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 γ-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 ∼50% of the 500−600 sites/platelet with K i values of 2.9 nM (G4−Q11), 24 nM (S3−L6), and 240 nM (F9−Q11), compared with native factor IXa (K i ∼2.5 nM). The two peptides S3−L6 and F9−Q11 added together at equimolar concentration demonstrated ∼50-fold synergism (K i = 2.4 nM). Although both factor IX and the Gla peptide (G4−Q11) displaced 100% of bound factor IX and ∼50% of bound factor IXa, factor IX was ineffective (at >1000-fold molar excess) and the Gla domain peptide (G4−Q11) was relatively ineffective (K i = 165 μM) 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.