Factor XI binding to activated platelets is mediated by residues R-250, K-255, F-260 and Q(263) within the apple 3 domain

To localize the platelet binding site on factor XII rationally designed, co nformationally constrained synthetic peptides were used to compete with [I- 125]factor XI binding to activated platelets. The major platelet binding en ergy resided within the sequence of amino acids T-249-F-260. Homology scann ing, using prekallikrein amino acid substitutions within the synthetic pept ide T-249-F-260 identified a major role for R-250 in platelet binding. Inhi bition of [I-125] factor XI binding to activated platelets by the recombina nt Apple 3 domain of factor XI and inhibition by unlabeled factor XI were i dentical, whereas the recombinant Apple 3 domain of prekallikrein had littl e effect, A "gain-of-function'' chimera in which the C-terminal amino acid sequence of the Apple 3 domain of prekallikrein was replaced with that of f actor XI was as effective as the recombinant Apple 3 domain of factor XI an d unlabeled Factor XI in inhibiting [I-125]factor XI binding to activated p latelets. Alanine scanning mutagenic analysis of the recombinant Apple 3 do main of factor XI indicated that amino acids R-250, K-255, F-260, and Q(263 ) (but not K-252 or K-253) are important for platelet binding, Thus, the bi nding energy mediating the interaction of factor XI with platelets is conta ined within the C-terminal amino acid sequence of the Apple 3 domain (T-249 -V-271) and is mediated in part by amino acid residues R-250, K-255, F-260, and Q(263).