The contribution of amino acid residues 1508-1515 of factor V to light chain generation

Abstract

Background: Factor (F) V is activated by α-thrombin following cleavages at Arg709, Arg1018 and Arg1545. Amino acid region 1490-1520 of FV is essential for procofactor activation. Aim: To ascertain which amino acid residues from this region are important for light chain formation and procofactor activation, site-directed mutagenesis was used to create recombinant FV molecules missing amino acid 1508-1510 (FVΔ1508-1510) and 1508-1515 (FVΔ1508-1515). We have also created recombinant FV molecules with mutations 1508DDY1510 → AAF (FVAAF), 1514DY1515 → AF (FVAF) and Y1510 → F (FVY1510F). Methods and results: The recombinant mutant molecules were expressed and purified to homogeneity. The clotting activities of all clotting recombinant mutant FV molecules were tested in a two-stage assay following activation by α-thrombin and were found to be impaired compared with the clotting activity observed with wild-type recombinant FV or plasma-derived FV, with the exception of FVY1510F, which had normal clotting activity. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with monoclonal antibodies to FV demonstrated that incubation of 100n m recombinant wild-type or plasma-derived FV with 1n m α-thrombin for 5min was sufficient to generate heavy and light chains and completely activate the procofactor. In contrast, similar experimental conditions were ineffective in fully activating the two deletion mutant molecules as well as FVaAAF and FVaAF, resulting in accumulation of a Mr 220000 fragment representing amino acids 1019-2195. Conclusion: Our data demonstrate that amino acid residues 1508-1515 of FV are required for efficient cleavage by α-thrombin and light chain formation. © 2007 International Society on Thrombosis and Haemostasis.