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.     

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.     

Binding sites for blood coagulation factor Xa and protein S involving residues 493-506 in factor Va.

Inactivation due to cleavage of Factor Va (FVa) at Arg 506 by activated protein C (APC) helps to downregulate blood coagulation. To identify potential functional roles of amino acids near Arg 506, synthetic overlapping pentadecapeptides comprising FVa heavy chain residues 481-525 were tested for their ability to inhibit prothrombin activation by prothrombinase complexes [Factor Xa (FXa):FVa:phospholipids:Ca2+]. The most potent inhibition was observed for peptide VP493 (residues 493-506), with 50% inhibition at 2.5 microM. VP493 also inhibited FXa in plasma in FXa-1-stage clotting assays by 50% at 3 microM. When the C-terminal carboxamide group of VP493 was replaced by a carboxyl group, most prothrombinase inhibitory activity was lost. VP493 preincubated with FXa inhibited prothrombinase with a pattern of mixed inhibition. Homologous peptides from Factor VIII sequences did not inhibit prothrombinase. Affinity-purified antibodies to VP493 inhibited prothrombinase activity and prolonged FXa-1-stage clotting times. VP493 also blocked the ability of protein S to inhibit prothrombinase independently of APC. Immobilized VP493 bound specifically with similar affinity to both FXa and protein S (Kd approximately 40 nM), but did not measurably bind prothrombin or APC. These studies suggest that FVa residues 493-506 contribute to binding sites for both FXa and protein S, providing a rationale for the ability of protein S to negate the protective effect of FXa toward APC cleavage of FVa. Possible loss of this FVa binding site for FXa due to cleavage at Arg 506 by APC may help explain why this cleavage causes 40% decrease in FVa activity and facilitates inactivation of FVa.     

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)     

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 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.     

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.     

Definition of a factor Va binding site in factor Xa

We reported previously that residue 347 in activated fX (fXa) contributes to binding of the cofactor, factor Va (fVa) (Rudolph, A. E., Porche-Sorbet, R. and Miletich, J. P. (2000) Biochemistry 39, 2861-2867). Four additional residues that participate in fVa binding have now been identified by mutagenesis. All five resulting fX species, fX(R306A), fX(E310N), fX(R347N), fX(K351A), and fX(K414A), are activated and inhibited normally. However, the rate of inhibition by antithrombin III in the presence of submaximal concentrations of heparin is reduced for all the enzymes. In the absence of fVa, all of the enzymes bind and activate prothrombin similarly except fXa(E310N), which has a reduced apparent affinity ( approximately 3-fold) for prothrombin compared with wild type fXa (fXa(WT)). In the absence of phospholipid, fVa enhances the catalytic activity of fXa(WT) significantly, but the response of the variant enzymes was greatly diminished. On addition of 100 nm PC:PS (3:1) vesicles, fVa enhanced fXa(WT), fXa(R306A), and fXa(E310N) similarly, whereas fXa(R347N), fXa(K351A), and fXa(K414A) demonstrated near-normal catalytic activity but reduced apparent affinity for fVa under these conditions. All enzymes function similarly to fXa(WT) on activated platelets, which provide saturating fVa on an ideal surface. Loss of binding affinity for fVa as a result of the substitutions in residues Arg-347, Lys-351, and Lys-414 was verified by a competition binding assay. Thus, Arg-347, Lys-351, and Lys-414 are likely part of a core fVa binding site, whereas Arg-306 and Glu-310 serve a less critical role.     

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.     

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.     

Secondary substrate-binding exosite in the serine protease domain of activated protein C important for cleavage at Arg-506 but not at Arg-306 in factor Va

Proteolytic inactivation of activated factor V (FVa) by activated protein C (APC) is a key reaction in the regulation of hemostasis. We now demonstrate the importance of a positive cluster in loop 37 of the serine protease (SP) domain of APC for the degradation of FVa. Lysine residues in APC at positions 37, 38, and 39 form a secondary binding site for FVa, which is important for cleavage of FVa at Arg-506 while having no effect on Arg-306 cleavage. In contrast, topological neighbors Lys-62, Lys-63, and Arg-74 in APC appear of minor importance in FVa degradation. This demonstrates that secondary binding exosites of APC specifically guide the proteolytic action of APC, resulting in a more favorable degradation of the 506-507 peptide bond as compared with the 306-307 bond.     

Identification of distinct sequences in human blood coagulation factor Xa and prothrombin essential for substrate and cofactor recognition in the prothrombinase complex

To identify amino acid sequences in factor Xa (fXa) and prothrombin (fII) that may be involved in prothrombinase complex (fXa.factor Va.fII.phospholipids) assembly, synthetic peptides based on fXa and fII sequences were prepared and screened for their ability to inhibit fXa-induced clotting of normal plasma. One fII peptide (PT557-571 homologous to chymotrypsin (CHT) residues 225-239) and two fXa peptides (X404-418, CHT231-244, and X415-429, CHT241-252C) potently inhibited plasma clotting and prothrombinase activity with 50% inhibition between 41 and 115 microM peptide. Inhibition of prothrombinase by PT557-571 and X415-429 was fVa-independent, whereas the inhibition by X404-418 was fVa-dependent. X404-418 inhibited the binding of fVa to fluorescein-labeled, inhibited fXai in the presence of phosphatidylcholine/phosphatidylserine vesicles, whereas X415-429 inhibited binding of fII to phospholipid-bound fluorescein-labeled, inhibited fXai. PT557-571 altered the fluorescence emission of fluorescein-labeled fXai, showing that PT557-571 binds to fXai. These data suggest that residues 404-418 in fXa provide fVa binding sites, whereas residues 557-571 in fII and 415-429 in fXa mediate interactions between fXa and fII in the prothrombinase complex.     

Factor Va is inactivated by activated protein C in the absence of cleavage sites at Arg-306, Arg-506, and Arg-679

Activated protein C (APC) exerts its anticoagulant activity via proteolytic degradation of the heavy chains of activated factor VIII (FVIIIa) and activated factor V (FVa). So far, three APC cleavage sites have been identified in the heavy chain of FVa: Arg-306, Arg-506, and Arg-679. To obtain more insight in the structural and functional implications of each individual cleavage, recombinant factor V (rFV) mutants were constructed in which two or three of the APC cleavage sites were mutated. After expression in COS-1 cells, rFV mutants were purified, activated with thrombin, and inactivated by APC. During this study we observed that activated rFV-GQA (rFVa-GQA), in which the arginines at positions 306, 506, and 679 were replaced by glycine, glutamine, and alanine, respectively, was still inactivated by APC. Further analysis showed that the inactivation of rFVa-GQA by APC was phospholipid-dependent and sensitive to an inhibitory monoclonal antibody against protein C. Inactivation proceeded via a rapid phase (kx1=5.4 x 10(4) M(-1) s(-1)) and a slow phase (kx2=3.2 x 10(3) M(-1) s(-1)). Analysis of the inactivation curves showed that the rapid phase yielded a reaction intermediate that retained approximately 80% of the original FVa activity, whereas the slow cleavage resulted in formation of a completely inactive reaction product. Inactivation of rFVa-GQA was accelerated by protein S, most likely via stimulation of the slow phase. Immunoblot analysis using a monoclonal antibody recognizing an epitope between Arg-306 and Arg-506 indicated that during the rapid phase of inactivation a fragment of 80 kDa was generated that resulted from cleavage at a residue very close to Arg-506. The slow phase was associated with the formation of fragments resulting from cleavage at a residue 1.5-2 kDa carboxyl-terminal to Arg-306. Our observations may explain the unexpectedly mild APC resistance associated with mutations at Arg-306 (FV HongKong and FV Cambridge) in the heavy chain of FV.     

Prothrombin residues 473-487 contribute to factor Va binding in the prothrombinase complex

To identify sequences in prothrombin (fII) involved in prothrombinase complex (fXa.fVa.fII.phospholipids) assembly, synthetic peptides based on fII sequences were prepared and screened for their ability to inhibit factor Xa (fXa)-induced clotting of normal plasma. The fII peptide (PT473-487, homologous to chymotrypsin residues 149D-163) potently inhibited plasma clotting assays and prothrombinase activity, with 50% inhibition of 12 and 10 microm peptide, respectively. Prothrombinase inhibition by PT473-487 was factor Va (fVa)-dependent and sequence-specific, because the peptide did not inhibit fII activation in the absence of fVa, and a scrambled sequence peptide, PT473-487SCR, was not inhibitory. Peptide PT473-487 did not inhibit the amidolytic activities of fXa and thrombin, suggesting that the peptide did not alter the integrity of their active sites. To determine whether PT473-487 interacted directly with fVa, fluorescein-labeled fVa (Fl-fVa) was prepared. When PT473-487 was titrated into samples containing phospholipid-bound Fl-fVa, the peptide increased fluorescein anisotropy (EC(50) at 3 microm peptide), whereas the control peptide PT473-487SCR did not alter the anisotropy, suggesting a direct binding interaction between PT473-487 and Fl-fVa. These functional and spectroscopic data suggest that fII residues 473-487 provide fVa-binding sites and mediate interactions between fVa and fII in the prothrombinase complex.     

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.     

Carbohydrate moieties on the procofactor factor V, but not the derived cofactor factor Va, regulate its inactivation by activated protein C

Factor V (FV) is a single-chain plasma protein containing 13-25% carbohydrate by mass. Studies were done to determine if these carbohydrate moieties altered the activated protein C (APC)-catalyzed cleavage and inactivation of both FV and the cofactor which results from its activation by alpha-thrombin, factor Va(IIa) (FVa(IIa)). Treatment of purified FV with N-glycanase and neuraminidase under nonprotein-denaturing conditions removed approximately 20-30% of the carbohydrate from the heavy chain region of the molecule. When glycosidase-treated FV was analyzed in an aPTT (activated partial thromboplastin time)-based APC sensitivity assay, the APC sensitivity ratio (APC-SR) increased from 2.34 to 3.33. In contrast, when glycosidase-treated FV was activated with alpha-thrombin, the addition of the resulting FVa(IIa) to the plasma-based APC sensitivity assay produced no substantial increase in the APC-SR. Additional functional analyses of the APC-catalyzed inactivation of FVa(IIa) in an assay consisting of purified components indicated that both glycosidase-treated and untreated FVa(IIa) expressed identical cofactor activities and were inactivated at identical rates. Analyses of the APC-catalyzed cleavage of glycosidase-treated FV at Arg(306), the initial cleavage site, revealed a 10-fold rate increase when compared to untreated FV. In contrast, and consistent with functional assays, similar analyses of FVa(IIa), derived from those FV species, revealed near-identical rates of APC-catalyzed cleavage at both the Arg(506) and Arg(306)sites. These combined results indicate that N-linked carbohydrate moieties play a substantial role in the APC-catalyzed cleavage and inactivation of FV but not FVa(IIa) at position Arg(306) and that the Arg(306) cleavage sites of FV and FVa(IIa) are distinct substrates for APC.     

Lyso-Sulfatide Binds Factor Xa and Inhibits Thrombin Generation by the Prothrombinase Complex

Blood coagulation reactions are strongly influenced by phospholipids, but little is known about the influence of sphingolipids on coagulation mechanisms. Lysosulfatide (lyso-SF) (sulfogalactosyl sphingosine) prolonged factor Xa (fXa) 1-stage plasma clotting assays, showing it had robust anticoagulant activity. In studies using purified clotting factors, lyso-SF inhibited >90% of prothrombin (II) activation for reaction mixtures containing fXa/factor Va (fVa)/II, and also inhibited II activation generation by fXa/ phospholipids and by Gla-domainless-fXa/fVa/phospholipids. When lyso-SF analogs were tested, results showed that N-acetyl-sulfatide was not anticoagulant, implying that the free amine group was essential for the anticoagulant effects of lyso-SF. Lyso-SF did not inhibit fXa enzymatic hydrolysis of small peptide substrates, showing it did not directly inhibit the fXa activity. In surface plasmon resonance studies, lyso-SF bound to immobilized inactivated fXa as well as inactivated Gla-domainless-fXa. Confirming this lyso-SF:fXa interaction, fluorescence studies showed that fluorescently-labeled-fXa in solution bound to lyso-SF. Thus, lyso-SF is an anticoagulant lipid that inhibits fXa when this enzyme is bound to either phospholipids or to fVa. Mechanisms for inhibition of procoagulant activity are likely to involve lyso-SF binding to fXa domain(s) that are distinct from the fXa Gla domain. This suggests that certain sphingolipids, including lyso-SF and some of its analogs, may down-regulate fXa activity without inhibiting the enzyme’s active site or binding to the fXa Gla domain.     

Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C

Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg⁵⁰⁶ and Arg³⁰⁶ and of factor VIIIa (FVIIIa) by cleavage at Arg³³⁶ and Arg⁵⁶². To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a K_D of 0.11 ± 0.05 nm and competed with active site-labeled Oregon Green activated coagulation factor X for binding to FVa. The binding of APC(S360A) to FVa was not affected by protein S but was inhibited by prothrombin. APC(S360A) binding to FVa was critically dependent upon the presence of Arg⁵⁰⁶ and not Arg³⁰⁶ and additionally required an active site accessible to substrates. Inhibition of FVIIIa activity by APC(S360A) was >100-fold less efficient than inhibition of FVa. Our results show that despite exosite interactions near the Arg⁵⁰⁶ cleavage site, binding of APC(S360A) to FVa is almost completely dependent on Arg⁵⁰⁶ interacting with APC(S360A) to form a nonproductive Michaelis complex. Because docking of APC to FVa and FVIIIa constitutes the first step in the inactivation of the cofactors, we hypothesize that the observed anticoagulant activity may be important for in vivo regulation of thrombin formation.     

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.     

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.