Effects of prothrombin on the individual activated protein C-mediated cleavages of coagulation factor Va

The factor Va (FVa) inactivation by activated protein C (APC), mediated by cleavages at Arg306 and Arg506 in FVa, is inhibited by both factor Xa (FXa) and prothrombin. Although FXa is known to specifically inhibit the Arg506 cleavage, the effect of prothrombin has not been confined to one cleavage site. We used recombinant FV variants, FV:R506Q/R679Q and FV:R306Q/R679Q, to investigate the effect of prothrombin on the individual cleavage sites. The APC-mediated FVa inhibition was monitored by a prothrombinase-based FVa assay, and apparent first order rate constants were calculated for each of the cleavage sites both in the presence and absence of prothrombin. Prothrombin impaired cleavages at both Arg306 and Arg506 and the inhibition correlated with a delayed appearance of proteolytic products on Western blots. Almost complete inhibition was obtained at around 3 microm prothrombin, whereas half-maximal inhibition was obtained at 0.7 microm prothrombin. After cleavage of prothrombin by thrombin, the inhibitory activity was lost. The inhibitory effect of prothrombin on APC-mediated inhibition of FVa was seen both in the presence and absence of protein S, but in particular for the Arg306 sites, it was more pronounced in the presence of protein S. Thus, prothrombin inhibition of APC inactivation of FVa appears to be due to both impaired APC function and decreased APC cofactor function of protein S. In conclusion, FVa, being part of the prothrombinase complex, is protected from APC by both FXa and prothrombin. Release of products of prothrombin activation from the prothrombinase complex would alleviate the protection, allowing APC-mediated inactivation of FVa.     

Characterization of a factor Xa binding site on factor Va near the Arg-506 activated protein C cleavage site

Prothrombin is proteolytically activated by the prothrombinase complex comprising the serine protease Factor (F) Xa complexed with its cofactor, FVa. Based on inhibition of the prothrombinase complex by synthetic peptides, FVa residues 493-506 were proposed as a FXa binding site. FVa is homologous to FVIIIa, the cofactor for the FIXa protease, in the FX-activating complex, and FVIIIa residues 555-561 (homologous to FVa residues 499-506) are recognized as a FIXa binding sequence. To test the hypothesis that FVa residues 499-505 contribute to FXa binding, we created the FVa loop swap mutant (designated 499-505(VIII) FV) with residues 499-505 replaced by residues 555-561 of FVIIIa, which differ at five of seven positions. Based on kinetic measurements and spectroscopic titrations, this FVa loop swap mutant had significantly reduced affinity for FXa. The fully formed prothrombinase complex containing this FVa mutant had fairly normal kinetic parameters (k(cat) and K(m)) for cleavage of prothrombin at Arg-320. However, small changes in both Arg-320 and Arg-271 cleavage rates result together in a moderate change in the pathway of prothrombin activation. Although residues 499-505 directly precede the Arg-506 cleavage site for activated protein C (APC), the 499-505(VIII) FVa mutant was inactivated entirely normally by APC. These results suggest that this A2 domain sequence of the FVa and FVIIIa cofactors evolved to have different specificity for binding FXa and FIXa while retaining compatibility as substrate for APC. In an updated three-dimensional model for the FVa structure, residues 499-505, along with Arg-506, Arg-306, and other previously suggested FXa binding sequences, delineate a continuous surface on the A2 domain that is strongly implicated as an extended FXa binding surface in the prothrombinase complex.     

Importance of individual activated protein C cleavage site regions in coagulation factor V for factor Va inactivation and for factor Xa activation.

Activated protein C (APC) cleavage of Factor Va (FVa) at residues R506 and R306 correlates with its inactivation. APC resistance and increased thrombotic risk are due to the mutation R506Q in Factor V (FV). To study the effects of individual cleavages in FVa by APC and the importance of regions near the cleavage sites, the following recombinant (r) human FVs were prepared and purified: wild-type, Q306-rFV, Q506-rFV, and Q306Q506-rFV. All had similar time courses for thrombin activation. Q506-rFVa was cleaved by APC at R306 and was moderately resistant to APC in plasma-clotting assays and in prothrombinase assays measuring FVa residual activity, in agreement with studies of purified plasma-derived Q506-FVa. Q306-rFVa was cleaved by APC at R506 and gave a low APC-resistance ratio similar to Q506-rFVa in clotting assays, whereas unactivated Q306-rFV gave a near-normal APC-resistance ratio. When FVa residual activity was measured after long exposure to APC, Q306-rFVa was inactivated by only < or = 40% under conditions where Q506-rFVa was inactivated > 90%, supporting the hypothesis that efficient inactivation of normal FVa by APC requires cleavage at R306. In addition, the heavy chain of Q306-rFVa was cleaved at R506 much more rapidly than activity was lost, suggesting that FVa cleaved at only R506 is partially active. Under the same conditions, Q306Q506-rFVa lost no activity and was not cleaved by APC. Therefore, cleavage at either R506 or R306 appears essential for significant inactivation of FVa by APC. Modest loss of activity, probably due to cleavage at R679, was observed for the single site rFVa mutants, as evidenced by a second phase of inactivation. Q306Q506-rFVa had a low activity-to-antigen ratio of 0.50-0.77, possibly due to abnormal Factor Xa (FXa) binding. Furthermore, Q306Q506-rFV was very resistant to cleavage and activation by FXa. Q306Q506-rFV appeared to bind FXa and inhibit FXa's ability to activate normal FV. Thus, APC may downregulate FV/Va partly by impairing FXa-binding sites upon cleavage at R306 and R506. This study shows that R306 is the most important cleavage site for normal efficient inactivation of FVa by APC and supports other studies suggesting that regions near R306 and R506 provide FXa-binding sites and that FVa cleaved at only R506 retains partial activity.     

Interdomain engineered disulfide bond permitting elucidation of mechanisms of inactivation of coagulation factor Va by activated protein C

Procoagulant factor Va (FVa) is inactivated via limited proteolysis at three Arg residues in the A2 domain by the anticoagulant serine protease, activated protein C (APC). Cleavage by APC at Arg306 in FVa causes dissociation of the A2 domain from the heterotrimeric A1:A2:A3 structure and complete loss of procoagulant activity. To help distinguish inactivation mechanisms involving A2 domain dissociation from inactivation mechanisms involving unfavorable changes in factor Xa (FXa) affinity, we used our FVa homology model to engineer recombinant FVa mutants containing an interdomain disulfide bond (Cys609-Cys1691) between the A2 and A3 domains (A2-SS-A3 mutants) in addition to cleavage site mutations, Arg506Gln and Arg679Gln. SDS-PAGE analysis showed that the disulfide bond in A2-SS-A3 mutants prevented dissociation of the A2 domain. In the absence of A2 domain dissociation from the A1:A2:A3 trimer, APC cleavage at Arg306 alone caused a sevenfold decrease in affinity for FXa, whereas APC cleavages at Arg306, Arg506, and Arg679 caused a 70-fold decrease in affinity for FXa and a 10-fold decrease in the k(cat) of the prothrombinase complex for prothrombin without any effect on the apparent K(m) for prothrombin. Therefore, for FVa inactivation by APC, dissociation of the A2 domain may provide only a modest final step, whereas the critical events are the cleavages at Arg506 and Arg306, which effectively inactivate FVa before A2 dissociation can take place. Nonetheless, for FVa Leiden (Gln506-FVa) inactivation by APC, A2 domain dissociation may become mechanistically important, depending on the ambient FXa concentration.     

Effects of factor Xa and protein S on the individual activated protein C-mediated cleavages of coagulation factor Va

Activated protein C inhibits the procoagulant function of activated factor V (FVa) through proteolytic cleavages at Arg-306, Arg-506, and Arg-679. The cleavage at Arg-506 is kinetically favored but protected by factor Xa (FXa). Protein S has been suggested to annihilate the inhibitory effect of FXa, a proposal that has been challenged. To elucidate the effects of FXa and protein S on the individual cleavage sites of FVa, we used recombinant FVa:Q306/Q679 and FVa:Q506/Q679 variants, which can only be cleaved at Arg-506 and Arg-306, respectively. In the presence of active site blocked FXa (FXa-1.5-dansyl-Glu-Gly-Arg), the FVa inactivation was followed over time, and apparent second order rate constants were calculated. Consistent with results on record, we observed that FXa-1.5-dansyl-Glu-Gly-Arg decreased the Arg-506 cleavage by 20-fold, with a half-maximum inhibition of approximately 2 nM. Interestingly and in contrast to the inhibitory effect of FXa on the 506 cleavage, FXa stimulated the Arg-306 cleavage. Protein S counteracted the inhibition by FXa of the Arg-506 cleavage, whereas protein S and FXa yielded additive stimulatory effect of the cleavage at Arg-306. This suggests that FXa and protein S interact with distinct sites on FVa, which is consistent with the observed lack of inhibitory effect on FXa binding to FVa by protein S. We propose that the apparent annihilation of the FXa protection of the Arg-506 cleavage by protein S is due to an enhanced rate of Arg-506 cleavage of FVa not bound to FXa, resulting in depletion of free FVa and dissociation of FXa-FVa complexes.     

Identification of surface epitopes of human coagulation factor Va that are important for interaction with activated protein C and heparin

Inactivation of factor Va (FVa) by activated protein C (APC) is a key reaction in the down-regulation of thrombin formation. FVa inactivation by APC is correlated with a loss of FXa cofactor activity as a result of three proteolytic cleavages in the FVa heavy chain at Arg306, Arg506, and Arg679. Recently, we have shown that heparin specifically inhibits the APC-mediated cleavage at Arg506 and stimulates cleavage at Arg306. Three-dimensional molecular models of APC docked at the Arg306 and Arg506 cleavage sites in FVa have identified several FVa amino acids that may be important for FVa inactivation by APC in the absence and presence of heparin. Mutagenesis of Lys320, Arg321, and Arg400 to Ala resulted in an increased inactivation rate by APC at Arg306, which indicates the importance of these residues in the FVa-APC interaction. No heparin-mediated stimulation of Arg306 cleavage was observed for these mutants, and stimulation by protein S was similar to that of wild type FVa. With this, we have now demonstrated that a cluster of basic residues in FVa comprising Lys320, Arg321, and Arg400 is required for the heparin-mediated stimulation of cleavage at Arg306 by APC. Furthermore, mutations that were introduced near the Arg506 cleavage site had a significant but modest effect on the rate of APC-catalyzed FVa inactivation, suggesting an extended interaction surface between the FVa Arg506 site and APC.     

Nonenzymatic anticoagulant activity of the mutant serine protease Ser360Ala-activated protein C mediated by factor Va.

The human plasma serine protease, activated protein C (APC), primarily exerts its anticoagulant function by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. A recombinant active site Ser 360 to Ala mutation of protein C was prepared, and the mutant protein was expressed in human 293 kidney cells and purified. The activation peptide of the mutant protein C zymogen was cleaved by a snake venom activator, Protac C, but the "activated" S360A APC did not have amidolytic activity. However, it did exhibit significant anticoagulant activity both in clotting assays and in a purified protein assay system that measured prothrombinase activity. The S360A APC was compared to plasma-derived and wild-type recombinant APC. The anticoagulant activity of the mutant, but not native APC, was resistant to diisopropyl fluorophosphate, whereas all APCs were inhibited by monoclonal antibodies against APC. In contrast to native APC, S360A APC was not inactivated by serine protease inhibitors in plasma and did not bind to the highly reactive mutant protease inhibitor M358R alpha 1 antitrypsin. Since plasma serpins provide the major mechanism for inactivating APC in vivo, this suggests that S360A APC would have a long half-life in vivo, with potential therapeutic advantages. S360A APC rapidly inhibited factor Va in a nonenzymatic manner since it apparently did not proteolyze factor Va. These data suggest that native APC may exhibit rapid nonenzymatic anticoagulant activity followed by enzymatic irreversible proteolysis of factor Va. The results of clotting assays and prothrombinase assays showed that S360A APC could not inhibit the variant Gln 506-FVa compared with normal Arg 506-FVa, suggesting that the active site of S360A APC binds to FVa at or near Arg 506.     

Factor Va residues 311-325 represent an activated protein C binding region

Activated protein C (APC) inactivates factor Va (fVa) by proteolytically cleaving fVa heavy chain at Arg(506), Arg(306), and Arg(679). Factor Xa (fXa) protects fVa from inactivation by APC. To test the hypothesis that fXa and APC share overlapping fVa binding sites, 15 amino acid-overlapping peptides representing the heavy chain (residues 1-709) of fVa were screened for inhibition of fVa inactivation by APC. As reported, VP311-325, a peptide comprising residues 311-325 in fVa, dose-dependently and potently inhibited fVa-dependent prothrombin activation by fXa in the absence of APC. This peptide also inhibited the inactivation of fVa by APC, suggesting that this region of fVa interacts with APC. The peptide inhibited the APC-dependent cleavage of both Arg(506) and Arg(306) because inhibition was observed with plasma-derived fVa and recombinant R506Q and RR306/679QQ fVa. VP311-325 altered the fluorescence emission of dansyl-active site-labeled APC(i) but not a dansyl-active site-labeled thrombin control, showing that the peptide binds to APC(i). This peptide also inhibited the resonance energy transfer between membrane-bound fluorescein-labeled fVa (donor) and rhodamine-active site-labeled S360C-APC (acceptor). These data suggest that peptide VP311-325 represents both an APC and fXa binding region in fVa.     

Structural investigation of the A domains of human blood coagulation factor V by molecular modeling.

Factor V (FV) is a large (2,196 amino acids) nonenzymatic cofactor in the coagulation cascade with a domain organization (A1-A2-B-A3-C1-C2) similar to the one of factor VIII (FVIII). FV is activated to factor Va (FVa) by thrombin, which cleaves away the B domain leaving a heterodimeric structure composed of a heavy chain (A1-A2) and a light chain (A3-C1-C2). Activated protein C (APC), together with its cofactor protein S (PS), inhibits the coagulation cascade via limited proteolysis of FVa and FVIIIa (APC cleaves FVa at residues R306, R506, and R679). The A domains of FV and FVIII share important sequence identity with the plasma copper-binding protein ceruloplasmin (CP). The X-ray structure of CP and theoretical models for FVIII have been recently reported. This information allowed us to build a theoretical model (994 residues) for the A domains of human FV/FVa (residues 1-656 and 1546-1883). Structural analysis of the FV model indicates that: (a) the three A domains are arranged in a triangular fashion as in the case of CP and the organization of these domains should remain essentially the same before and after activation; (b) a Type II copper ion is located at the A1-A3 interface; (c) residues R306 and R506 (cleavage sites for APC) are both solvent exposed; (d) residues 1667-1765 within the A3 domain, expected to interact with the membrane, are essentially buried; (e) APC does not bind to FVa residues 1865-1874. Several other features of factor V/Va, like the R506Q and A221V mutations; factor Xa (FXa) and human neutrophil elastase (HNE) cleavages; protein S, prothrombin and FXa binding, are also investigated.     

Altered inactivation pathway of factor Va by activated protein C in the presence of heparin.

Inactivation of factor Va (FVa) by activated protein C (APC) is a predominant mechanism in the down-regulation of thrombin generation. In normal FVa, APC-mediated inactivation occurs after cleavage at Arg306 (with corresponding rate constant k'306) or after cleavage at Arg506 (k506) and subsequent cleavage at Arg306 (k306). We have studied the influence of heparin on APC-catalyzed FVa inactivation by kinetic analysis of the time courses of inactivation. Peptide bond cleavage was identified by Western blotting using FV-specific antibodies. In normal FVa, unfractionated heparin (UFH) was found to inhibit cleavage at Arg506 in a dose-dependent manner. Maximal inhibition of k506 by UFH was 12-fold, with the secondary cleavage at Arg306 (k306) being virtually unaffected. In contrast, UFH stimulated the initial cleavage at Arg306 (k'306) two- to threefold. Low molecular weight heparin (Fragmin) had the same effects on the rate constants of FVa inactivation as UFH, but pentasaccharide did not inhibit FVa inactivation. Analysis of these data in the context of the 3D structures of APC and FVa and of simulated APC-heparin and FVa-APC complexes suggests that the heparin-binding loops 37 and 70 in APC complement electronegative areas surrounding the Arg506 site, with additional contributions from APC loop 148. Fewer contacts are observed between APC and the region around the Arg306 site in FVa. The modeling and experimental data suggest that heparin, when bound to APC, prevents optimal docking of APC at Arg506 and promotes association between FVa and APC at position Arg306.     

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)     

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.     

Defining the factor Xa-binding site on factor Va by site-directed glycosylation

Activated Factor V (FVa) functions as a membrane-bound cofactor to the enzyme Factor Xa (FXa) in the conversion of prothrombin to thrombin, increasing the catalytic efficiency of FXa by several orders of magnitude. To map regions on FVa that are important for binding of FXa, site-directed mutagenesis resulting in novel potential glycosylation sites on FV was used as strategy. The consensus sequence for N-linked glycosylation was introduced at sites, which according to a computer model of the A domains of FVa, were located at the surface of FV. In total, thirteen different regions on the FVa surface were probed, including sites that are homologous to FIXa-binding sites on FVIIIa. The interaction between the FVa variants and FXa and prothrombin were studied in a functional prothrombin activation assay, as well as in a direct binding assay between FVa and FXa. In both assays, the four mutants carrying a carbohydrate side chain at positions 467, 511, 652, or 1683 displayed attenuated FXa binding, whereas the prothrombin affinity was unaffected. The affinity toward FXa could be restored when the mutants were expressed in the presence of tunicamycin to inhibit glycosylation, indicating the lost FXa affinity to be caused by the added carbohydrates. The results suggested regions surrounding residues 467, 511, 652, and 1683 in FVa to be important for FXa binding. This indicates that the enzyme:cofactor assembly of the prothrombinase and the tenase complexes are homologous and provide a useful platform for further investigation of specific structural elements involved in the FVa.FXa complex assembly.     

Importance of protein S and phospholipid for activated protein C-mediated cleavages in factor Va

The procoagulant function of activated factor V (FVa) is inhibited by activated protein C (APC) through proteolytic cleavages at Arg306, Arg506, and Arg679. The effect of APC is potentiated by negatively charged phospholipid membranes and the APC cofactor protein S. Protein S has been reported to selectively stimulate cleavage at Arg306, an effect hypothesized to be related to reorientation of the active site of APC closer to the phospholipid membrane. To investigate the importance of protein S and phospholipid in the APC-mediated cleavages of individual sites, recombinant FV variants FV(R306Q/R679Q) and FV(R506Q/R679Q) (can be cleaved only at Arg506 and Arg306, respectively) were created. The cleavage rate was determined for each cleavage site in the presence of varied protein S concentrations and phospholipid compositions. In contrast to results on record, we found that protein S stimulated both APC cleavages in a phospholipid composition-dependent manner. Thus, on vesicles containing both phosphatidylserine and phosphatidylethanolamine, protein S increased the rate of Arg306 cleavage 27-fold and that of Arg506 cleavage 5-fold. Half-maximal stimulation was obtained at approximately 30 nm protein S for both cleavages. In conclusion, we demonstrate that APC-mediated cleavages at both Arg306 and Arg506 in FVa are stimulated by protein S in a phospholipid composition-dependent manner. These results provide new insights into the mechanism of APC cofactor activity of protein S and the importance of phospholipid composition.     

Molecular characterization of an extended binding site for coagulation factor Va in the positive exosite of activated protein C

The anticoagulant human plasma serine protease, activated protein C (APC), inhibits blood coagulation by specific inactivation of the coagulation cofactors factor Va (FVa) and factor VIIIa. Site-directed mutagenesis of residues in three surface loops of a positive exosite located on APC was used to identify residues that play a significant role in binding to FVa. Eighteen different residues were mutated to alanine singly, in pairs, or in triple mutation combinations. Mutant APC proteins were purified and characterized for their inactivation of FVa. Three APC residues were identified that provide major contributions to FVa interactions: Lys(193), Arg(229), and Arg(230). In addition, four residues made significant minor contributions to FVa interactions: Lys(191), Lys(192), Asp(214), and Glu(215). All of these residues primarily contribute to APC cleavage at Arg(506) in FVa and play a small role in the interaction of APC with the Arg(306) cleavage site. In conjunction with previously published work, these results define an extensive FVa binding site in the positive exosite of APC that is primarily involved in binding and cleaving at Arg(506) on FVa.     

Role of the alpha-helix 163-170 in factor Xa catalytic activity

Factor Xa (FXa) is a key protease of the coagulation pathway whose activity is known to be in part modulated by binding to factor Va (FVa) and sodium ions. Previous investigations have established that solvent-exposed, charged residues of the FXa alpha-helix 163-170 (h163-170), Arg(165) and Lys(169), participate in its binding to FVa. In the present study we aimed to investigate the role of the other residues of h163-170 in the catalytic functions of the enzyme. FX derivatives were constructed in which point mutations were made or parts of h163-170 were substituted with the corresponding region of either FVIIa or FIXa. Purified FXa derivatives were compared with wild-type FXa. Kinetic studies in the absence of FVa revealed that, compared with wild-type FXa, key functional parameters (catalytic activity toward prothrombin and tripeptidyl substrates and non-enzymatic interaction of a probe with the S1 site) were diminished by mutations in the NH(2)-terminal portion of h163-170. The defective amidolytic activity of these FXa derivatives appears to result from their impaired interaction with Na(+) because using a higher Na(+) concentration partially restored normal catalytic parameters. Furthermore, kinetic measurements with tripeptidyl substrates or prothrombin indicated that assembly of these FXa derivatives with an excess of FVa in the prothrombinase complex improves their low catalytic efficiency. These data indicate that residues in the NH(2)-terminal portion of the FVa-binding h163-170 are energetically linked to the S1 site and Na(+)-binding site of the protease and that residues Val(163) and Ser(167) play a key role in this interaction.     

Role of Hirudin-like factor Va heavy chain sequences in prothrombinase function

Proexosite I on prothrombin has been implicated in providing a recognition site for factor Va within prothrombinase. To examine whether hirudin-like sequences (659-698) on the cofactor contribute to this interaction, we expressed and purified two-chain FVa derivatives that were intracellularly truncated at the C terminus of the heavy chain: FVa709 (des710-1545), FVa699 (des700-1545), FVa(692 (des693-1545), FVa678 (des679-1545), and FVa658 (des659-1545). We found that FVa709, FVa699, FVa692, and FVa678 exhibited specific clotting activities that were comparable with plasma-derived and recombinant FVa. Additionally, kinetic studies using prothrombin revealed that the Km and kcat values for these derivatives were unaltered. Fluorescent measurements and chromatography studies indicated that FVa709, FVa699, FVa692, and FVa678 bound to FXa membranes and thrombin-agarose in a manner that was comparable with the wild-type cofactors. In contrast, FVa658 had an approximately 1% clotting activity and reduced affinity for FXa membranes (approximately 20-fold) and did not bind to thrombin-agarose. Surprisingly, however, FVa(658) exhibited essentially normal kinetic parameters for prothrombin when the variant was fully saturated with FXa membranes. Overall our results are consistent with the interpretation that any possible binding interactions between prothrombin and the C-terminal region of the FVa heavy chain do not contribute in a detectable way to the enhanced function of prothrombinase.     

C4b-binding protein protects coagulation factor Va from inactivation by activated protein C

We investigated the effect of C4BP on APC-mediated inactivation of factor Va (FVa) in the absence and presence of protein S. FVa inactivation was biphasic (k(506) = 4.4 x 10(8) M(-)(1) s(-)(1), k(306) = 2.7 x 10(7) M(-)(1) s(-)(1)), and protein S accelerated Arg(306) cleavage approximately 10-fold. Preincubation of protein S with C4BP resulted in a total abrogation of protein S cofactor activity. C4BP also protected FVa from inactivation by APC in the absence of protein S. Control experiments with CLB-PS13, a monoclonal anti-protein S antibody, indicated that inhibition of FVa inactivation by C4BP was not mediated through contaminating traces of protein S in our reaction systems. Protection of FVa was prevented by a monoclonal antibody directed against the C4BP alpha-chain. Recombinant rC4BPalpha comprised of only alpha-chains also protected FVa, but in the presence of protein S, the level of protection was decreased, since rC4BPalpha lacks the beta-chain responsible for C4BP binding to protein S. A truncated C4BP beta-chain (SCR-1+2) inhibited protein S cofactor activity, but had no effect on FVa inactivation by APC in the absence of protein S. In conclusion, C4BP protects FVa from APC-catalyzed cleavage in a protein S-independent way through direct interactions of the alpha-chaims of C4BP with FVa and/or APC.     

Identification of a sequence of human activated protein C (residues 390-404) essential for its anticoagulant activity.

Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. To identify the regions on the surface that mediate anticoagulant activity, 26 synthetic peptides were prepared representing 90% of the human protein C heavy chain primary structure and tested for their ability to inhibit APC anticoagulant activity. Peptide-(390-404) specifically inhibited APC activity in activated partial thromboplastin time and Xa-1-stage coagulation assays in normal, in protein S-depleted and Factor VIII-deficient plasma with 50% inhibition at 5 microM peptide. Polyclonal antibodies raised against this peptide and immunoaffinity-purified on a protein C-Sepharose column inhibited APC anticoagulant activity in activated partial thromboplastin time and Xa-1-stage assays in normal, protein S-depleted, and Factor VIII-deficient plasma with half-maximal inhibition at 30 nM anti-(390-404) antibody. Neither the peptide-(390-404) nor the anti-(390-404) antibodies inhibited APC amidolytic activity or the reaction of APC with recombinant [Arg358] alpha 1-antitrypsin. Furthermore, in a purified system, peptide-(390-404) inhibited APC-catalyzed inactivation of Factor Va in the presence as well as in the absence of phospholipids with 50% inhibition at 4 microM peptide. These data suggest that the region containing residues 390-404 in APC is essential for anticoagulant activity and is available to interact with antibodies or with other proteins such as the macromolecular substrates Factors Va or VIIIa.     

Thrombin-mediated proteolysis of factor V resulting in gradual B-domain release and exposure of the factor Xa-binding site

To investigate the relationship between the individual thrombin cleavages in factor V (FV) and the generation of activated factor X (FXa) cofactor activity, recombinant FV mutants having the cleavage sites eliminated separately or in combination were used. After thrombin incubation, the ability of the FV variants to bind FXa and support prothrombin activation was tested. The interaction between FVa and FXa on the surface of phospholipid was investigated with a direct binding assay as well as in a functional prothrombin activation assay. FV mutated at all cleavage sites functioned poorly as FXa cofactor in prothrombin activation, the apparent K(d) for FXa being approximately 10 nm. Fully activated wild type FVa, yielded an apparent K(d) of around 0.2 nm. The Arg(709) and Arg(1018) cleavages occurred at low thrombin concentrations and decreased the K(d) for FXa binding 5- and 3-fold, respectively. The Arg(1545) cleavage, being less sensitive to thrombin, decreased the K(d) for FXa binding approximately 20-fold. The K(m) for prothrombin was the same for all FV variants, demonstrating B-domain dissociation to result in exposure of binding site for FXa but not for prothrombin. In conclusion, we demonstrate FV activation to be associated with the stepwise release of the B-domain, which results in a gradual exposure of the FXa-binding site.