Kinetics of coagulation factor X activation by platelet-bound factor IXa.
Thrombin-activated human platelets, in the presence of factors VIIIa and X, have specific, high-affinity (Kd approximately 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 microM) was greater than 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 microM) with no significant effect on kcat (0.052 min-1) and allowed factor VIIIa to decrease the Km further to a concentration (0.16 microM) 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 x 10(6))-fold.
Rawala-Sheikh
R
R
Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Ahmad
S S
SS
Ashby
B
B
Walsh
P N
PN
D005164:Factor IX / Q000096:biosynthesis / Q000378:metabolism*; D005169:Factor VIII / Q000378:metabolism; D005170:Factor X / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010743:Phospholipids / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects; D013917:Thrombin / Q000378:metabolism*
D010743:Phospholipids; D005169:Factor VIII; D005164:Factor IX; D005170:Factor X; D013917:Thrombin