Activation of porcine factor VIII:C by thrombin and factor Xa

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Abstract
The activation of porcine factor VIII:C by thrombin and by factor Xa was studied by a chromogenic substrate assay and by sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis of 125I-labeled factor VIII:C activation products. In the chromogenic assay, the kinetics of factor VIII:C dependent activation of factor X by factor IXa in the presence of calcium and phosphatidylserine/phosphatidylcholine vesicles were measured with N-benzoyl-L-isoleucyl-L-glutamylglycyl-L-arginine p-nitroanilide (S2222) as substrate. Substrate dependence of initial rates of the reaction at fixed factor IXa, factor VIII:C, lipid, and calcium obeyed Michaelis-Menten kinetics. At fixed factor IXa, factor X, lipid, and calcium the initial rates of the reaction varied linearly with lower factor VIII:C concentrations and plateaued at higher concentrations. The linear initial rate dependence formed the basis of a rapid, plasma-free assay of activated factor VIII:C. The activation of factor VIII:C by thrombin or factor Xa and the enzyme-independent rate of spontaneous inactivation were studied under conditions of excess enzyme. A model of the activation kinetics was developed and fit to the data by a nonlinear least-squares technique. From the model, the catalytic efficiencies (kcat/Km) of factor VIII:C activation by thrombin and factor Xa were 5.0 .times. 106 M-1 s-1 and 1.1 .times. 106 M-1 s-1, respectively. By comparison with published values of the catalytic efficiencies of several other coagulation enzymes for various substrates, both thrombin and factor Xa are efficient enznymes toward factor VIII:C. Additionally, the model allows calculation of the relative cofactor activities of thrombin-activated factor VIII:C (factor VIII:CaIIa) vs. factor Xa activated factor VIII:C (factor VIII:CaXa). The ratio of cofactor activities (VIII:CaIIa/VIII:CaXa) is 3.0. This indicates that significantly more activity is generated when factor VIII is fully activates by thrombin than when factor VIII is fully activated by factor Xa. The formation of cofactor activity by both enzymes is closely paralleled by proteolysis of factor VIII:C polypeptides although thrombin and factor Xa give distinctly different products.