Role of Hirudin-like factor Va heavy chain sequences in prothrombinase function.
Proexosite I on prothrombin has been implicated in providing a recognition site for factor Va within prothrombinase. To examine whether hirudin-like sequences (659-698) on the cofactor contribute to this interaction, we expressed and purified two-chain FVa derivatives that were intracellularly truncated at the C terminus of the heavy chain: FVa709 (des710-1545), FVa699 (des700-1545), FVa(692 (des693-1545), FVa678 (des679-1545), and FVa658 (des659-1545). We found that FVa709, FVa699, FVa692, and FVa678 exhibited specific clotting activities that were comparable with plasma-derived and recombinant FVa. Additionally, kinetic studies using prothrombin revealed that the Km and kcat values for these derivatives were unaltered. Fluorescent measurements and chromatography studies indicated that FVa709, FVa699, FVa692, and FVa678 bound to FXa membranes and thrombin-agarose in a manner that was comparable with the wild-type cofactors. In contrast, FVa658 had an approximately 1% clotting activity and reduced affinity for FXa membranes (approximately 20-fold) and did not bind to thrombin-agarose. Surprisingly, however, FVa(658) exhibited essentially normal kinetic parameters for prothrombin when the variant was fully saturated with FXa membranes. Overall our results are consistent with the interpretation that any possible binding interactions between prothrombin and the C-terminal region of the FVa heavy chain do not contribute in a detectable way to the enhanced function of prothrombinase.
Toso
Raffaella
R
Department of Pediatrics, Division of Hematology, The Children's Hospital of Philadelphia and University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Camire
Rodney M
RM
D000595:Amino Acid Sequence; D001780:Blood Coagulation Tests / Q000379:methods; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D015943:Factor Va / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism; D005453:Fluorescence; D014644:Genetic Variation; D006629:Hirudins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D011516:Prothrombin / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism*
D006629:Hirudins; D011994:Recombinant Proteins; D015943:Factor Va; D011516:Prothrombin; D013925:Thromboplastin; D015951:Factor Xa