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.
Highlights
Cofactor factor Va (FVa), assembled on the surface of negatively charged phospholipid membranes, in the presence of divalent metal ions [1, 2]
To delineate the factor Xa (FXa) binding site in further detail, recombinant FV variants were created after site-directed mutagenesis of 25 selected positions (Table 1)
Multiple protein-protein and protein-phospholipid interactions between FXa, FVa, prothrombin, and phospholipid are involved in the formation of the prothrombinase complex and the activation of prothrombin
Summary
Cofactor factor Va (FVa), assembled on the surface of negatively charged phospholipid membranes, in the presence of divalent metal ions [1, 2]. FX is converted to its active form by either the extrinsic pathway factor VIIa/tissue factor complex or the intrinsic pathway tenase complex composed of the phospholipid-bound factor IXa (FIXa) and activated FVIII (FVIIIa) [1, 2] Both FVa and FXa bind to negatively charged phospholipid membranes via their light chains; the FXa binding mediated by the Gla domain, while the FVa binding depends on the C2 domain with additional contribution from the C1 domain [8, 9]. Taken together with previous peptide studies, the results suggested that residues 499 –505 of FVa formed part of an extended FXa binding surface This is very close to one of the cleavage sites for activated protein C (APC), which inhibits FVa by cleaving at Arg-306, Arg-506, and Arg-679 [2, 7, 24]. Cleavage at Arg-506 results in partial loss of FVa activity due to decreased affinity for FXa, whereas cleavages at all sites result in the complete loss of FVa activity
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