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.     

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.     

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.     

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.     

Neutral glycosphingolipid-dependent inactivation of coagulation factor Va by activated protein C and protein S.

To test whether neutral glycosphingolipids can serve as anticoagulant cofactors, the effects of incorporation of neutral glycosphingolipids into phospholipid vesicles on anticoagulant and procoagulant reactions were studied. Glucosylceramide (GlcCer), lactosylceramide (LacCer), and globotriaosylceramide (Gb(3)Cer) in vesicles containing phosphatidylserine (PS) and phosphatidylcholine (PC) dose dependently enhanced factor Va inactivation by the anticoagulant factors, activated protein C (APC) and protein S. Addition of GlcCer to PC/PS vesicles enhanced protein S-dependent APC cleavage in factor Va at Arg-506 by 13-fold, whereas PC/PS vesicles alone minimally affected protein S enhancement of this reaction. Incorporation into PC/PS vesicles of GlcCer, LacCer, or Gb(3)Cer, but not galactosylceramide or globotetraosylceramide, dose dependently prolonged factor Xa-1-stage clotting times of normal plasma in the presence of added APC without affecting baseline clotting times in the absence of APC, showing that certain neutral glycosphingolipids enhance anticoagulant but not procoagulant reactions in plasma. Thus, certain neutral glycosphingolipids (e.g. GlcCer, LacCer, and Gb(3)Cer) can enhance anticoagulant activity of APC/protein S by mechanisms that are distinctly different from those of phospholipids alone. We speculate that under some circumstances certain neutral glycosphingolipids either in lipoprotein particles or in cell membranes may help form antithrombotic microdomains that might enhance down-regulation of thrombin by APC in vivo.     

Proteolysis of factor Va by factor Xa and activated protein C

Bovine Factor Va, produced by selective proteolytic cleavage of Factor V by thrombin, consists of a heavy chain (D chain) of Mr = 94,000 and a light chain (E chain) of Mr = 74,000. These peptides are noncovalently associated in the presence of divalent metal ion(s). Each chain is susceptible to proteolysis by activated protein C and by Factor Xa. Sodium dodecyl sulfate electrophoretic analysis indicates that cleavage of the E chain by either activated protein C or Factor Xa yields two major fragments: Mr = 30,000 and Mr = 48,000. Amino acid sequence analysis indicates that the Mr = 30,000 fragments have identical NH2-terminal sequences and that this sequence corresponds to that of intact E chain. The Mr = 48,000 fragments also have identical NH2-terminal sequences, indicating that activated protein C and Factor Xa cleave the E chain at the same position. Sodium dodecyl sulfate electrophoretic analysis indicates that activated protein C cleavage of the D chain yields two products: Mr = 70,000 and Mr = 24,000. Amino acid sequence analysis indicates that the Mr = 70,000 fragment has the same NH2-terminal sequence as intact D chain, whereas the Mr = 24,000 fragment does not. Factor Xa cleavage of the D chain also yields two products: Mr = 56,000 and Mr = 45,000. The Mr = 56,000 fragment corresponds to the NH2-terminal end of the D chain and Factor V. Functional studies have shown that both chains of Factor Va may be entirely cleaved to products by Factor Xa without loss of activity, whereas activated protein C cleavage results in loss of activity. Since activated protein C and Factor Xa cleave the E chain at the same position, the cleavage of the D chain by activated protein C is responsible for the inactivation of Factor Va.     

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.     

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)     

Homocysteine inhibits inactivation of factor Va by activated protein C

We report the effect of homocysteine on the inactivation of factor Va by activated protein C (APC) using clotting assays, immunoblotting, and radiolabeling experiments. Homocysteine, cysteine, or homocysteine thiolactone have no effect on factor V activation by alpha-thrombin. Factor Va derived from homocysteine-treated factor V was inactivated by APC at a reduced rate. The inactivation impairment increased with increasing homocysteine concentration (pseudo first order rate k = 1.2, 0.9, 0.7, 0.4 min(-1) at 0, 0.03, 0.1, 1 mm homocysteine, respectively). Neither cysteine nor homocysteine thiolactone treatment of factor V affected APC inactivation of derived factor Va. Western blot analyses of APC inactivation of homocysteine-modified factor Va are consistent with the results of clotting assays. Factor Va, derived from factor V treated with 1 mm beta-mercaptoethanol was inactivated more rapidly than the untreated protein sample. Factor V incubated with [(35)S]homocysteine (10-450 micrometer) incorporated label within 5 min, which was found only in those fragments that contained free sulfhydryl groups: the light chain (Cys-1960, Cys-2113), the B region (Cys-1085), and the 26/28-kDa (residues 507-709) APC cleavage products of the heavy chain (Cys-539, Cys-585). Treatment with beta-mercaptoethanol removed all radiolabel. Plasma of patients assessed to be hyperhomocysteinemic showed APC resistance in a clot-based assay. Our results indicate that homocysteine rapidly incorporates into factor V and that the prothrombotic tendency in hyperhomocysteinemia may be related to impaired inactivation of factor Va by APC due to homocysteinylation of the cofactor by modification of free cysteine(s).     

The association of coagulation factor Xa and factor Va

The binding of factor Xa to factor Va in the presence of Ca2+ ions and phospholipid is fundamental for the activation of prothrombin to thrombin. Nevertheless, the biochemistry of the intrinsic association between factors Xa and Va is poorly understood. In the present study we have measured the formation of the protein-protein complex in the absence of phospholipid by using analytical ultracentrifugation. Factor Xa or factor Va were respectively modified with a chromophore-peptidyl-chloromethyl ketone or a thiol-specific chromophore, which permitted selective evaluation of the sedimentation of either component by virtue of its unique absorbance properties. Regardless of which protein was labeled, a factor Xa-Va complex (s20,w = 9.8) was formed. The interaction is specific and reversible. In 2 mM Ca2+ and at 20 degrees C, the dissociation constant for the binding of factor Xa to factor Va is 0.8 microM with a 1:1 stoichiometry. The association has multiphasic Ca2+ dependence. At concentrations of Ca2+ below 1 mM or above 2 mM, a weaker protein-protein equilibrium is maintained.     

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.     

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.     

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.     

Functional properties of factor Va subunits after proteolytic alterations by activated protein C

The two-subunit structure of the factor Va molecule is essential to its function in the prothrombinase complex. In the presence of phospholipids, the cleavage of the light chain of bovine factor Va by activated protein C proceeded at the same rate as the cleavage of the heavy chain. The limited proteolysis of factor Va is accompanied by a parallel loss of factor Va activity. Evidence that loss of activity was solely the result of the cleavage of the heavy chain, was obtained from reconstitution experiments utilizing cleaved and intact chains. The pseudo first-order rate constant of factor Va inactivation by activated protein C was found to be dependent on the amount of phospholipid-bound activated protein C and not on the amount of phospholipid-bound factor Va. However, phospholipids enhance the rate of proteolysis of the phospholipid-binding subunit, i.e. the light chain, and not the cleavage of the heavy chain. Cleavage of the heavy chain and as a consequence the inactivation of factor Va by activated protein C is mediated by phospholipid-bound light chain. After cleavage of the light chain, the 'two-subunit' structure, as well as the phospholipid-binding properties of factor Va were found to be conserved.     

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.     

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.     

Regulation of activated protein C by protein S. The role of phospholipid in factor Va inactivation

Protein S enhances the rate of Factor Va inactivation by activated Protein C (Walker, F. J. (1980) J. Biol. Chem. 255, 5521-5524). The activity of protein S is saturable, appearing to interact stoichiometrically with activated Protein C. Diisopropylphosphate-modified activated Protein C reversed the effect of Protein S, further indicating that a Protein S-activated Protein C interaction is required for expression of the activity of Protein S. In the absence of phospholipid, Protein S had no effect on the rate of activated Protein C-catalyzed inactivation of Factor Va. The activity of Protein S was only expressed in the presence of phospholipid vesicles, where it appeared to increase the affinity of the inactivation system for phospholipid. Protein S had no effect upon the rate of Factor Va inactivation in the presence of saturating levels of phospholipid vesicles. The effects of Protein S on the kinetics of Factor Va inactivation corresponded with its effect on the interaction between activated Protein C and phospholipid vesicles, measured by light scattering. In the presence of Protein S, the binding of activated Protein C to phospholipid vesicles was enhanced. Protein S had no effect upon the binding on the zymogen (Protein C to phospholipid vesicles). In conclusion, the stimulatory effect of Protein S on the inactivation of Factor Va by activated Protein C can be attributed, in part, to the enhancement of the binding of activated Protein C to phospholipid vesicles.     

A novel anticoagulant protein with high affinity to blood coagulation factor Va from Tegillarca granosa

A novel inhibitory protein against blood coagulation factor Va (FVa) was purified from muscle protein of granulated ark (Tegillarca granosa, order Arcoida, marine bivalvia) by consecutive FPLC method using anion exchange and gel permeation chromatography. In the results of ESIQTOF tandem mass analysis and database research, it was revealed that the purified T. granosa anticoagulant protein (TGAP) has 7.7 kDa of molecular mass and its partial sequence, HTHLQRAPHPNALGYHGK, has a high identity (64%) with serine/threonine kinase derived from Rhodopirellula baltica (order Planctomycetales, marine bacteria). TGAP could potently prolong thrombin time (TT), corresponding to inhibition of thrombin (FIIa) formation. Specific factor inhibitory assay showed that TGAP inhibits FVa among the major components of prothrombinase complex. In vitro assay for direct-binding affinity using surface plasmon resonance (SPR) spectrometer indicated that TGAP could be directly bound with FVa. In addition, the binding affinity of FVa to FII was decreased by addition of TGAP in dose-dependant manner (IC50 value = 77.9 nM). These results illustrated that TGAP might interact with a heavy chain of FVa (FVa(H)) bound to FII in prothrombin complex. The present study elucidated that non-cytotoxic T. granosa anticoagulant protein (TGAP) bound to FVa can prolong blood coagulation time by inhibiting conversion of FII to FIIa in blood coagulation cascade. In addition, TGAP did not significantly (P < 0.05) show fibrinolytic activity and cytotoxicity on venous endothelial cell line (ECV 304).     

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.