Activation of porcine factor VIII:C by thrombin and factor Xa.
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 X 10(6) M-1 s-1 and 1.1 X 10(6) 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 enzymes toward factor VIII:C.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001779:Blood Coagulation Factors / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D004789:Enzyme Activation; D005169:Factor VIII / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa; D007700:Kinetics; D008433:Mathematics; D013552:Swine; D013917:Thrombin / Q000378:metabolism*
D001779:Blood Coagulation Factors; D005169:Factor VIII; D005170:Factor X; D013917:Thrombin; D015951:Factor Xa; D002118:Calcium