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Altered inactivation pathway of factor Va by activated protein C in the presence of heparin

På svenska

Author

  • GAF Nicolaes
  • KW Sorensen
  • Ute Friedrich
  • G Tans
  • J Rosing
  • L Autin
  • Björn Dahlbäck
  • BO Villoutreix

Summary, in English

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.

Publishing year

2004

Language

English

Pages

2724-2736

Publication/Series

European Journal of Biochemistry

Volume

271

Issue

13

Document type

Journal article

Publisher

Wiley-Blackwell

Topic

  • Medicinal Chemistry

Keywords

  • heparin
  • protein docking
  • factor V
  • coagulation
  • protein C

Status

Published

Research group

  • Clinical Chemistry, Malmö

ISBN/ISSN/Other

  • ISSN: 0014-2956