Coagulation factor IX residues G4-Q11 mediate its interaction with a shared factor IX/IXa binding site on activated platelets but not the assembly of the functional factor X activating complex.

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.