Regulation of activated protein C by thrombin-modified protein S

Protein S, a vitamin K-dependent plasma protein having Gla-residues, increases the rate of inactivation of Factor Va by activated protein C by enhancing the binding of activated protein C to phospholipid [Walker, J.F. (1981) J. Biol. Chem. 256, 11128-11131]. The present study aimed at elucidating the effect of thrombin-modified protein S on Factor Va inactivation by activated protein C. Nondigested protein S consisted 81% of intact form and 19% of modified form, and thrombin-digested protein S had 96% modified form. Protein S, both nondigested and digested, did not show any effects on the amidolytic activity of activated protein C towards synthetic peptide substrate. Nondigested protein S stimulated the Factor Va inactivation by activated protein C, whereas the digested protein appeared to suppress the inactivation. Protein-phospholipid binding experiments showed that although nondigested protein S enhanced the binding of activated protein C to phospholipid stoichiometrically, digested protein S appeared to not only suppress the complex formation, but also dissociate the complex. This evidence suggested that protein S modified by thrombin regulates the action of activated protein C towards Factor Va on phospholipid.     

Protein S is essential for the activated protein C-catalyzed inactivation of platelet-associated factor Va

The prothrombin-converting activity of Factor Xa was enhanced by thrombin-stimulated Factor V-deficient platelets and supplementary extraneous Factor Va, and also by thrombin-stimulated normal human platelets. Both extraneous Factor Va and intra-platelet Factor Va were equally inactivated by a gamma-carboxyglutamic acid-containing plasma protease, activated protein C. However, a relatively larger amount of activated protein C was required for efficient inactivation of platelet-associated Factor Va as compared with the amount of activated protein C needed for inactivation of phospholipid vesicle-associated Factor Va. Protein S, another gamma-carboxyglutamic acid-containing plasma protein, increased the rate of the inactivation of platelet-associated Factor Va about 25-fold. This stimulating effect was observed only slightly with the thrombin-modified protein S. Thus, it was concluded that protein S is essential for the process of inactivation of platelet-associated Factor Va by activated protein C.     

Kinetics of inactivation of membrane-bound factor Va by activated protein C. Protein S modulates factor Xa protection

Kinetic analyses were done to determine what effect factor Xa and protein S had on the activated protein C (APC)-catalyzed inactivation of factor Va bound to phospholipid vesicles or human platelets. In the presence of optimal concentrations of phospholipid vesicles and Ca2+, a Km of 19.7 +/- 0.6 nM factor Va and a kcat of 23.7 +/- 10 mol of factor Va inactivated/mol of APC/min were obtained. Added purified plasma protein S increased the maximal rate of factor Va inactivation only 2-fold without effect on the Km. Protein S effect was unaltered when the phospholipid concentration was varied by 2 orders of magnitude. The reaction on unactivated human platelets yielded a Km = 12.5 +/- 2.6 nM and kcat = 6.2 +/- 0.6 mol of factor Va inactivated/mol of APC/min. Added purified plasma protein S or release of platelet protein S by platelet activation doubled the kcat value without affecting the Km. Addition of a neutralizing anti-protein S antibody abrogated the effect of plasma protein S or platelet-released protein S, but was without effect in the absence of plasma protein S or platelet activation. Studies with factor Xa indicated that factor Xa protects factor Va from APC-catalyzed inactivation by lowering the effective concentration of factor Va available to interact with APC. From these data a dissociation constant of less than 0.5 nM was calculated for the interaction of factor Xa with membrane-bound factor Va. Protein S abrogated the ability of factor Xa to protect factor Va from inactivation by APC without affecting the interaction of factor Xa with factor Va. These combined data suggest that one physiological function of protein S is to allow the APC-catalyzed inactivation of factor Va in the presence of factor Xa.     

Cultured bovine aortic endothelial cells promote activated protein C-protein S-mediated inactivation of factor Va

Previous studies have demonstrated that protein S is required for optimal activated protein C-mediated inactivation of Factor Va on the surface of either the platelet or phospholipid vesicles. In this report we demonstrate assembly of the activated protein C-protein S complex on the surface of cultured bovine aortic endothelial cells. Endothelial cell surface acceleration of Factor Va inactivation by activated protein C required the presence of protein S. Kinetic studies indicated that the rate of Factor Va inactivation was half-maximal at a protein S concentration of 0.2 nM and an activated protein C concentration of 0.05 nM. Binding of 125I-activated protein C to endothelial cell monolayers was absolutely dependent on the presence of protein S. At saturating levels of protein S, activated protein C binding was saturable with Kd = 0.04 nM. In contrast, specific, time-dependent, and saturable binding of 125I-protein S to endothelium occurred in the absence of activated protein C. Addition of activated protein C increased the affinity of protein S from Kd = 11 nM to 0.2 nM, but did not change the number of molecules bound per cell at saturation (85,000 molecules/cell). These studies suggest that activated protein C increases the affinity of protein S for pre-existing sites on the endothelial cell surface. The close correlation between the parameters of protein S-activated protein C binding to endothelium and Factor Va inactivation supports the concept that it is bound protein S and activated protein C that are the active species. Formation of functional activated protein C-protein S complexes thus occurs effectively on the endothelial cell surface and represents a new addition to the list of vessel wall anticoagulant properties.     

Inactivation of factor Va by plasmin

The inactivation of Factor Va by plasmin was studied in the presence and absence of phospholipid vesicles and calcium ions. The cleavage patterns of bovine Factor Va and its isolated subunits were analyzed using polyacrylamide gel electrophoresis, and the progress of inactivation was monitored by clotting assays and measurements of prothrombin activation using 5-dimethylaminonaphthalene-1-sulfonylarginine-N-(3-ethyl-1,5-penta nediyl)amide. In addition, the ability of prothrombin and Factor Xa to protect Factor Va from inactivation by human plasmin was examined. The data presented indicate that the cofactor Factor Va is inactivated rapidly upon its interaction with human plasmin. The rate of inactivation is significantly enhanced in the presence of phospholipid vesicles, suggesting that the inactivation process is a membrane-bound phenomenon. The isolated D component (heavy chain of factor Va) was found to be slowly degraded by human plasmin, giving rise to cleavage products different from those obtained with activated protein C and Factor Xa. However, the 48- and 30-kDa fragments obtained from human plasmin degradation of component E (light chain of Factor Va) appear to be similar to those obtained following the proteolysis of the same subunit by activated protein C and Factor Xa.     

The effect of phospholipids, calcium ions and protein S on rate constants of human factor Va inactivation by activated human protein C.

Rate constants for human factor Va inactivation by activated human protein C (APC) were determined in the absence and presence of Ca2+ ions, protein S and varying concentrations of phospholipid vesicles of different lipid composition. APC-catalyzed factor Va inactivation in free solution (in the presence of 2 mM Ca2+) was studied under first-order reaction conditions with respect to both APC and factor Va and was characterized by an apparent second-order rate constant of 6.1 x 10(5) M-1 s-1. Stimulation of APC-catalyzed factor Va inactivation by phospholipids was dependent on the concentration and composition of the phospholipid vesicles. Optimal acceleration (230-fold) of factor Va inactivation was observed with 10 microM phospholipid vesicles composed of 20 mol% dioleoylglycerophosphoserine (Ole2GroPSer) and 80 mol% dioleoylglycerophosphocholine (Ole2GroPCho). At higher vesicle concentrations and at higher molar fractions of Ole2GroPSer some inhibition of APC-catalyzed factor Va inactivation was observed. Membranes that contained anionic phospholipids other than phosphatidylserine also promoted factor Va inactivation. The ability of different anionic lipids to enhance factor Va inactivation increased in the order phosphatidylethanolamine less than oleic acid less than phosphatidic acid less than phosphatidylglycerol less than phosphatidylmethanol less than phosphatidylserine. APC-catalyzed factor Va inactivation in the presence of phospholipid vesicles could be saturated with respect to factor Va and the reaction obeyed Michaelis-Menten kinetics. Both the Km for factor Va and the Vmax of factor Va inactivation were a function of the phospholipid concentration. The Km increased from 1 nM at 2.5 microM phospholipid (Ole2GroPSer/Ole2GroPCho 20:80, mol/mol) to 65 nM at 250 microM phospholipid. The Vmax increased from 20 mol factor Va inactivated.min-1.mol APC-1 at 2.5 microM phospholipid to 62 mol factor Va inactivated.min-1.mol APC-1 at 10 microM phospholipid and remained constant at higher phospholipid concentrations. Protein S appeared to be a rather poor stimulator of APC-catalyzed factor Va inactivation. Protein-S-dependent rate enhancements were only observed in reaction mixtures that contained negatively charged phospholipid vesicles. Independent of the concentration and the lipid composition of the vesicles, protein S caused a twofold stimulation of APC-catalyzed factor Va inactivation. This suggests that, in the human system, enhancement of APC binding to phospholipid vesicles by protein S is of minor importance. Considering that protein S is a physiologically essential antithrombotic agent, it is likely that other factors or phenomena contribute to the in vivo antithrombotic action of protein S.     

C-terminal residues 621-635 of protein S are essential for binding to factor Va

Protein S is anticoagulant in the absence of activated protein C because of direct interactions with coagulation Factors Xa and Va. Synthetic peptides corresponding to amino acid sequences of protein S were tested for their ability to inhibit prothrombinase activity. The peptide containing the C-terminal sequence of protein S, residues 621-635 (PSP14), reversibly inhibited prothrombinase activity in the presence but not in the absence of Factor Va (K(i) approximately 2 microM). PSP14 inhibition of prothrombinase was independent of phospholipids but could be competitively overcome by increasing Factor Xa concentrations, suggesting that the C-terminal region of protein S may compete for a Factor Xa binding site on Factor Va. Studies using peptides with amino acid substitutions suggested that lysines 630, 631, and 633 were critical residues. PSP14 inhibited Factor Va activity in Factor Xa-one-stage clotting assays. PSP14 inhibited protein S binding to immobilized Factor Va. When preincubated with protein S, antibodies raised against PSP14 inhibited binding of protein S to Factor Va and blocked inhibition of prothrombinase activity by protein S. These results show that the C-terminal region of protein S containing residues 621-635 is essential for binding of protein S to Factor Va and that this interaction contributes to anticoagulant action.     

The binding of activated protein C to factors V and Va

Activated protein C has been derivatized with the active site-directed fluorophore 2-(dimethylamino)-6-naphthalenesulfonylglutamylglycylarginyl chloromethyl ketone (2,6-DEGR-APC). Covalently modified activated protein C has been used to investigate the binding interactions of the protein to factors V and Va in the presence of phospholipid vesicles. The fluorescence polarization of the 6-dimethylaminonaphthalene-2-sulfonyl moiety increased saturably with increasing phospholipid concentrations in the presence or absence of factor V or Va. Differences in the limiting polarization values indicated distinguishable differences in the interactions between 2,6-DEGR-APC and phospholipid in the presence of factor V or Va. The dissociation constant calculated for the 2,6-DEGR-APC/phospholipid interaction (7.3 X 10(-8) M) was not significantly altered by factor V but was decreased to 7 X 10(-9) M in the presence of factor Va. The interaction between 2,6-DEGR-APC and factor V or Va was characterized by a 1:1 stoichiometry. The binding of 2,6-DEGR-APC to factor V or Va in the presence of phospholipid could be reduced in a competitive manner by diisopropylphosphofluoridate-treated activated protein C. An analysis of the displacement curves indicated that the binding of 2,6-DEGR-APC was indistinguishable from the binding of diisopropylphosphofluoridate-treated activated protein C. The interaction between 2,6-DEGR-APC and phospholipid-bound factor Va was further examined using the isolated subunits of factor Va. Fluorescence polarization changes observed with component E of Va (light chain) closely corresponded with the changes observed with factor Va, whereas isolated component D (heavy chain) had little influence on the binding of 2,6-DEGR-APC to phospholipid vesicles. The data presented are consistent with the interpretation that component E of factor Va contains a binding site for activated protein C.     

The contribution of bovine Factor V and Factor Va to the activity of prothrombinase.

The rates of prothrombin activation under initial conditions of invariant concentrations of prothrombin and Factor Xa were studied in the presence of various combinations of Ca2+, homogeneous bovine Factor V, Factor Va, phosphatidylcholine-phosphatidylserine vesicles, and activated bovine platelets. Reactions were monitored continuously through the enhanced fluorescence accompanying the interaction of newly formed thrombin with dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide. The complete prothrombinase (Factor Xa, Ca2+, phospholipid, and Factor Va) behaved as a "typical" enzyme and catalyzed the activation of prothrombin with an apparent Vmax of 2100 mol of thrombin/min/mol of Factor Va or Factor Xa, whichever was the rate-limiting component. Regardless of whether the enzymatic complex was composed of Factor Xa, Ca2+, and plasma Factor Va plus phospholipid vesicles, or activated platelets in the place of the latter components, similar specific activity values were observed. The combination of Factor Va, Ca2+, and phospholipid enhanced the rate of the Factor Xa-catalyzed activation of prothrombin by a factor of 278,000. Factor Va itself when added to Factor Xa, Ca2+, and phospholipid, enhanced the rate of prothrombin activation by a factor of 13,000. Unactivated Factor V appears to possess 0.27% of the procoagulant activity of thrombin-activated Factor Va. From the kinetics of prothrombinase activity, an interaction between Factor Xa and both Factor V and Factor Va was observed, with apparent 1:1 stoichiometries and dissociation constants of 7.3 x 10(-10) M for Factor Va and 2.7 x 10(-9) M for Factor V. The present data, combined with data on the equilibrium binding of prothrombinase components to phospholipid, indicate that the model prothrombinase described in this paper consists of a phospholipid-bound, stoichiometric complex of Factor Va and Factor Xa, with bound Factor Va serving as the "binding site" for Factor Xa, in concert with its proposed role in platelets.     

The effect of Arg306-->Ala and Arg506-->Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S.

Factor Va (fVa) is inactivated by activated protein C (APC) by cleavage of the heavy chain at Arg306, Arg506, and Arg679. Site-directed mutagenesis of human factor V cDNA was used to substitute Arg306-->Ala (rfVa306A) and Arg506-->Gln (rfVa506Q). Both the single and double mutants (rfVa306A/506Q) were constructed. The activation of these procofactors by alpha-thrombin and their inactivation by APC were assessed in coagulation assays using factor V-deficient plasma. All recombinant and wild-type proteins had similar initial cofactor activity and identical activation products (a factor Va molecule composed of light and heavy chains). Inactivation of factor Va purified from human plasma (fVaPLASMA) in HBS Ca2+ +0.5% BSA or in conditioned media by APC in the presence of phospholipid vesicles resulted in identical inactivation profiles and displayed identical cleavage patterns. Recombinant wild-type factor Va (rfVaWT) was inactivated by APC in the presence of phospholipid vesicles at an overall rate slower than fVaPLASMA. The rfVa306A and rfVa506Q mutants were each inactivated at rates slower than rfVaWT and fVaPLASMA. Following a 90-min incubation with APC, rfVa306A and rfVa506Q retain approximately 30-40% of the initial cofactor activity. The double mutant, rfVa306A/506Q, was completely resistant to cleavage and inactivation by APC retaining 100% of the initial cofactor activity following a 90-min incubation in the presence of APC. Recombinant fVaWT, rfVa306A, rfVa506Q, and rfVa306A/506Q were also used to evaluate the effect of protein S on the individual cleavage sites of the cofactor by APC. The initial rates of rfVaWT and rfVa306A inactivation in the presence of protein S were unchanged, indicating cleavage at Arg506 is not affected by protein S. The initial rate of rfVa506Q inactivation was increased, suggesting protein S slightly accelerates the cleavage at Arg306. Overall, the data demonstrate high specificity with respect to cleavage sites for APC on factor Va and demonstrate that cleavages of the cofactor at both Arg306 and Arg506 are required for efficient factor Va inactivation.     

The effect of membrane composition on the hemostatic balance

The phospholipid composition requirements for optimal prothrombin activation and factor Va inactivation by activated protein C (APC) anticoagulant were examined. Vesicles composed of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) supported factor Va inactivation relatively well. However, optimal factor Va inactivation still required relatively high concentrations of phosphatidylserine (PS). In addition, at a fixed concentration of phospholipid, PS, and APC, vesicles devoid of PE never attained a rate of factor Va inactivation achievable with vesicles containing PE. Polyunsaturation of any vesicle component also contributed significantly to APC inactivation of factor Va. Thus, PE makes an important contribution to factor Va inactivation that cannot be mimicked by PS. In the absence of polyunsaturation in the other membrane constituents, this contribution was dependent upon the presence of both the PE headgroup per se and unsaturation of the 1,2 fatty acids. Although PE did not affect prothrombin activation rates at optimal PS concentrations, PE reduced the requirement for PS approximately 10-fold. The Km(app) for prothrombin and the Kd(app) for factor Xa-factor Va decreased as a function of increasing PS concentration, reaching optimal values at 10-15% PS in the absence of PE but only 1% PS in the presence of PE. Fatty acid polyunsaturation had minimal effects. A lupus anticoagulant immunoglobulin was more inhibitory to both prothrombinase and factor Va inactivation in the presence of PE. The degree of inhibition of APC was significantly greater and much more dependent on the phospholipid composition than that of prothrombinase. Thus, subtle changes in the phospholipid composition of cells may control procoagulant and anticoagulant reactions differentially under both normal and pathological conditions.     

Regulation of vitamin K-dependent protein S. Inactivation by thrombin

Thrombin treatment of the vitamin K-dependent protein S resulted in the loss of the activated protein C cofactor activity associated with protein S. The addition of phospholipid vesicles inhibited the inactivation. Thrombin treatment did not alter the molecular weight of the native protein. However, upon reduction, a peptide of approximately 3000 daltons was released from the treated protein. The interaction between calcium and protein S was reduced by thrombin treatment. When the calcium interaction was determined by the quenching of the intrinsic fluorescence of protein S, thrombin treatment appeared to inhibit the interaction between calcium and the protein. When the calcium interaction was observed by measuring the effect on the electrophoretic mobility of the protein, thrombin treatment reduced the interaction between calcium and protein S. However, the effect of thrombin treatment on the interaction between calcium and protein S was less than observed by the fluorescent method. This observation suggests that fluorescence quenching may be a result of a structural change induced by calcium binding. Thrombin treatment of protein S appears to uncouple the calcium binding from the structural change. In addition, the interaction between protein S and phospholipid vesicles was reduced by thrombin treatment. These results suggest that the thrombin conversion of protein S into a two-chain protein causes the loss of a calcium-induced change in protein structure, loss of the lipid-binding properties, and the loss of cofactor activity.     

The interaction of bone Gla protein (osteocalcin) with phospholipid vesicles

The binding interaction of bone Gla protein (BGP), or osteocalcin, to phospholipid vesicles in the presence of calcium has been investigated. Two separate indirect methodologies involving displacement of pyrene-modified Factor Va bound to phospholipid vesicles, and competition with several coagulation proteins in a prothrombin activation assay were performed. Titration of BGP into a cuvette containing phospholipid vesicles (75:25, L-alpha-phosphatidylcholine/L-alpha-phosphatidylserine (PCPS] saturated with pyrene-modified Factor Va resulted in a systematic decrease in steady-state anisotropy, suggesting competition for membrane binding sites with pyrene-modified Factor Va. BGP was also found to inhibit thrombin generation in the prothrombin activation assay. Approximately 50% inhibition was observed at 3 microM BGP under phospholipid-limiting (0.5 microM PCPS) concentrations. No inhibition was observed under phospholipid excess (30 microM PCPS) concentrations. Direct measurement of phospholipid binding was measured using equilibrium gel filtration. Elution profiles using fixed lipid (3.4 mumol of PCPS) and varying BGP concentrations (1-17 microM) in the presence of 3 mM CaCl2 showed a BGP-phospholipid association. Quantitation of determined isotherm yielded a dissociation constant of 6 +/- 1 microM with a stoichiometry of 102 +/- 9 BGP molecules/vesicle at saturation (35 PCPS lipids/BGP) in the presence of 3 mM CaCl2. These results support the hypothesis that protein gamma-carboxylation events are coincident with membrane binding potential.     

Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function

The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits APC anticoagulant activity. In this study, we investigate the mechanisms by which sEPCR modulates APC function. Soluble EPCR inhibited the inactivation of factor Va by APC only in the presence of phospholipid vesicles. By using flow cytometric analysis in the presence of 3 mM CaCl(2) and 0. 6 mM MgCl(2), sEPCR inhibited the binding of protein C and APC to phospholipid vesicles (K(i) = 40 +/- 7 and 33 +/- 4 nM, respectively). Without MgCl(2), the K(i) values increased approximately 4-fold. Double label flow cytometric analysis using fluorescein-APC and Texas Red-sEPCR indicated that the APC.sEPCR complex does not interact with phospholipid vesicles. By using surface plasmon resonance, we found that sEPCR also inhibited binding of protein C to phospholipid in a dose-dependent fashion (K(i) = 32 nM). To explore the possibility that sEPCR evokes structural changes in APC, fluorescence spectroscopy studies were performed to monitor sEPCR/Fl-APC interactions. sEPCR binds saturably to Fl-APC (K(d) = 27 +/- 13 nM) with a maximum decrease in Fl-APC fluorescence of 10.8 +/- 0.6%. sEPCR also stimulated the amidolytic activity of APC toward synthetic substrates. We conclude that sEPCR binding to APC blocks phospholipid interaction and alters the active site of APC.     

Interactions and inhibition of blood coagulation factor Va involving residues 311-325 of activated protein C.

Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. The synthetic peptide-(311-325) (KRNRTFVLNFIKIPV), derived from the heavy chain sequence of APC, potently inhibited APC anticoagulant activity in activated partial thromboplastin time (APTT) and Xa-1-stage coagulation assays in normal and in protein S-depleted plasma with 50% inhibition at 13 microM peptide. In a system using purified clotting factors, peptide-(311-325) inhibited APC-catalyzed inactivation of factor Va in the presence or absence of phospholipids with 50% inhibition at 6 microM peptide. However, peptide-(311-325) had no effect on APC amidolytic activity or on the reaction of APC with the serpin, recombinant [Arg358]alpha 1-antitrypsin. Peptide-(311-325) surprisingly inhibited factor Xa clotting activity in normal plasma, and in a purified system it inhibited prothrombinase activity in the presence but not in the absence of factor Va with 50% inhibition at 8 microM peptide. The peptide had no significant effect on factor Xa or thrombin amidolytic activity and no effect on the clotting of purified fibrinogen by thrombin, suggesting it does not directly inhibit these enzymes. Factor Va bound in a dose-dependent manner to immobilized peptide-(311-325). Peptide-(311-315) inhibited the binding of factor Va to immobilized APC or factor Xa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of prothrombinase complex by plasma proteinase inhibitors

The rate of inactivation of human coagulation factor Xa by the plasma proteinase inhibitors antithrombin III and alpha 1-antitrypsin has been studied in the presence of the accessory components which constitute the prothrombinase complex. The rate of inactivation of factor Xa by antithrombin III was found to be decreased in the presence of phospholipid vesicles with high affinity for factor Xa. The second-order rate constant for the reaction fell from 6.21 X 10(4) to 3.40 X 10(4) M-1 min-1 in the presence of 20 microM phospholipid. Purified factor Va had no effect on the rate of inactivation of factor Xa in the absence of phospholipid. In the presence of phospholipid, factor Va increased the protective effect displayed by phospholipid, further reducing the rate constant to 2.20 X 10(4) M-1 min-1. The rate of inactivation of factor Xa by alpha 1-antitrypsin was unaffected under these conditions. Platelet-bound prothrombinase complex was formed by incubation of factor Xa with washed human platelets activated by a mixture of collagen and thrombin. The prothrombinase activity was inhibited by antithrombin III was a second-order rate constant of 0.85 X 10(4) M-1 min-1. This rate was obtained in both the presence and absence of exogenous factor Va. Platelet factor 3 vesicles, isolated from platelet aggregation supernatants, also formed prothrombinase complex in the presence of factor Va, and this was inhibited by antithrombin III at the same rate as the platelet-bound complex. There was no protection of the platelet-bound prothrombinase complex from inhibition by alpha 1-antitrypsin.     

Platelet-derived microparticles express high affinity receptors for factor VIII.

Factor VIII is a cofactor in the tenase enzyme complex which assembles on the membrane of activated platelets. A critical step in tenase assembly is membrane binding of factor VIII. Platelet membrane factor VIII-binding sites were characterized by flow cytometry using either fluorescein maleimide-labeled recombinant factor VIII or a fluorescein-labeled monoclonal antibody against factor VIII. Following activation by thrombin, most platelets bound factor VIII within 90 s. In addition, over the course of several minutes, membranous vesicles (microparticles) were shed from the platelet plasma membrane and each microparticle bound as much factor VIII as a stimulated platelet. Over 30 min, stimulated platelets (but not microparticles) lost the capacity to bind factor VIII. Factor VIII bound saturably to microparticles from platelets stimulated with thrombin, thrombin plus collagen, or the complement proteins C5b-9. The binding of factor VIII was compared to factor V, a structurally homologous coagulation cofactor. Analysis of microparticle binding kinetics yielded similar on and off rates for factor VIII and factor Va and KD values of 2-10 nM. In the presence of 20 nM factor Va, the binding of factor VIII to microparticles was increased, and there was a comparable increase in platelet tenase activity. At higher factor Va concentrations, factor VIII binding and tenase activity were inhibited. Conversely, factor VIII had a similar dose-dependent effect on factor Va binding and platelet prothrombinase activity. Synthetic phospholipid vesicles containing phosphatidylserine competed with microparticles for binding of factor VIII and factor Va. These studies indicate that activated platelets express a transient increase in high affinity receptors for factor VIII, whereas platelet-derived microparticles express a sustained increase in receptors. The binding characteristics of platelet membrane receptors for factor VIII are similar to those for factor Va.     

Inactivation of factor VIII by activated protein C and protein S

Factor VIII was inactivated by activated protein C in the presence of calcium and phospholipids. Analysis of the activated protein C-catalyzed cleavage products of factor VIII indicated that inactivation resulted from the cleavage of the heavy chains. The heavy chains appeared to be converted into 93- and 53-kDa peptides. Inactivation of factor VIII that was only composed of the 93-kDa heavy chain and 83-kDa light chain indicated that the 93-kDa polypeptide could be degraded into a 68-kDa peptide that could be subsequently cleaved into 48- and 23-kDa polypeptides. Thus, activated protein C catalyzed a minimum of four cleavages in the heavy chain. Activated protein C did not appear to alter the factor VIII light chain. The addition of protein S accelerated the rate of inactivation and the rate of all of the cleavages. The effect of protein S could be observed on the cleavage of the heavy chains and on secondary cleavages of the smaller products, including the 93-, 68-, and 53-kDa polypeptides. The addition of factor IX to the factor VIII-activated protein C reaction mixture resulted in the inhibition of factor VIII inactivation. The effect of factor IX was dose dependent. Factor VIII was observed to compete with factor Va for activated protein C. The concentration dependence of factor VIII inhibition of factor Va inactivation suggested that factor VIII and factor Va were equivalent substrates for activated protein C.     

The role of the membrane in the inactivation of factor va by plasmin. Amino acid region 307-348 of factor V plays a critical role in factor Va cofactor function

The mechanism of inactivation of bovine factor Va by plasmin was studied in the presence and absence of phospholipid vesicles (PCPS vesicles). Following 60-min incubation with plasmin (4 nm) membrane-bound factor Va (400 nm) is completely inactive, whereas in the absence of phospholipid vesicles following a 1-h incubation period, the cofactor retains 90% of its initial cofactor activity. Amino acid sequencing of the fragments deriving from cleavage of factor Va by plasmin demonstrated that while both chains of factor Va are cleaved by plasmin, only cleavage of the heavy chain correlates with inactivation of the cofactor. In the presence of a membrane surface the heavy chain of the bovine cofactor is first cleaved at Arg(348) to generate a fragment of M(r) 47,000 containing the NH(2)-terminal part of the cofactor (amino acid residues 1-348) and a M(r) 42,000 fragment (amino acid residues 349-713). This cleavage is associated with minimal loss in cofactor activity. Complete loss of activity of the membrane-bound cofactor coincides with three cleavages at the COOH-terminal portion of the M(r) 47,000 fragment: Lys(309), Lys(310), and Arg(313). These cleavages result in the release of the COOH terminus of the molecule and the production of a M(r) 40,000 fragment containing the NH(2)-terminal portion of the factor Va molecule. Factor Va was treated with plasmin in the absence of phospholipid vesicles followed by the addition of PCPS vesicles and activated protein C (APC). A rapid inactivation of the cofactor was observed as a result of cleavage of the M(r) 47,000 fragment at Arg(306) by APC and appearance of a M(r) 39,000 fragment. These data suggest a critical role of the amino acid sequence 307-348 of factor Va. A 42-amino acid peptide encompassing the region 307-348 of human factor Va (N42R) was found to be a good inhibitor of factor Va clotting activity with an IC(50) of approximately 1.3 microm. These data suggest that plasmin is a potent inactivator of factor Va and that region 307-348 of the cofactor plays a critical role in cofactor function and may be responsible for the interaction of the cofactor with factor Xa and/or prothrombin.     

Binding of protein S to C4b-binding protein. Mutagenesis of protein S.

Protein S and C4b-binding protein (C4BP) form a tight complex (Kd approximately 0.6 nM) the physiologic purpose of which is unknown. The participation of protein S in this complex was investigated using site-specific mutagenesis. Normal recombinant human protein S (rHPS) and five specifically mutated protein S analogs were expressed in transformed human kidney 293 cells and the following properties were characterized: solution-phase C4BP binding, ability to be cleaved by thrombin, ability to act as a cofactor in the activated protein C-catalyzed inactivation of factor Va, and gamma-carboxyglutamic acid content. In some cases, beta-hydroxyaspartic acid plus beta-hydroxyasparagine content was also determined. Binding studies indicated that while clearly important for a high affinity interaction, the amino acid sequence Gly605-Ile614 identified by Walker (Walker, F J. (1989) J. Biol. Chem. 264, 17645-17648) does not account for all the binding energy of the HPS-C4BP interaction. All mutants perturbed in this region or lacking it altogether displayed reduced C4BP binding, and some retained anticoagulant cofactor function. Neither human factor X nor human steroid-binding protein had any measurable ability to compete with plasma HPS for C4BP binding. Furthermore, bovine protein S and a rHPS analog with bovine sequence from Gly597-Trp629 bound to human C4BP with the same affinity as did HPS, and both proteins substituted effectively for HPS as a cofactor for activated protein C in an otherwise human anticoagulation system. Together these results suggest that optimal binding of protein S to C4BP requires the putative alpha-helix Gly605-Ile614, as well as other undetermined regions of protein S, and that the regions of HPS responsible for C4BP binding and activated protein C cofactor function are structurally isolated.