High-affinity, specific factor IX/IXa binding to platelets is mediated at least in part by amino acids (G4-Q11) exposed on the surface of the gamma-carboxyglutamic acid (Gla) domain. Rationally designed, conformationally constrained synthetic peptides were screened for their capacity to inhibit factor IXa binding to platelets. Each of these peptides (G4-Q11, S3-L6, and F9-Q11) acted alone to inhibit factor IXa binding to approximately 50% of the 500-600 sites/platelet with Ki values of 2.9 nM (G4-Q11), 24 nM (S3-L6), and 240 nM (F9-Q11), compared with native factor IXa (Ki approximately 2.5 nM). The two peptides S3-L6 and F9-Q11 added together at equimolar concentration demonstrated approximately 50-fold synergism (Ki = 2.4 nM). Although both factor IX and the Gla peptide (G4-Q11) displaced 100% of bound factor IX and approximately 50% of bound factor IXa, factor IX was ineffective (at > 1000-fold molar excess) and the Gla domain peptide (G4-Q11) was relatively ineffective (Ki = 165 microM) in inhibiting platelet receptor-mediated factor X activation by factor IXa. We conclude that the Gla domain (G4-Q11) of factor IXa contains two conformationally constrained loop structures that mediate binding of factor IX/IXa to a shared site on activated human platelets which is separate and distinct from the site used by the enzyme, factor IXa, for assembly of the factor X activating complex.

To study the structural requirements for factor IXa binding to platelets, we have carried out equilibrium binding studies with human factor IXa after replacing the second epidermal growth factor (EGF) domain by the corresponding polypeptide region of factor X. The chimeric protein, factor IX(Xegf2), and the wild-type, factor IXwt, produced in embryonic kidney cells 293 were radiolabelled with 125I and activated with factor XIa. Direct binding studies with thrombin-activated platelets showed normal stoichiometry and affinity of binding of factor IXawt in the presence of factor VIIIa (2 units/ml) and factor X (1.5 microM). However, under similar experimental conditions, factor IXa(Xegf2) was bound to a smaller number of sites (396 sites/platelet) with decreased affinity, i.e. a dissociation constant (Kd) of 1.4 nM, compared with normal factor IXa, factor IXaN (558 sites/platelet; Kd 0.67 nM), or factor IXawt (590 sites/platelet; Kd 0.61 nM). The concentrations of factor IXaN and factor IXawt required for half-maximal rates of factor-X activation were 0.63 nM and 0.7 nM, indicating a close correspondence of the Kd,app. for binding of factor IXawt to the factor-X activating complex on activated platelets to the Kd obtained in equilibrium binding studies. In contrast, kinetic parameters for factor-X activation by factor IXa(Xegf2) showed a decreased affinity (Kd 1.5 nM), in agreement with results of binding studies. These studies with factor IX(Xegf2) suggest that the EGF-2 domain may be important for specific high-affinity factor IXa binding to platelets in the presence of factor VIIIa and factor X.

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