TAILIEUCHUNG - Báo cáo khoa học: 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 corres-pondingrate constantk¢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 inac-tivation. Peptide bond cleavage was identified by Western blottingusingFV-specific antibodies. . | Eur. J. Biochem. 271 2724-2736 2004 FEBS 2004 doi Altered inactivation pathway of factor Va by activated protein C in the presence of heparin Gerry A. F. Nicolaes1 Kristoffer W. Sorensen1 2 Ute Friedrich2 3 Guido Tans1 Jan Rosing1 Ludovic Autin4 Bjorn Dahlback2 and Bruno O. Villoutreix4 1 Department of Biochemistry Cardiovascular Research Institute Maastricht the Netherlands department of Clinical Chemistry University Hospital Malmo Sweden 3Immunochemistry Department Novo Nordisk A S Gentofte Denmark 4INSERM U428 University of Paris V France 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 conttant 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-specifir anlioodies. 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 vitlLirllly Llnaftected. m conri atl. 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 ehe thth addiáanal 0 0011 4x11001 from APC loop 148. Fewer contacts are observed between APC and the region around the Arg306 site in FVa. The modeling and experimental .

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