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

Nicolaes, GAF; Sorensen, KW; Friedrich, Ute LU ; Tans, G; Rosing, J; Autin, L; Dahlbäck, Björn LU and Villoutreix, BO (2004) In European Journal of Biochemistry 271(13). p.2724-2736
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
heparin, protein docking, factor V, coagulation, protein C
in
European Journal of Biochemistry
volume
271
issue
13
pages
2724 - 2736
publisher
Wiley-Blackwell
external identifiers
  • wos:000222176500015
  • pmid:15206937
  • scopus:3042720703
ISSN
0014-2956
DOI
10.1111/j.1432-1033.2004.04201.x
language
English
LU publication?
yes
id
65eeab9a-2781-4c03-bdb3-b10785f1da33 (old id 898881)
date added to LUP
2008-01-15 15:30:07
date last changed
2017-01-01 06:52:39
@article{65eeab9a-2781-4c03-bdb3-b10785f1da33,
  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.},
  author       = {Nicolaes, GAF and Sorensen, KW and Friedrich, Ute and Tans, G and Rosing, J and Autin, L and Dahlbäck, Björn and Villoutreix, BO},
  issn         = {0014-2956},
  keyword      = {heparin,protein docking,factor V,coagulation,protein C},
  language     = {eng},
  number       = {13},
  pages        = {2724--2736},
  publisher    = {Wiley-Blackwell},
  series       = {European Journal of Biochemistry},
  title        = {Altered inactivation pathway of factor Va by activated protein C in the presence of heparin},
  url          = {http://dx.doi.org/10.1111/j.1432-1033.2004.04201.x},
  volume       = {271},
  year         = {2004},
}