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.     

Activation of human factor VII by factors IXa and Xa on human bladder carcinoma cells

Single chain factor VII is converted by limited proteolysis to its activated form, factor VIIa, by a number of blood coagulation proteases including factor IXa and factor Xa. We have determined the relative rate of human factor VII activation by human factors IXa and Xa in two different systems: one containing Ca++ and human bladder carcinoma (J82) cells, and the other containing Ca++ and mixed brain phospholipids. The rate of factor VII activation was determined by a one stage coagulation assay, and proteolytic cleavage of factor VII was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting techniques. On a molar basis, factor Xa was sixfold more efficient than factor IXa beta in activating factor VII when the activation reaction occurs on J82 cell surfaces. In contrast, when incubation takes place in a suspension of mixed phospholipids, factor Xa was 18-fold more efficient in activating factor VII than factor IXa beta. In addition, factor IXa alpha activated factor VII at a rate approximately one-half that observed using factor IXa beta. In the absence of cells or phospholipids, no activation of factor VII by either factors IXa or Xa was observed. The addition of stoichiometric amounts of either recombinant human factor VIII (des B-domain) or plasma-derived factor VIIIa failed to augment the rate of factor VII activation by either factors IXa alpha or IXa beta. Likewise, purified human factor Va failed to influence the rate of factor VII activation by factor Xa in either system. Collectively, our studies reveal that J82 cells possess procoagulant phospholipid capable of readily supporting the activation of factor VII by either factors IXa beta or Xa. Our data also demonstrate that the relative ability of factor IXa beta and Xa to activate factor VII is significantly different when these reactions occur on tumor cell surfaces as compared with suspensions of mixed phospholipids.     

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.     

Flow cytometric analysis of agonist-induced annexin V, factor Va and factor Xa binding to human platelets

Activated platelets provide a procoagulant surface for the assembly and expression of prothrombinase complex. Expression of activity is associated with the binding of the protease factor Xa (FXa) and the co-factor Va (FVa) to the procoagulant surface. A flow cytometric methodology to measure annexin V-FITC as well as FVa and FXa binding to ionophore A 23187 activated platelets is described. Annexin V-FITC was used to determine platelet exposure of phosphatidylserine. The binding was calcium-dependent and excess of unlabelled annexin V (10-fold) prevented the binding of the labelled protein. The binding of FVa and FXa to platelets was measured using specific FITC-labelled monoclonal antibodies. The FITC labelled antibodies were displaced by 10-to 20-fold excess of unlabelled antibodies. Binding was strictly Ca2+-dependent. Fixation of platelets by formaldehyde caused artificial binding of annexin V, FVa and FXa as well, irrespective of the platelet activation status. Using gel-filtered platelets, the binding of FVa increased with alpha -granule secretion but the amount of stored FVa was not sufficient to saturate the available platelet binding sites. Exogenous FVa was needed for maximal FVa binding to occur. No binding of FXa from internal platelet stores was observed. Addition of exogenous FVa and FXa resulted in FXa binding to the platelet surface. The methodology might be of use for the study of platelets from patients with bleeding disorders.     

Platelet coagulation factor Va: the major secretory platelet phosphoprotein

Platelet-derived coagulation factor Va is the primary secreted substrate for a thrombin-stimulation-dependent platelet kinase. Human platelet factor Va, consisting of a molecular weight (M(r)) 105,000 heavy chain and an M(r) 74,000 light chain, incorporates phosphate in at least two sites on the light chain. Phosphorylated factor Va represents 50% of the secreted protein-associated phosphate. This modification occurs exclusively at serine residues and is inhibited by H-7 and staurosporine, which suggests a protein kinase C (PKC)-mediated event. Purified plasma factor V and Va are phosphorylated in the light chain region by rat brain PKC. The activity of platelet factor Va in prothrombinase on platelets is not altered when phosphorylation is inhibited by staurosporine. Plasma-derived factor Va in the presence of thrombin stimulated platelets is phosphorylated on both the heavy chain and the light chain. Plasma factor V and factor Va heavy chain phosphorylation occurs without light chain phosphorylation in the presence of added 32P gamma-ATP and non-stimulated or collagen- stimulated platelets or casein kinase II. This differential phosphorylation of factor Va heavy and light chain shows two independent platelet kinase activities that act on factor Va. The heavy chain factor V/Va kinase activity is similar to casein kinase II, which we have demonstrated previously to act on factor Va and accelerate activated protein C inactivation of the cofactor. Our data show platelet-dependent phosphorylation of platelet and plasma factor V and Va resulting in significant covalent modifications of the cofactor. These modifications may play a role in directing the extracellular distribution of factor V and factor Va.     

Factor Xa interacts with two sites on monocytes with different functional activities.

Studies were performed to elucidate the functional significance of factor Xa interactions at the monocyte membrane in the presence and absence of factor Va, with respect to prothrombin and factor IX cleavage. Factor Xa-catalyzed prothrombin activation at the monocyte surface was absolutely dependent on the addition of factor Va, indicating that thrombin was generated solely by a membrane-bound complex of factors Va and Xa. In contrast, in the absence of added factor Va, factor Xa bound to monocytes catalyzed the cleavage of factor IX to the nonenzymatic intermediate factor IX alpha through a reaction that was dependent on both monocyte and factor Xa concentration. At limiting factor Xa concentration, added factor Va inhibited the factor Xa-catalyzed cleavage of factor IX, suggesting that a monocyte-bound complex of factors Va and Xa did not recognize factor IX as a substrate. These combined data suggest that factor Xa interacts with the monocyte through two sites which can be distinguished by their requirement for added factor Va and their expression of different functional activities. Both functional sites could be distinguished also by their differential susceptibility to inhibition by a monoclonal antibody directed against the light chain of factor Va (alpha-HFV1). At the monocyte surface, the factor Va/Xa-catalyzed activation of prothrombin was maximally inhibited with 0.25 mumol/L alpha-HFV1, whereas 1.0 mumol/L alpha-HFV1 was required to effect 50% inhibition of the factor Xa-catalyzed cleavage of factor IX. The ability of factor Va to modulate factor Xa substrate specificity was investigated further. Factor Xa bound to thrombin-activated platelets either through platelet-released factor Va or added factor Va did not cleave factor IX. Consistent with this result, a plasma concentration of factor IX had no effect on thrombin generation catalyzed by a platelet-bound complex of factors Va and Xa. In marked contrast, factor Xa bound to phospholipid vesicles either independently or in complex with factor Va catalyzed factor IX cleavage with equal efficiency. These combined data indicate that factor Va bound to cell surfaces modulates factor Xa substrate specificity, whereas no discriminatory effect is conferred by factor Va bound to phospholipid vesicles. Thus, by providing two distinct sites at its membrane surface, the monocyte modulates factor Xa binding and the functional activity expressed by the bound enzyme, depending on the availability of factor Va.     

Protease-activated receptor 1 is the primary mediator of thrombin-stimulated platelet procoagulant activity

The activation of human platelets by thrombin is mediated primarily by protease-activated receptors (PARs). PAR1 and PAR4 are present on human platelets and are activated by the hexapeptides SFLLRN and GYPGQV, respectively. To further characterize the involvement of PAR1 and PAR4 in platelet activation, the ability of SFLLRN or GYPGQV to generate annexin V binding to newly exposed phospholipids on the platelet surface and generate procoagulant activity has been examined. Exposure of phosphatidylserine and phosphatidylethanolamine on platelets, as determined by an increase in annexin V binding, was strongly stimulated by SFLLRN, thrombin, and collagen, but only to a minor extent by GYPGQV. In a clotting assay initiated with factor VIIa, soluble tissue factor, and calcium, the clotting time in the absence of platelets was >5 min. In the presence of unstimulated platelets, the clotting time was 200 +/- 20 sec. In the presence of platelets activated with SFLLRN or collagen, the clotting time decreased to 100 +/- 10 sec. This shortening of the clotting time is equivalent to about a 5-fold increase in coagulant activity when stimulated platelets are compared with unstimulated platelets and activated platelets are used as a reference. These results indicate that thrombin initiates a very strong response in platelets through PAR1, leading to exposure of anionic phospholipids that support blood clotting. The response mediated by PAR4, however, was limited to platelet aggregation and similar to that triggered in platelets by weaker agonists such as ADP or epinephrine.     

Plasma lipoproteins enhance tissue factor-independent factor VII activation

The effect of plasma lipoprotein fractions (large very-low-density lipoprotein, small very-low-density lipoprotein, intermediate-density lipoprotein, and low-density lipoprotein) on initiation of blood coagulation by supporting factor VII activation or by stimulating monocytes to express tissue factor was investigated in vitro. Endotoxin-free preparations of lipoprotein fractions did not induce functional tissue factor in monocytes, whereas all lipoprotein fractions enhanced tissue factor-independent activation of factor VII by factor Xa and by factors Xa/Va. In contrast, no or only slight enhancement of factor IXa-, factor IXa/VIIIa-, factor XIa-, or thrombin-mediated factor VII activation was observed. The effect of small very-low-density lipoprotein was less than that of large very-low-density lipoprotein, and intermediate-density and low-density lipoproteins caused an even lower but still significant increase of factor Xa- and factor Xa/Va-mediated factor VII activation. When the data were normalized for apolipoprotein B-100 content, differences remained between lipoprotein fractions. In contrast, when phospholipid content was used for normalization, differences between lipoprotein fractions in factor Xa- and factor Xa/Va-mediated factor VII activation disappeared, indicating that phospholipids were involved in factor VII activation. This was supported by enhancement of factor Xa-mediated factor VII activation by synthetic phospholipid vesicles containing negatively charged phospholipids.     

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.     

The role of activated human platelets in prothrombin and factor X activation

The effect of activated human platelets in intrinsic factor X activation was compared with their effect in prothrombin activation. Compared with unstimulated platelets, platelets triggered by the combined action of collagen plus thrombin showed a tenfold activity increase in prothrombin activation, and a 20-fold rate enhancement in factor X activation. Treatment of collagen plus thrombin-stimulated platelets with N.naja phospholipase A2 almost completely abolished their activity in prothrombin and factor X activation. Since no significant cell lysis occurs during phospholipase treatment, this indicates that platelet phospholipids, exposed at the membrane exterior, play an essential role in the interaction of platelets with the proteins of the prothrombin and factor X-activating complexes. The time course of generation of the procoagulant platelet surface was different when the amount of coagulation factors present in the assay systems was varied. At suboptimal concentrations of coagulation factors, maximum platelet activity was reached after a shorter time period than at saturating concentrations. When measured at suboptimal amounts of coagulation factors, the platelet activity in prothrombin and factor X activation is also more sensitive to phospholipase treatment. Experiments with synthetic phospholipid mixtures show that prothrombin and factor X activation are optimal at low mol% phosphatidylserine when high concentrations of factor Va and factor VIIIa are employed. The optimal mol% phosphatidylserine increases when the concentrations of nonenzymatic protein cofactors are lowered. These findings are discussed in relation to a model in which phosphatidylserine, exposed at the outer surface of activated platelets, plays an essential role in prothrombin and factor X activation. It is proposed that this phosphatidylserine is not homogeneously distributed in the platelet outer membrane, but that areas with different phosphatidylserine density participate in coagulation factor activation.     

Subcellular localization and secretion of factor V from human platelets.

Factor V, a plasma protein cofactor necessary for optimal conversion of prothrombin to thrombin, is also present in considerable concentration in blood platelets (9.9 units per 10(9) platelets). Subcellular fractionation by two methods has localized factor V in the alpha granules of unstimulated platelets. ADP and epinephrine cause release of 4.6% and 6.4%, respectively, of the total factor V, a process completely inhibited by cyclooxygenase alkylation by aspirin. In contrast, collagen causes release of 25% of platelet factor V, a process only partially suppressed by aspirin. Secretion of factor V depends on the availability of metabolic energy, because antimycin A, an inhibitor of aerobic metabolism, and 2-deoxyglucose, an inhibitor of anaerobic glycolysis, together almost totally inhibited the secretion of factor V induced by collagen. The data establish that factor V is not normally available on unstimulated platelets but can be secreted from alpha granules upon stimulation with physiological agents such as ADP, epinephrine, and collagen. Because factor V is known to serve as a receptor for factor Xa, the exposure of factor V on platelets consequent to release would accelerate the process of blood coagulation.     

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.     

Physiological concentrations of tissue factor pathway inhibitor do not inhibit prothrombinase

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa dependent manner, inhibits the factor VIIa/tissue factor catalytic complex. The inhibitory effect of TFPI in prothrombin activation assays using purified components of the prothrombinase complex was examined. When factor Xa is added to mixtures containing TFPI, prothrombin, calcium ions, and nonactivated platelets or factor V and phospholipids, TFPI significantly reduces subsequent thrombin generation, and the inhibitory effect is enhanced by heparin. If factor Xa is preincubated with calcium ions and thrombin-activated platelets or factor Va and phospholipids to permit formation of prothrombinase before the addition of prothrombin and physiologic concentrations of TFPI (< 8 nmol/L), minimal inhibition of thrombin generation occurs, even in the presence of heparin. Thus, contrary to results in amidolytic assays with chromogenic substrates, prothrombinase is resistant to inhibition by TFPI in the presence of its physiological substrate, prothrombin. Higher concentrations of TFPI (approximately 100 nmol/L), similar to those used in animal studies testing for therapeutic actions of TFPI, do effectively block prothrombinase activity.     

Tumor cells augment the factor Xa-catalyzed conversion of prothrombin to thrombin

Activation of blood coagulation and local fibrin deposition may contribute to tumor metastasis. We have examined the ability of four human tumor cell lines (COLO 205, HepG2, J82 and CAPAN-2) to augment the conversion of prothrombin to thrombin by factor Xa and calcium in the presence and absence of exogenous factor Va. Using a chromogenic substrate assay to assess thrombin formation, we observed that all the above cell lines accelerated prothrombin activation in the absence of exogenous factor Va. The order of effectiveness was COLO 205 greater than HepG2 greater than J82 greater than CAPAN-2. In the absence of cells, no detectable thrombin formation occurred. Pretreatment of COLO 205 and HepG2 cells with anti-human factor V IgG inhibited prothrombin activation in a dose-dependent manner, but was without effect in J82 and CAPAN-2 incubation mixtures. Factor V coagulant activity was observed in COLO 205 and HepG2 cells as well as their culture media, but was not detected in J82 and CAPAN-2 cells or their culture media. Biosynthetic labeling and immunoprecipitation studies revealed that COLO 205 and HepG2 cells constitutively synthesized factor V or a factor-V-like molecule that comigrated with human factor V/Va on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All four tumor cell lines exhibited saturation binding of exogenous human factor Va resulting in a dose-dependent enhancement of their ability to augment prothrombin activation. Our results indicate that these tumor cells can readily assemble a functional cell surface prothrombinase complex that may be important in fibrin deposition associated with the growth and metastatic progression of these, and perhaps, other tumors.     

The isolation of human platelet factor V [published erratum appears in Blood 1987 Jul;70(1):339]

Human platelet factor V has been isolated using either a monoclonal or polyclonal antibody directed against human plasma factor V. The largest peptide observed upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified human platelet factor V comigrates with purified human plasma factor V. However, a significant portion of the isolated protein is represented by peptides of lower apparent molecular weight (Mr). These lower Mr species that copurify with platelet factor V have been shown to be platelet factor V components by their immunological cross-reactivity with monoclonal and polyclonal antibodies to purified human plasma factor V. Platelets isolated from whole blood drawn directly into inhibitors to prevent proteolysis and platelet activation demonstrate the pattern of fragmented platelet factor V. The components of purified platelet factor V demonstrate apparent Mr ranging between 115 K and 330 K and are detectably different from the intermediates and end products observed during the thrombin cleavage of single-chain plasma factor V. Upon treatment with thrombin the platelet factor V components are cleaved and the end products are indistinguishable from those obtained upon thrombin activation of plasma factor V to plasma factor Va. Examination of the components by immunoblotting demonstrates that some of the cleavages which have occurred in the platelet factor V molecule are within the 150-K activation peptide. Bioassay indicates that platelet factor V exists as a procofactor and cleavage by thrombin yields the active cofactor, platelet factor Va. These data suggest that human platelet factor V is stored in the platelet as a partially fragmented procofactor that can be activated by thrombin to yield human platelet factor Va, the active cofactor in the human prothrombinase complex.     

Phospholipase abolishes the effect of stimulated platelets on the thrombin activation of factor VIII

Factor VIII functions as a cofactor in the intrinsic coagulation pathway and must first be activated to function optimally in this capacity. Low concentrations of thrombin activate factor VIII, and the presence of stimulated platelets is known to enhance the activation of factor VIII complexed to von Willebrand factor. The current studies show that platelets stimulated by thrombin, collagen, or calcium ionophore will increase the activation of isolated factor VIII by thrombin. Ongoing platelet release is not necessary for the enhanced factor VIII activation, nor is platelet von Willebrand factor or platelet membrane glycoproteins Ib or IIb/IIIa. Platelet membrane phospholipids, on the other hand, are important for the enhanced activation of factor VIII by thrombin because the effect of stimulated platelets is abolished by incubation of the stimulated platelets with phospholipases. These results suggest that the enhanced activation of factor VIII by thrombin in the presence of stimulated platelets may be mediated by factor VIII binding to platelet phospholipid or to a receptor whose functional integrity is dependent on surrounding membrane phospholipid.     

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.     

Complement proteins C5b-9 stimulate procoagulant activity through platelet prothrombinase

The capacity of platelets treated with nonlytic concentrations of the C5b-9 proteins to catalyze prothrombin activation and thereby trigger clot formation has been investigated. When suspended in the presence of exogenous factors Xa and Va, gel-filtered platelets treated with purified C5b-9 proteins catalyzed prothrombin to thrombin conversion at rates up to tenfold above controls, and exceeded by up to fourfold the prothrombinase activity observed for thrombin-stimulated platelets. In the absence of added factor Va, C5b-9 assembly on the platelet surface significantly shortened the lag period before prothrombinase expression that was observed for untreated platelets and increased the maximum catalytic rate of thrombin formation. A comparison with other platelet stimuli revealed that the C5b-9-induced activation of platelet prothrombinase closely paralleled the effects mediated by calcium ionophore A23187. Our data suggest that the C5b-9 proteins promote the release of platelet factor V and the assembly of the prothrombinase complex, thereby potentiating the effects of thrombin on the activation of prothrombinase. Membrane assembly of the C5b-9 proteins was also observed to markedly accelerate the rate of platelet-catalyzed plasma clotting, suggesting a direct link between C5b-9-mediated prothrombinase activation and procoagulant activity accompanying immunologic damage to the platelet.     

Enhanced prothrombin-converting activity and factor Xa binding of platelets activated by the alternative complement pathway

Platelet prothrombin-converting activity and factor Xa binding were studied after exposure of human platelet rich plasma (PRP) to various conditions leading to platelet activation. Zymosan resulted in increased platelet-bound C3, enhanced prothrombin-converting activity and increased factor Xa binding. Similar findings were observed with normal platelets resuspended in factor XII-deficient plasma. The combined use of zymosan and thrombin to activate platelets resulted in synergistic prothrombin-converting activity and factor Xa binding. In contrast, no synergism was obtained with the concomitant use of zymosan and collagen, suggesting that collagen and zymosan share the same pathway for platelet activation. Heterologous antibody to factor V completely inhibited the platelet prothrombin-converting activity for all modes of platelet activation, indicating that this activity is mediated by factor V.     

Binding of thrombin-activated human factor VIII to platelets

To study association of platelets with factor VIII, the purified protein was 125I-labelled with Bolton-Hunter reagent to a specific activity of 243,000-360,000 cpm/U. Autoradiographs of SDS polyacrylamide gels revealed polypeptides of VIII at Mr about 240 kDa, 90 kDa and intermediate values, as well as some radioactive contaminants, but the light chain (Mr 78/76) seen with silver stain was not labelled. After 2.5-5-fold activation with thrombin, the higher radioactive Mr band disappeared, the band at 90 kDa became more intense, and a band appeared at about 45 kDa. The radioactivity associated with platelets, studied in the presence of haemophilic BaSO4-treated plasma, was maximal after 6-8 min and increased 3-15-fold on activation with thrombin. With activated VIII, autoradiographs of platelet pellets showed only VIIIa but results are expressed as units of unactivated VIII bound. At 0.3-0-0.7 U/ml, 10(8) platelets bound 0.0008-0.004 U VIIIa. The amount bound was not affected by the ratio of unlabelled VIII to VIII labelled in the presence of a 50-fold molar excess of unlabelled Bolton-Hunter reagent. Binding increased to 1.5 U VIIIa/10(8) platelets (about 13,600 molecules per platelet) at 140 U/ml, with no evidence of saturation. Binding was not affected by monoclonal antibodies to platelet glycoproteins IIb/IIIa or Ib, quenching the thrombin before adding platelets, or aggregating the platelets with A23187 in the presence of thrombin. Qualitatively, binding of labelled VIIIa and factor Va studied by others are similar. Binding of 125I-VIIIa to aggregated and unaggregated platelets was normal in patient M.S. whose platelets were shown by others to be deficient in their ability to bind radiolabelled factor Xa and generate coagulant activity. This difference. and the fact that platelet coagulant activity is increased by platelet activation and/or aggregation, suggest that binding of Va or VIIIa alone does not determine the assembly of active proteolytic complexes on the platelet surface.