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

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Author

Summary, in English

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.

Publishing year

2008

Language

English

Pages

20805-20812

Publication/Series

Journal of Biological Chemistry

Volume

283

Issue

30

Document type

Journal article

Publisher

American Society for Biochemistry and Molecular Biology

Topic

  • Medicinal Chemistry

Status

Published

Research group

  • Clinical Chemistry, Malmö

ISBN/ISSN/Other

  • ISSN: 1083-351X