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- Jeimy, SB, et al.
(författare)
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Identification of the MMRN1 binding region within the C2 domain of human factor V
- 2004
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 279:49, s. 51466-51471
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Tidskriftsartikel (refereegranskat)abstract
- In platelets, coagulation cofactor V is stored in complex with multimerin 1 in alpha-granules for activation- induced release during clot formation. The molecular nature of multimerin 1 factor V binding has not been determined, although multimerin 1 is known to interact with the factor V light chain. We investigated the region in factor V important for multimerin 1 binding using modified enzyme-linked immunoassays and recombinant factor V constructs. Factor V constructs lacking the C2 region or entire light chain had impaired and absent multimerin 1 binding, respectively, whereas the B domain deleted construct had modestly reduced binding. Analyses of point mutated constructs indicated that the multimerin 1 binding site in the C2 domain of factor V partially overlaps the phosphatidylserine binding site and that the factor V B domain enhances multimerin 1 binding. Multimerin 1 did not inhibit factor V phosphatidylserine binding, and it bound to phosphatidylserine independently of factor V. There was a reduction in factor V in complex with multimerin 1 after activation, and thrombin cleavage significantly reduced factor V binding to multimerin 1. In molar excess, multimerin 1 minimally reduced factor V procoagulant activity in prothrombinase assays and only if it was added before factor V activation. The dissociation of factor V-multimerin 1 complexes following factor V activation suggests a role for multimerin 1 in delivering and localizing factor V onto platelets prior to prothrombinase assembly.
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| 3. |
- Nicolaes, GAF, et al.
(författare)
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Altered inactivation pathway of factor Va by activated protein C in the presence of heparin
- 2004
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:13, s. 2724-2736
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Tidskriftsartikel (refereegranskat)abstract
- 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 (k(506)) and subsequent cleavage at Arg306 (k(306)). 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 k(506) by UFH was 12-fold, with the secondary cleavage at Arg306 (k(306)) being virtually unaffected. In contrast, UFH stimulated the initial cleavage at Arg306 (k'(306)) two- to threefold. Low molecular weight heparin (Fragmin(R)) 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.
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