Comparative platelet binding and kinetic studies with normal and variant factor IXa molecules.

Abstract

We have recently shown that thrombin-stimulated human platelets have specific, saturable receptors for factor IXa, occupancy of which promotes factor X activation (Ahmad, S. S., Rawala-Sheikh, R., and Walsh, P. N. (1989) J. Biol. Chem. 264: 3244-3251, 20012-20016; Rawala-Sheikh, R., Ahmad, S. S., and Walsh, P. N. (1990) Biochemistry 29, 2606-2611). To study the structural requirements for factor IXa binding to platelets, equilibrium binding studies and kinetic studies of factor X activation were carried out with normal factor IXa and with two variant proteins: factor IXaAlabama (FIXaAL; Asp47----Gly substitution) and factor IXaChapel Hill (FIXaCH; Arg145----His substitution). In the absence of factors VIIIa and X, there were 331 binding sites/platelet for FIXaCH (Kdapp = 2.8 nM), and 540 sites/platelet for FIXaAL (Kdapp = 3.2 nM), compared with 540 sites/platelet (Kdapp = 2.3 nM) for normal factor IXa. The addition of factors VIIIa and X, both at saturating concentrations, had no effect on the number of binding sites for either normal or variant factor IXa, resulted in a decrease in the Kd for normal factor IXa to 0.67 nM, resulted in a suboptimal decrease in Kd for FIXaAL (1.4 nM), and had no effect on the Kd for FIXaCH. Kinetic studies of factor X activation at variable factor IXa concentration confirmed these values of Kd in the presence of factors VIIIa and X. Determination of rates of factor X activation at variable substrate concentrations yielded normal values of catalytic efficiency (kcat/Km) for the variant proteins, thereby indicating that the abnormally low rates of factor X activation obtained were a consequence of the low affinity binding of FIXaAL and FIXaCH to thrombin-activated platelets in the presence of factors VIIIa and X. These studies suggest that the presence of Asp47 and the cleavage of factor IX at Arg145-Ala146 are important structural features required for specific, high affinity factor IXa binding to platelets in the presence of factors VIIIa and X.