Kinetics of coagulation factor-X activation by platelet-bound factor IXa

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Abstract
Thrombin-activated human platelets, in the presence of factors VIIIa and X, have specific, high-affinity (Kd .apprx. 0.5 nM), saturable binding sites for factor IXa that are involved in factor X activation [Ahmad, S. S., Rawala-Sheikh, R., and 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 .mu.M) 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 .mu.M) with no significant effect on kcat (0.052 min-1) and allowed factor VIIIa to decrease the Km further to a concentration (0.16 .mu.M) near that of factor X in plasma and to increase the kcat 24,000-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 VIIIa, 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 VIIIa was (17.4 .times. 106)-fold.