ABSTRACT: Factor (F) V is activated by alpha-thrombin following cleavages at Arg(709), Arg(1,018) and Arg(1,545). Amino acid region 1,490-1,520 of FV is essential for procofactor activation.
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 1,508-1,510 (FV(Delta1,508-1,510)) and 1,508-1,515 (FV(Delta1508-1515)). We have also created recombinant FV molecules with mutations (1508)DDY(1510)-->AAF (FV(AAF)), (1514)DY(1515)-->AF (FV(AF)) and Y(1510)-->F (FV(Y1510F)).
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 alpha-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 FV(Y1510F), which had normal clotting activity. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with monoclonal antibodies to FV demonstrated that incubation of 100 nm recombinant wild-type or plasma-derived FV with 1 nmalpha-thrombin for 5 min 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 FVa(AAF) and FVa(AF), resulting in accumulation of a M(r) 220,000 fragment representing amino acids 1,019-2,195.
Our data demonstrate that amino acid residues 1,508-1,515 of FV are required for efficient cleavage by alpha-thrombin and light chain formation.
Journal of Thrombosis and Haemostasis 02/2008; 6(1):118-24. · 5.73 Impact Factor