors Va and VIIIa bind to the platelets and facilitate subsequent binding of factors IXa and Xa to form functional procoagulant complexes.     

Neutralization of factor X activity by factor X-specific monoclonal antibodies.

Factor X, a vitamin K-dependent protein, is the plasma zymogen for the active serine protease factor Xa. Factor Xa is the proteolytic enzyme for prothrombinase, the multi-protein membrane complex that catalyses the cleavage of prothrombin to thrombin. A panel of 10 monoclonal antibodies (identified by their corresponding clone numbers: 1, 2, 3, 5, 7, 26, 27, 54, 73, and 79) to factor X were produced by immunizing mice with purified factor X. All of the antibodies bound both human factor X and factor Xa in a solid-phase ELISA and binding of the antibodies was not affected by removal of Ca2+ with EDTA. In immunoblot analysis, antibody alpha HFX-54 bound to the light chain and antibodies alpha HFX-1, -5, -7, and -26 bound to the heavy chain of reduced factor X. Antibodies alpha BFX-2b, alpha HFX-27, -54, and -73 prolonged both the factor X-dependent clotting time and activated partial thromboplastin time (APTT) of normal plasma while antibody alpha HFX-1 only prolonged the APTT. None of the antibodies significantly inhibited factor X activation by purified Russell's viper venom factor X activator. In prothrombin activation assays using purified factor Xa, factor Va, prothrombin, Ca2+ and phospholipid vesicles, seven of the antibodies (alpha HFX-1, -3, -26, -27, -54, -73 and alpha BFX2b) showed some inhibition of thrombin generation ranging from 18 to 60% of the control. The decrease in factor X plasma clotting activity was most likely due to inhibition of factor Xa activity in prothrombinase, although some antibody-dependent inhibition of factor X activation may contribute to the observed inhibition of plasma clotting. Prothrombinase activity on platelets was inhibited in an identical manner by the monoclonal antibodies. When prothrombin was activated in the absence of factor Va, only antibody alpha BFX-2b inhibited activation. Calcium-independent determinants on both the heavy chain (determinants 1 and 26) and light chain (determinant 54) of factor X may play a role in prothrombin activation by prothrombinase. Other epitopes (antibodies alpha HFX-3, -27, -73) appeared to be influenced by association of factor Xa with factor Va. Topographic regions on factor X important for factor X activation and factor Xa function may be identified by the use of these monoclonal antibodies.     

Mechanistic coupling of protease signaling and initiation of coagulation by tissue factor

The crucial role of cell signaling in hemostasis is clearly established by the action of the downstream coagulation protease thrombin that cleaves platelet-expressed G-protein-coupled protease activated receptors (PARs). Certain PARs are cleaved by the upstream coagulation proteases factor Xa (Xa) and the tissue factor (TF)--factor VIIa (VIIa) complex, but these enzymes are required at high nonphysiological concentrations and show limited recognition specificity for the scissile bond of target PARs. However, defining a physiological mechanism of PAR activation by upstream proteases is highly relevant because of the potent anti-inflammatory in vivo effects of inhibitors of the TF initiation complex. Activation of substrate factor X (X) by the TF--VIIa complex is here shown to produce enhanced cell signaling in comparison to the TF--VIIa complex alone, free Xa, or Xa that is generated in situ by the intrinsic activation complex. Macromolecular assembly of X into a ternary complex of TF--VIIa--X is required for proteolytic conversion to Xa, and product Xa remains transiently associated in a TF--VIIa--Xa complex. By trapping this complex with a unique inhibitor that preserves Xa activity, we directly show that Xa in this ternary complex efficiently activates PAR-1 and -2. These experiments support the concept that proinflammatory upstream coagulation protease signaling is mechanistically coupled and thus an integrated part of the TF--VIIa-initiated coagulation pathway, rather than a late event during excessive activation of coagulation and systemic generation of proteolytic activity.     

Deficiency of factor Xa-factor Va binding sites on the platelets of a patient with a bleeding disorder.

Factor V (Va) is essential for binding of factor Xa to the surface of platelets. After thrombin treatment, normal platelets release at least five times more factor Va activity than is required for maximal factor Xa binding. The concentration of factor V activity obtained after thrombin stimulation of 10(7) normal platelets is sufficient to allow half-maximal factor Xa binding to 10(8) platelets (10% normal, 90% factor-V deficient). Therefore, factor Va activity is not limiting in platelet-surface factor Xa binding and prothrombin activation in normal platelets; some other components limit the number of binding sites. We report studies of a patient (M.S.) with a moderate to severe bleeding abnormality whose platelets are deficient in the platelet-surface component required for the factor Va-factor Xa binding. The patient's platelet factor Va activity released after thrombin treatment is normal, but factor Xa binding is 20%-25% of control values at saturation. Abnormal prothrombin consumption in a patient with normal plasma coagulation factors and platelet function suggests a disorder in platelet-surface thrombin formation.     

Effects of storage-induced platelet microparticles on the initiation and propagation phase of blood coagulation

Platelets shed microparticles, which support haemostasis via adherence to the damaged vasculature and by promoting blood coagulation. We investigated mechanisms through which storage-induced microparticles might support blood coagulation. Flow cytometry was used to determine microparticle number, cellular origin and surface expression of tissue factor (TF), procoagulant phosphatidylserine (PtdSer) and glycoprotein (GP) Ib-alpha. The influence of microparticles on initiation and propagation of coagulation were examined in activated factor X (factor Xa; FXa) and thrombin generation assays and compared with that of synthetic phospholipids. About 75% of microparticles were platelet derived and their number significantly increased during storage of platelet concentrates. About 10% of the microparticles expressed functionally active TF, as measured in a FXa generation assay. However, TF-driven thrombin generation was only found in plasma in which tissue factor pathway inhibitor (TFPI) was neutralised, suggesting that microparticle-associated TF in platelet concentrates is of minor importance. Furthermore, 60% of all microparticles expressed PtdSer. In comparison with synthetic procoagulant phospholipids, the maximal rate of thrombin formation in TF-activated plasma was 15-fold higher when platelet-free plasma was titrated with microparticles. This difference could be attributed to the ability of microparticles to propagate thrombin generation by thrombin-activated FXI. Collectively, our findings indicate a role of microparticles in supporting haemostasis by enhancement of the propagation phase of blood coagulation.     

An inhibitory monoclonal antibody to factor X that blocks prothrombin activation but not prothrombinase enzyme assembly

A monoclonal antibody (designated alpha BFX-2b) prepared against bovine factor X inhibited factor X activity in human, bovine, porcine, rabbit, and canine plasma. In assays using purified prothrombinase components, factor Xa, factor Va, phospholipid vesicles, and calcium ion with the fluorescent active site thrombin inhibitor dansylarginyl-N-(3-ethyl-1,5- pentanediyl)amide, the antibody inhibited the conversion of prothrombin to thrombin. Antibody alpha BFX-2b also blocked prothrombinase cleavage of the macromolecular substrates prethrombin 1 and prethrombin 2 but did not inhibit factor Xa hydrolysis of the synthetic substrate benzoyl- Ile-Glu-Gly-Arg-p-nitroanilide. The antibody also prevented the inactivation of factor Xa by antithrombin III but did not prevent the inactivation by soybean trypsin inhibitor. Antibody alpha BFX-2b bound factor Xa with a stoichiometry of 1:1 and an apparent dissociation constant of 9.0 x 10(-11) mol/L as estimated from its inhibition of prothrombinase activity. Antibody alpha BFX-2b did not prevent binding of factor Xa to factor Va-phospholipid as measured by using fluorescence polarization or high-pressure liquid gel chromatography with the fluorescent Factor Xa analogue dansyl-glutamyl-glycyl-arginyl- Xa. Immunoblotting of factor X following electrophoresis on sodium dodecyl sulphate-polyacrylamide gels and transfer to nitrocellulose indicated that the antigenic determinant recognized by antibody alpha BFX-2b was found on the heavy chain of factors X and Xa. From these observations it can be concluded that antibody alpha BFX-2b recognizes a highly conserved epitope on the factor X heavy chain that is remote from the topographic sites required for prothrombinase complex assembly and substrate hydrolysis but may be located at or near a portion of the macromolecular substrate binding site.     

Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa

Protease-activated receptor 2 (PAR2) is expressed by vascular endothelial cells and other cells in which its function and physiological activator(s) are unknown. Unlike PAR1, PAR3, and PAR4, PAR2 is not activatable by thrombin. Coagulation factors VIIa (FVIIa) and Xa (FXa) are proteases that act upstream of thrombin in the coagulation cascade and require cofactors to interact with their substrates. These proteases elicit cellular responses, but their receptor(s) have not been identified. We asked whether FVIIa and FXa might activate PARs if presented by their cofactors. Co-expression of tissue factor (TF), the cellular cofactor for FVIIa, together with PAR1, PAR2, PAR3, or PAR4 conferred TF-dependent FVIIa activation of PAR2 and, to lesser degree, PAR1. Responses to FXa were also observed but were independent of exogenous cofactor. The TF/FVIIa complex converts the inactive zymogen Factor X (FX) to FXa. Strikingly, when FX was present, low picomolar concentrations of FVIIa caused robust signaling in cells expressing TF and PAR2. Responses in keratinocytes and cytokine-treated endothelial cells suggested that PAR2 may be activated directly by TF/FVIIa and indirectly by TF/FVIIa-generated FXa at naturally occurring expression levels of TF and PAR2. These results suggest that PAR2, although not activatable by thrombin, may nonetheless function as a sensor for coagulation proteases and contribute to endothelial activation in the setting of injury and inflammation. More generally, these findings highlight the potential importance of cofactors in regulating PAR function and specificity.     

Internal duplication and sequence homology in factors V and VIII.

Blood coagulation factors V and VIII each serve cofactor functions with different vitamin K-dependent serine proteases of the coagulation cascade. Physical, physiologic, and kinetic data suggest analogous structures and functions for these two proteins. Proteolytically activated factor V (factor Va) is required for the efficient production of thrombin from prothrombin by factor Xa. Similarly, activated factor VIII (factor VIIIa) performs its cofactor activity with factor IXa to produce the activated form of factor X (factor Xa). The studies reported here on the sequences from the thrombin-activated and unactivated cofactors provide evidence that factor V and factor VIII are chemically related and that the structures of both cofactors involve some tandem duplication.     

Human coagulation factor Va is a cofactor for the activation of protein C.

During blood clotting in vitro, protein C is converted in part to protein Ca, Protein Ca, in turn, inactivates factor Va. This is evidenced by the rapid inactivation of factor Va coagulant activity after clot formation which is associated with the cleavage of the Mr 110,000 peptide of factor Va. When exogenous factor Va is added to serum, it is inactivated only after a lag of 10-20 min. Using purified coagulation factors in the presence of EDTA, we demonstrated that factor Va enhances the rate of protein C activation by thrombin by 50-fold. The Km for factor Va in the reaction is 14 nM, 100 times higher than its Km for accelerating platelet surface prothrombin activation by factor Xa. By this mechanism, factor Va can act as a procoagulant as well as limit dissemination of the coagulation process through the activation of protein C and the subsequent inactivation of both factor Va and factor VIIIa.     

Structural requirements for TFPI-mediated inhibition of neointimal thickening after balloon injury in the rat.

The intimal thickening that follows vascular injury is inhibited by periprocedural tissue factor pathway inhibitor (TFPI) treatment in animal models. TFPI is a multivalent Kunitz-type protease inhibitor that inhibits factor Xa via its second Kunitz domain and the factor VIIa/tissue factor (TF) complex via its first Kunitz domain. The basic C-terminus of TFPI is required for the binding of TFPI to cell surfaces and cell-bound TFPI mediates the internalization and degradation of factor X and the down regulation of surface factor VIIa/TF activity. The C-terminus of TFPI is also required for its reported direct inhibition of smooth muscle cell proliferation in vitro. To examine the structural requirements for the inhibition of neointimal formation by TFPI, several TFPI-related proteins were tested in the rat carotid angioplasty model: 1) XK(1), a hybrid protein containing the N-terminal portion of factor X and the first Kunitz domain of TFPI that directly inhibits factor VIIa/TF; 2) TFPI(WT), the full-length TFPI molecule that inhibits factor Xa and factor VIIa/TF and binds cell surfaces; 3) TFPI(K36I), an altered form of TFPI that inhibits factor Xa, but not factor VIIa/TF, and binds cell surfaces; 4) TFPI(13-161), a truncated form of TFPI that inhibits factor VIIa/TF but interacts with factor Xa poorly and does not bind to cell surfaces. Seven day infusions of XK(1), TFPI(WT), and high levels of TFPI(K36I) begun the day before balloon-induced vascular injury produced a significant reduction in the intimal hyperplasia measured 28 days after angioplasty. The infusion of high concentrations of TFPI(13-161) was ineffective in this model. These in vivo results directly mirror the ability of each TFPI-related protein to inhibit tissue thromboplastin-induced coagulation in rat plasma: XK(1) approximately TFPI(WT)>TFPI(K36I)>TFPI(13-161). The studies confirm the important role of TF-mediated coagulation in the smooth muscle proliferation and neointimal thickening that follows vascular injury and suggest that the anticoagulant effect alone of TFPI and TFPI-related proteins is sufficient to explain their therapeutic action.     

Inhibition of thrombin generation by the zymogen factor VII: implications for the treatment of hemophilia A by factor VIIa

Factor VII circulates as a single chain inactive zymogen (10 nmol/L) and a trace ( approximately 10-100 pmol/L) circulates as the 2-chain form, factor VIIa. Factor VII and factor VIIa were studied in a coagulation model using plasma concentrations of purified coagulation factors with reactions initiated with relipidated tissue factor (TF). Factor VII (10 nmol/L) extended the lag phase of thrombin generation initiated by 100 pmol/L factor VIIa and low TF. With the coagulation inhibitors TFPI and AT-III present, factor VII both extended the lag phase of the reaction and depressed the rate of thrombin generation. The inhibition of factor Xa generation by factor VII is consistent with its competition with factor VIIa for TF. Thrombin generation with TF concentrations >100 pmol/L was not inhibited by factor VII. At low tissue factor concentrations (<25 pmol/L) thrombin generation becomes sensitive to the absence of factor VIII. In the absence of factor VIII, factor VII significantly inhibits TF-initiated thrombin generation by 100 pmol/L factor VIIa. In this hemophilia A model, approximately 2 nmol/L factor VIIa is needed to overcome the inhibition of physiologic (10 nmol/L) factor VII. At 10 nmol/L, factor VIIa provided a thrombin generation response in the hemophilia model (0% factor VIII, 10 nmol/L factor VII) equivalent to that observed with normal plasma, (100% factor VIII, 10 nmol/L factor VII, 100 pmol/L factor VIIa). These results suggest that the therapeutic efficacy of factor VIIa in the medical treatment of hemophiliacs with inhibitors is, in part, based on overcoming the factor VII inhibitory effect. (Blood. 2000;95:1330-1335)     

The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action

Blood coagulation is initiated when plasma factor VII(a) binds to its essential cofactor tissue factor (TF) and proteolytically activates factors X and IX. Progressive inhibition of TF activity occurs upon its addition to plasma. This process is reversible and requires the presence of VII(a), catalytically active Xa, Ca2+, and another component that appears to be associated with the lipoproteins in plasma, a lipoprotein-associated coagulation inhibitor (LACI). A protein, LACI(HG2), possessing the same inhibitory properties as LACI, has recently been isolated from the conditioned media of cultured human liver cells (HepG2). Rabbit antisera raised against a synthetic peptide based on the N-terminal sequence of LACI(HG2) and purified IgG from a rabbit immunized with intact LACI(HG2) inhibit the LACI activity in human serum. In a reaction mixture containing VIIa, Xa, Ca2+, and purified LACI(HG2), the apparent half-life (t1/2) for TF activity was 20 seconds. The presence of heparin accelerated the initial rate of inhibition threefold. Antithrombin III alpha alone had no effect, but antithrombin III alpha with heparin abrogated the TF inhibition. LACI(HG2) also inhibited Xa with an apparent t1/2 of 50 seconds. Heparin enhanced the rate of Xa inhibition 2.5-fold, whereas phospholipids and Ca2+ slowed the reaction 2.5-fold. Xa inhibition was demonstrable with both chromogenic substrate (S-2222) and bioassays, but no complex between Xa and LACI(HG2) could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Nondenaturing PAGE, however, showed that LACI(HG2) bound to Xa but not to X or Xa inactivated by diisopropyl fluorophosphate. Thus, LACI(HG2) appears to bind to Xa at or near its active site. Bovine factor Xa lacking its gamma-carboxyglutamic acid-containing domain, BXa(-GD), through treatment with alpha-chymotrypsin, was used to further investigate the Xa requirement for VIIa/TF inhibition by LACI(HG2). LACI(HG2) bound to BXa(-GD) and inhibited its catalytic activity against a small molecular substrate (Spectrozyme Xa), though at a rate approximately sevenfold slower than native BXa. Preincubation of LACI(HG2) with saturating concentrations of BXa(-GD) markedly retarded the subsequent inhibition of BXa. The VII(a)/TF complex was not inhibited by LACI(HG2) in the presence of BXa(-GD), and further, preincubation of LACI(HG2) with BXa(-GD) slowed the inhibition of VIIa/TF after the addition of native Xa. The results are consistent with the hypothesis that inhibition of VII(a)/TF involves the formation of a VIIa-TF-XA-LACI complex that requires the GD of XA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690–2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.     

Prothrombinase complex assembly on the platelet surface is mediated through the 74,000-dalton component of factor Va.

The blood coagulation protein factor Va forms the receptor for the serine protease factor Xa on the platelet surface. This membrane-bound complex of factor Va and factor Xa plus Ca2+ comprises the prothrombinase complex, the enzyme that catalyzes the proteolytic conversion of prothrombin to the clotting enzyme thrombin. Factor Va is a two-subunit protein composed of component D (Mr = 94,000) and component E (Mr = 74,000); subunit interaction is Ca2+ dependent. Factor Va bound to platelets consists of three peptides: component D, component E, and component D'(Mr = 90,000) which appears as the result of a platelet-associated protease cleavage of component D. The present studies were undertaken to determine which peptide(s) mediates the binding of factor Va to the platelet membrane surface and which peptide(s) serves as the binding site for factor Xa. These interactions were assessed by direct measurements of radiolabeled factor Va and factor Xa binding to platelets as well as autoradiographic visualization of the factor Va peptides associated with the platelet. Experiments were performed to determine the interaction of components D and E with platelets under reaction conditions in which components D and E were present as either the intact, functional two-subunit protein or as nonfunctional discrete peptides dissociated by the addition of Na2EDTA. The results suggest that component E mediates the binding of factor Va to the platelet and also serves as the binding site for the interaction of factor Xa with platelet-bound factor Va.     

Protein S binds to and inhibits factor Xa.

Although human protein S binds to human factor Va and inhibits prothrombinase activity, this inhibition is not totally dependent on factor Va. Hence, we investigated possible interaction of protein S with human factor Xa. Factor Xa, diisopropylphospho-factor Xa and their biotin derivatives ligand blotted specifically to protein S and protein S ligand blotted specifically to factor X and factor Xa. Biotinylated factors X and Xa bound to immobilized protein S and, reciprocally, protein S bound to immobilized factor Xa with a Kd of approximately 19 nM. In fluid phase, protein S bound to factor Xa with a Kd of approximately 18 nM. Protein S at 33 nM reversibly inhibited 50% of factor Xa amidolytic activity. Protein S inhibition of prothrombin conversion to thrombin by factor Xa was phospholipid-independent and was 1.6 times stimulated by Ca2+ ions. Inhibition of prothrombinase activity by protein S was 2.3-fold more potent in the presence of factor Va, with 50% inhibition at approximately 8 nM protein S. Protein S prolonged the factor Xa one-stage clotting time of protein S-depleted plasma in a dose-dependent manner. These data demonstrate mechanisms of anticoagulant action for protein S that are independent of activated protein C and that involve direct binding to factors Xa and Va and direct inhibition of factor Xa.     

Orchestration of coagulation protease signaling by tissue factor

Protease-activated receptors (PARs) are vascular sensors for signaling of the trypsinlike coagulation serine proteases that play key roles in cardiovascular medicine. In the initiation phase of coagulation, tissue factor (TF) orchestrates the assembly of VIIa with substrate X, forming a ternary complex in which product Xa is generated. The resulting TF-VIIa-Xa complex is an efficient activator of PAR1 and PAR2. TF initiation of the coagulation cascade is thus intimately linked to inflammatory cell signaling. Inflammation is an increasingly appreciated component of the vulnerable atherosclerotic plaque. Targeting inflammatory cell signaling events of the coagulation system may become an important aspect of efforts to improve antithrombotic therapy.