Structure of bovine blood coagulation factor Va. Determination of the subunit associations, molecular weights, and asymmetries by analytical ultracentrifugation.
Thrombin-activated bovine factor V (factor Va), an essential component of the blood clotting cascade pro thrombinase complex, is composed of two nonidentical subunits (Vl and Vh) and Ca2+ in tight association. We have examined Vl, Vh, and factor Va using analytical ultracentrifugation. At pH 7.65 in 50 mM tris(hydroxymethyl)aminomethane, 0.1 M NaCl, 1 mM benzamidine, and 10 mM Ca2+, the Vl subunit has a molecular weight (Mr) of 82 500, an S0(20) ,w = 5. 0(2)S , and, assuming a model of a prolate ellipsoid with 0.3 g of H2O/g of protein, an axial ratio of 5:1. The corresponding values for the Vh subunit are an Mr of 92 300, an S0(20) ,w = 5.2(9) S, and an axial ratio of 5:1. We found these same values for Vl and for Vh in a buffer that contained 2 mM ethylenediaminetetraacetate (EDTA) rather than the 10 mM Ca2+. The Vl subunit undergoes a weak, reversible self-association at 9 degrees C with an apparent monomer-dimer association constant of 5.6 X 10(3) M-1 in the presence of 2 mM EDTA and 2.3 X 10(3) M-1 in the presence of 10 mM Ca2+. Our data indicate that the Vl self-association includes dimer and higher oligomers. Factor Va, examined in the presence of 10 mM Ca2+ and at 20 degrees C, has an Mr of 174 000, and S0(20) ,w = 8.1(8)S, an axial ratio of 5:1, and an apparent Vl-Vh association constant of at least 2.7 X 10(8) M-1. Our results suggest that factor Va self-associates to form higher multimers. When solutions of Va are dialyzed against a buffer that contains no Ca2+ and 2 mM EDTA, the apparent Vl-Vh subunit association constant is reduced to 9.4 X 10(3) M-1. Our hydrodynamic data indicate that there is a substantial decrease in molecular asymmetry when factor V is proteolytically activated by thrombin to form factor Va and that Vl and Vh are arranged "side by side" rather than "end to end" in factor Va.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D004492:Edetic Acid / Q000494:pharmacology; D005165:Factor V; D015943:Factor Va; D046911:Macromolecular Substances; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D014461:Ultracentrifugation
D046911:Macromolecular Substances; D015943:Factor Va; D005165:Factor V; D004492:Edetic Acid; D002118:Calcium