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Mapping of the factor Xa-binding site on factor Va by site-directed mutagenesis.

Steen, Mårten LU ; Tran, Sinh LU ; Autin, Ludovic; Villoutreix, Bruno O; Tholander, Ann-Louise LU and Dahlbäck, Björn LU (2008) In Journal of Biological Chemistry 283(30). p.20805-20812
Abstract
Activated coagulation factor V functions as a cofactor to factor Xa in the conversion of prothrombin to thrombin. Based on introduction of extra carbohydrate side chains in recombinant factor V, we recently proposed several regions in factor Va to be important for factor Xa binding. To further define which residues are important for factor Xa binding, we prepared fifteen recombinant factor V variants in which clusters of charged amino acid residues were mutated, mainly to alanines. The factor V variants were expressed in COS-1 cells and their functional properties evaluated in a prothrombinase-based assay, as well as in a direct binding test. Four of the factor V variants, 501A/510A/511D, 501A/510A/511D/513A, 513A/577A/578A, and... (More)
Activated coagulation factor V functions as a cofactor to factor Xa in the conversion of prothrombin to thrombin. Based on introduction of extra carbohydrate side chains in recombinant factor V, we recently proposed several regions in factor Va to be important for factor Xa binding. To further define which residues are important for factor Xa binding, we prepared fifteen recombinant factor V variants in which clusters of charged amino acid residues were mutated, mainly to alanines. The factor V variants were expressed in COS-1 cells and their functional properties evaluated in a prothrombinase-based assay, as well as in a direct binding test. Four of the factor V variants, 501A/510A/511D, 501A/510A/511D/513A, 513A/577A/578A, and 501A/510A/511D/513A/577A/578A exhibited markedly reduced factor Xa-cofactor activity tested in the prothrombinase assay, and reduced binding affinity as judged by the direct binding assay. These factor Va variants were normally cleaved at Arg506 by activated protein C and the interaction between the factor Xa - factor Va complex and prothrombin was unaffected by the introduced mutations. Based on the integration of all available data we propose a key factor Xa-binding surface to be centered on Arg501, Arg510, Ala511, Asp513, Asp577 and Asp578 in the factor Va A2 domain. These residues form an elongated charged factor Xa-binding cluster on the factor Va surface. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
283
issue
30
pages
20805 - 20812
publisher
ASBMB
external identifiers
  • wos:000257746100022
  • pmid:18502757
  • scopus:51049092534
ISSN
1083-351X
DOI
10.1074/jbc.M802703200
language
English
LU publication?
yes
id
28edec01-4175-4b54-bd35-57332f79d223 (old id 1153772)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/18502757?dopt=Abstract
date added to LUP
2008-06-03 11:11:23
date last changed
2017-07-23 04:55:15
@article{28edec01-4175-4b54-bd35-57332f79d223,
  abstract     = {Activated coagulation factor V functions as a cofactor to factor Xa in the conversion of prothrombin to thrombin. Based on introduction of extra carbohydrate side chains in recombinant factor V, we recently proposed several regions in factor Va to be important for factor Xa binding. To further define which residues are important for factor Xa binding, we prepared fifteen recombinant factor V variants in which clusters of charged amino acid residues were mutated, mainly to alanines. The factor V variants were expressed in COS-1 cells and their functional properties evaluated in a prothrombinase-based assay, as well as in a direct binding test. Four of the factor V variants, 501A/510A/511D, 501A/510A/511D/513A, 513A/577A/578A, and 501A/510A/511D/513A/577A/578A exhibited markedly reduced factor Xa-cofactor activity tested in the prothrombinase assay, and reduced binding affinity as judged by the direct binding assay. These factor Va variants were normally cleaved at Arg506 by activated protein C and the interaction between the factor Xa - factor Va complex and prothrombin was unaffected by the introduced mutations. Based on the integration of all available data we propose a key factor Xa-binding surface to be centered on Arg501, Arg510, Ala511, Asp513, Asp577 and Asp578 in the factor Va A2 domain. These residues form an elongated charged factor Xa-binding cluster on the factor Va surface.},
  author       = {Steen, Mårten and Tran, Sinh and Autin, Ludovic and Villoutreix, Bruno O and Tholander, Ann-Louise and Dahlbäck, Björn},
  issn         = {1083-351X},
  language     = {eng},
  number       = {30},
  pages        = {20805--20812},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Mapping of the factor Xa-binding site on factor Va by site-directed mutagenesis.},
  url          = {http://dx.doi.org/10.1074/jbc.M802703200},
  volume       = {283},
  year         = {2008},
}