Structure of bovine blood-coagulation factor Va. Determination of the subunit associations, molecular weights, and asymmetries by analytical ultracentrifugation

Abstract

Thrombin-activated bovine factor V (factor Va), an essential component of the blood clotting cascade prothrombinase complex, is composed of 2 nonidentical subunits (Vl and Vh) and Ca2+ in tight association. Vl, Vh and factor Va were examined 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 MW of 82,500, an .**GRAPHIC**. = 5.02 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 MW of 92,300, an .**GRAPHIC**. = 5.29 S, and an axial ratio of 5:1. These same values were found 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.degree. C with an apparent monomer-dimer association constant of 5.6 .times. 103 M-1 in the presence of 2 mM EDTA and 2.3 .times. 103 M-1 in the presence of 10 mM Ca2+. The Vl self-association includes dimer and higher oligomers. Factor Va, examined in the presence of 10 mM Ca2+ and at 20.degree. C, has an MW of 174,000, an .**GRAPHIC**. = 8.18 S, an axial ratio of 5:1, and an apparent Vl-Vh association constant of at least 2.7 .times. 108 M-1. Factor Va apparently 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 .times. 103 M-1. 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.