Factor XI Interacts with the Leucine-rich Repeats of Glycoprotein Ibα on the Activated Platelet*

Factor XI (FXI) binds specifically and reversibly to high affinity sites on the surface of stimulated platelets (K_{d app} of ∼10 nm; B_max of ∼1,500 sites/platelet) utilizing residues exposed on the Apple 3 domain in the presence of high molecular weight kininogen and Zn²⁺ or prothrombin and Ca²⁺. Because the FXI receptor in the platelet membrane is contained within the glycoprotein Ibα subunit of the glycoprotein Ib-IX-V complex (Baglia, F. A., Badellino, K. O., Li, C. Q., Lopez, J. A., and Walsh, P. N. (2002) J. Biol. Chem. 277, 1662–1668), we utilized mocarhagin, a cobra venom metalloproteinase, to generate a fragment (His¹–Glu²⁸²) of glycoprotein Ibα that contains the leucine-rich repeats of the NH₂-terminal globular domain and excludes the macroglycopeptide portion of glycocalicin, the soluble extracytoplasmic portion of glycoprotein Ibα. This fragment was able to compete with FXI for binding to activated platelets (K_i of 3.125 ± 0.25 nm) with a potency similar to that of intact glycocalicin (K_i of 3.72 ± 0.30 nm). However, a synthetic glycoprotein Ibα peptide, Asp²⁶⁹–Asp²⁸⁷, containing a thrombin binding site had no effect on the binding of FXI to activated platelets. Moreover, the binding of ¹²⁵I-labeled thrombin to glycocalicin was unaffected by the presence of FXI at concentrations up to 10^-5m. The von Willebrand factor A1 domain, which binds the leucine-rich repeats, inhibited the binding of FXI to activated platelets. Thus, we examined the effect of synthetic peptides of each of the seven leucine-rich repeats on the binding of ¹²⁵I-FXI to activated platelets. All leucine-rich repeat (LRR) peptides derived from glycoprotein Ibα were able to inhibit FXI binding to activated platelets in the following order of decreasing potency: LRR7, LRR1, LRR4, LRR5, LRR6, LRR3, and LRR2. However, the leucine-rich repeat synthetic peptides derived from glycoprotein Ibβ and Toll protein had no effect. We conclude that FXI binds to glycoprotein Ibα at sites comprising the leucine-rich repeat sequences within the NH₂-terminal globular domain that are separate and distinct from the thrombin-binding site.