Cofactor dependence of factor Xa incorporation into the prothrombinase complex.

The blood coagulation serine protease, factor Xa, was dansylated with the active site-directed inhibitor, dansyl-glutamyl-glycyl-arginyl chloromethyl ketone. The Ca2+-dependent interactions of the inactivated factor Xa with its cofactors, phospholipid and activated factor V (factor Va), were studied through alterations of fluorescence polarization values of dansyl moiety of the modified enyme. In the presence of phospholipid and Ca2+, factor Va and factor Xa interacted with 1:1 stoichiometry, an interaction characterized by markedly enhanced polarization. The factor Va-independent interaction of factor Xa with phospholipid was also observed, characterized by dissociation constant Kd=2.7 x 10-6 M and stoichiometry of 66 mol of phospholipid/mol of factor Xa. The interaction of factor Xa with vesicles in the absence of factor Va exhibited considerably lower polarization values than in the presence of factor Va. These data obtained by direct spectral measurements are in agreement with the inferences drawn previously from studies of kinetics that the prothrombinase complex consists of 1:1 stoichiometric complex of factor Xa and phospholipid-bound factor Va, and that the enzymatic complex assembles in the absence of the natural substrate, prothrombin.