KINETICS OF COAGULATION FACTOR-X ACTIVATION BY PLATELET-BOUND FACTOR-1XA

Thrombin-activated human platelets, in the presence of factors Villa and X, have specific, high-affinity (Kd ~ 0.5 nM), saturable binding sites for factor IXa that are involved in factor X activation [Ahmad, S. S., Rawala-Sheikh, R., & Walsh, P. N. (1989) J. Biol. Chem. 264, 3244–3251], To determine the functional consequences of factor IXa binding to platelets, a detailed kinetic analysis of the effects of platelets, phospholipids, and factor VIII on factor IXa catalyzed factor X activation was done. In the absence of platelets, phospholipids, or factor VIII, the Michaelis constant (Km = 81 µM) was >500-fold higher than the factor X concentration in human plasma. Unactivated platelets and thrombin-activated factor VIII, alone or in combination, had no effect on the kinetic parameters, whereas thrombin-activated platelets caused a major decrease in Km (0.39 µM) with no significant effect on kat (0.052 min−1) and allowed factor Villa to decrease the Km further to a concentration (0.16 µM) near that of factor X in plasma and to increase the kcat 24000-fold to 1240 min−1. Sonicated mixed phosphatidylserine/phosphatidylcholine vesicles (25/75, mol/mol) had kinetic effects similar to those of activated platelets. When factor IXa binding to thrombin-activated platelets and rates of factor X activation were measured simultaneously at saturating concentrations of factor X and factor Villa, the kcat was independent of factor IXa concentration, and the mean kcat value was 2391 min−1. The increase in catalytic efficiency (kcat/Km) in the presence of thrombin-activated platelets and factor Villa was (17.4 ? 106)-fold. © 1990, American Chemical Society. All rights reserved.