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 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 VI subunit has a molecular weight (Mr) of 82 500, an S°20,w = 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 Mr of 92 300, an S°20,w = 5.29 S, and an axial ratio of 5:1. We found these same values for VI and for Vh in a buffer that contained 2 mM ethylenediaminetetraacetate (EDTA) rather than the 10 mM Ca2+. The VI subunit undergoes a weak, reversible self-association at 9 °C with an apparent monomer-dimer association constant of 5.6 X 103 M″1 in the presence of 2 mM EDTA and 2.3 X 103 M″1 in the presence of 10 mM Ca2+. Our data indicate that the VI self-association includes dimer and higher oligomers. Factor Va, examined in the presence of 10 mM Ca2+ and at 20 °C, has an Mr of 174000, an s°20,w = 8.18 S, an axial ratio of 5:1, and an apparent Vl-Vh association constant of at least 2.7 × 108 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 × 103 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 VI and Vh are arranged “side by side” rather than “end to end” in factor Va. © 1984, American Chemical Society. All rights reserved.