Mapping of the Factor Xa Binding Site on Factor Va by Site-directed Mutagenesis Export

@article{citeulike:3022626, abstract = {Activated coagulation factor V functions as a cofactor to factor Xa in the conversion of prothrombin to thrombin. Based on the 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 Arg-506 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 Arg-501, Arg-510, Ala-511, Asp-513, Asp-577, and Asp-578 in the factor Va A2 domain. These residues form an elongated charged factor Xa binding cluster on the factor Va surface. 10.1074/jbc.M802703200}, author = {Steen, Marten and Tran, Sinh and Autin, Ludovic and Villoutreix, Bruno O. and Tholander, Ann-Louise and Dahlback, Bjorn}, citeulike-article-id = {3022626}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M802703200}, citeulike-linkout-1 = {http://www.jbc.org/cgi/content/abstract/283/30/20805}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/18502757}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=18502757}, day = {25}, doi = {10.1074/jbc.M802703200}, journal = {J. Biol. Chem.}, keywords = {binding-sites}, month = {July}, number = {30}, pages = {20805--20812}, posted-at = {2008-07-20 16:14:13}, priority = {2}, 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} }

TY - JOUR ID - citeulike:3022626 L3 - citeulike-article-id:3022626 N2 - Activated coagulation factor V functions as a cofactor to factor Xa in the conversion of prothrombin to thrombin. Based on the 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 Arg-506 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 Arg-501, Arg-510, Ala-511, Asp-513, Asp-577, and Asp-578 in the factor Va A2 domain. These residues form an elongated charged factor Xa binding cluster on the factor Va surface. 10.1074/jbc.M802703200 IS - 30 JF - J. Biol. Chem. EP - 20812 TI - Mapping of the Factor Xa Binding Site on Factor Va by Site-directed Mutagenesis VL - 283 SP - 20805 KW - binding-sites AU - Steen, Marten AU - Tran, Sinh AU - Autin, Ludovic AU - Villoutreix, Bruno AU - Tholander, Ann-Louise AU - Dahlback, Bjorn PY - 2008/07/25/ UR - http://dx.doi.org/10.1074/jbc.M802703200 ER -