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-Q(11)) 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-Q(11), S-3-L-6, and F-9-Q(11)) acted alone to inhibit factor IXa binding to similar to 50% of the 500-600 sites/platelet with K-i values of 2.9 nM (G(4)-Q(11)), 24 nM (S-3-L-6), and 240 nM (F-9-Q(11)), compared with native factor IXa (K-i similar to 2.5 nM). The two peptides S-3-L-6 and F-9-Q(11) added together at equimolar concentration demonstrated similar to 50-fold synergism (K-i = 2.4 nM). Although both factor IX and the Gla peptide (G(4)-Q(11)) displaced 100% of bound factor IX and similar to 50% of bound factor IXa, factor IX was ineffective (at >1000-fold molar excess) and the Gla domain peptide (G(4)-Q(11)) was relatively ineffective (K-i = 165 mu M) in inhibiting platelet receptor-mediated factor X activation by factor IXa. We conclude that the Gla domain (G(4)-Q(11)) 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.