Targeting Platelet–Leukocyte Interactions: Identification of the Integrin Mac-1 Binding Site for the Platelet Counter Receptor Glycoprotein Ibα

The firm adhesion and transplatelet migration of leukocytes on vascular thrombus are dependent on the interaction of the leukocyte integrin Mac-1 (α(M)β(2), CD11b/CD18) and the platelet counter receptor glycoprotein (GP) Ibα. Previous studies have established a central role for the I domain, a stretch of ∼200 amino acids within the α(M) subunit, in the binding of GP Ibα. This study was undertaken to establish the molecular basis of GP Ibα recognition by α(M)β(2). The P(201)–K(217) sequence, which spans an exposed loop and amphipathic α4 helix in the three-dimensional structure of the α(M)I domain, was identified as the binding site for GP Ibα. Mutant cell lines in which the α(M)I domain segments P(201)–G(207) and R(208)–K(217) were switched to the homologous, but non-GP Ibα binding, α(L) domain segments failed to support adhesion to GP Ibα. Mutation of amino acid residues within P(201)–K(217), H(210)–A(212), T(213)–I(215), and R(216)–K(217) resulted in the loss of the binding function of the recombinant α(M)I domains to GP Ibα. Synthetic peptides duplicating the P(201)–K(217), but not scrambled versions, directly bound GP Ibα and inhibited α(M)β(2)-dependent adhesion to GP Ibα and adherent platelets. Finally, grafting critical amino acids within the P(201)–K(217) sequence onto α(L), converted α(L)β(2) into a GP Ibα binding integrin. Thus, the P(201)–K(217) sequence within the α(M)I domain is necessary and sufficient for GP Ibα binding. These observations provide a molecular target for disrupting leukocyte–platelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplasty-related restenosis.