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1.
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. 相似文献
2.
M J Heeb A Rehemtulla M Moussalli Y Kojima R J Kaufman 《European journal of biochemistry》1999,260(1):64-75
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. 相似文献
3.
Interdomain engineered disulfide bond permitting elucidation of mechanisms of inactivation of coagulation factor Va by activated protein C
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Gale AJ Xu X Pellequer JL Getzoff ED Griffin JH 《Protein science : a publication of the Protein Society》2002,11(9):2091-2101
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. 相似文献
4.
Segers K Dahlbäck B Rosing J Nicolaes GA 《The Journal of biological chemistry》2008,283(33):22573-22581
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. 相似文献
5.
Binding sites for blood coagulation factor Xa and protein S involving residues 493-506 in factor Va. 总被引:7,自引:2,他引:5
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M. J. Heeb Y. Kojima T. M. Hackeng J. H. Griffin 《Protein science : a publication of the Protein Society》1996,5(9):1883-1889
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. 相似文献
6.
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. 相似文献
7.
Structural investigation of the A domains of human blood coagulation factor V by molecular modeling. 总被引:16,自引:1,他引:15
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B. O. Villoutreix B. Dahlbck 《Protein science : a publication of the Protein Society》1998,7(6):1317-1325
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. 相似文献
8.
Gerry A F Nicolaes Kristoffer W S?rensen Ute Friedrich Guido Tans Jan Rosing Ludovic Autin Bj?rn Dahlb?ck Bruno O Villoutreix 《European journal of biochemistry》2004,271(13):2724-2736
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. 相似文献
9.
Gale AJ Yegneswaran S Xu X Pellequer JL Griffin JH 《The Journal of biological chemistry》2007,282(30):21848-21855
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. 相似文献
10.
A Gla domain-mutated protein C variant, QGNSEDY, modified at positions 10-12, 23, 32-33, and 44, having enhanced affinity for negatively charged phospholipid and increased anticoagulant potential, was used to elucidate the importance of the interaction between the Gla domain and the phospholipid for the ability of activated protein C (APC) to inactivate factor Va (FVa). FVa degradation by wild type (WT)-APC and QGNSEDY-APC yielded similar fragments on Western blotting; QGNSEDY-APC was, however, considerably more efficient. The kinetic parameters for individual APC-mediated cleavages in FVa, i.e. at Arg-306 and Arg-506, were investigated at high and low phospholipid concentrations in the presence and absence of protein S. FVa variants 306Q679Q and 506Q679Q, which can only be cleaved at Arg-506 and Arg-306, respectively, were used. In the absence of protein S, QGNSEDY-APC was 17.8- and 4-fold more efficient than WT-APC in cleaving at Arg-306 and Arg-506, respectively, at high phospholipid. Similar values were obtained at low phospholipid. In the presence of protein S, QGNSEDYAPC was 6.8- and 3.2-fold more active than WT-APC in cleaving at Arg-306 and Arg-506, respectively, at high phospholipid. At low phospholipid, the corresponding values were 14- and 6.5-fold. In conclusion, the modification of the Gla domain in QGNSEDY-APC yielded increased rates of cleavage at both sites in FVa, the increase being particularly pronounced for the Arg-306 site in the absence of protein S. The results obtained with QGNSEDY-APC provide insights into the importance of the APC-phospholipid interaction for the APC-mediated cleavages at Arg-306 and Arg-506 in FVa. 相似文献
11.
The effect of Arg306-->Ala and Arg506-->Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S.
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J. O. Egan M. Kalafatis K. G. Mann 《Protein science : a publication of the Protein Society》1997,6(9):2016-2027
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. 相似文献
12.
Yegneswaran S Kojima Y Nguyen PM Gale AJ Heeb MJ Griffin JH 《The Journal of biological chemistry》2007,282(39):28353-28361
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. 相似文献
13.
van der Neut Kolfschoten M Dirven RJ Vos HL Tans G Rosing J Bertina RM 《The Journal of biological chemistry》2004,279(8):6567-6575
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. 相似文献
14.
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. 相似文献
15.
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. 相似文献
16.
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. 相似文献
17.
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. 相似文献
18.
Friedrich U Nicolaes GA Villoutreix BO Dahlbäck B 《The Journal of biological chemistry》2001,276(25):23105-23108
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. 相似文献
19.
Gerry A. F. Nicolaes Paul E. Bock Kenneth Segers Karin C. A. A. Wildhagen Bj?rn Dahlb?ck Jan Rosing 《The Journal of biological chemistry》2010,285(30):22890-22900
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 KD 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. 相似文献
20.
Newell-Caito JL Laha M Tharp AC Creamer JI Xu H Maddur AA Tans G Bock PE 《The Journal of biological chemistry》2011,286(44):38286-38297
Notecarin D (NotD) is a prothrombin (ProT) activator in the venom of the tiger snake, Notechis scutatus, and a factor Xa (FXa) homolog. NotD binds specifically to the FXa binding site expressed on factor V (FV) upon activation to factor Va (FVa) by thrombin. NotD active site-labeled with 5-fluorescein ([5F]FFR-NotD) binds FV and FVa with remarkably high affinity in the absence of phospholipids (K(D) 12 and ≤ 0.01 nm, respectively). In the presence of membranes, the affinity of [5F]FFR-NotD for FVa is similar, but increased ~55-fold for FV. Binding of FXa active site-labeled with Oregon Green to FV and FVa in the presence of phospholipids is ~5,000- and ~80-fold weaker than [5F]FFR-NotD, respectively. NotD reports FVa and not FV binding by a 3-fold increase in tripeptide substrate hydrolysis, demonstrating allosteric regulation by FVa. The NotD·FVa·membrane complex activates ProT with K(m)((app)) similar to prothrombinase, and ~85-fold weaker without membranes. Active site-blocked NotD exhibits potent anticoagulant activity in plasma thrombin generation assays, representing inhibition of productive prothrombinase assembly and possible disruption of FXa inhibition by the tissue factor pathway inhibitor. The results show that high affinity binding of NotD to FVa is membrane-independent, unlike the strict membrane dependence of FXa for high affinity FVa binding. 相似文献