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Thrombin-mediated proteolysis of factor V resulting in gradual B-domain release and exposure of the factor Xa-binding site.

Steen, Mårten LU and Dahlbäck, Björn LU (2002) In Journal of Biological Chemistry 277(41). p.38424-38430
Abstract
To investigate the relationship between the individual thrombin-cleavages in factor V (FV) and the generation of 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 Kd for FXa being approximately 10 nM. Fully activated wt-FVa, yielded an apparent Kd of around 0.2 nM. The Arg709 and... (More)
To investigate the relationship between the individual thrombin-cleavages in factor V (FV) and the generation of 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 Kd for FXa being approximately 10 nM. Fully activated wt-FVa, yielded an apparent Kd of around 0.2 nM. The Arg709 and Arg1018 cleavages occurred at low thrombin concentrations, and decreased the Kd for FXa binding 5- and 3-fold, respectively. The Arg1545 cleavage, being less sensitive to thrombin, decreased the Kd for FXa binding approximately 20-fold. The Km 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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
277
issue
41
pages
38424 - 38430
publisher
ASBMB
external identifiers
  • wos:000178529600056
  • scopus:0037064117
ISSN
1083-351X
DOI
10.1074/jbc.M204972200
language
English
LU publication?
yes
id
6767304f-f03a-4535-b0f1-d0e33361effd (old id 109761)
alternative location
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12163491&dopt=Abstract
date added to LUP
2007-07-27 09:26:07
date last changed
2017-10-22 03:34:24
@article{6767304f-f03a-4535-b0f1-d0e33361effd,
  abstract     = {To investigate the relationship between the individual thrombin-cleavages in factor V (FV) and the generation of 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 Kd for FXa being approximately 10 nM. Fully activated wt-FVa, yielded an apparent Kd of around 0.2 nM. The Arg709 and Arg1018 cleavages occurred at low thrombin concentrations, and decreased the Kd for FXa binding 5- and 3-fold, respectively. The Arg1545 cleavage, being less sensitive to thrombin, decreased the Kd for FXa binding approximately 20-fold. The Km 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.},
  author       = {Steen, Mårten and Dahlbäck, Björn},
  issn         = {1083-351X},
  language     = {eng},
  number       = {41},
  pages        = {38424--38430},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Thrombin-mediated proteolysis of factor V resulting in gradual B-domain release and exposure of the factor Xa-binding site.},
  url          = {http://dx.doi.org/10.1074/jbc.M204972200},
  volume       = {277},
  year         = {2002},
}