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_Mβ₂, 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_Mβ₂. The P²⁰¹–K²¹⁷ sequence, which spans an exposed loop and amphipathic α4 helix in the three-dimensional structure of the α_MI domain, was identified as the binding site for GP Ibα. Mutant cell lines in which the α_MI domain segments P²⁰¹–G²⁰⁷ and R²⁰⁸–K²¹⁷ 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 P²⁰¹–K²¹⁷, H²¹⁰–A²¹², T²¹³–I²¹⁵, and R²¹⁶–K²¹⁷ resulted in the loss of the binding function of the recombinant α_MI domains to GP Ibα. Synthetic peptides duplicating the P²⁰¹–K²¹⁷, but not scrambled versions, directly bound GP Ibα and inhibited α_Mβ₂-dependent adhesion to GP Ibα and adherent platelets. Finally, grafting critical amino acids within the P²⁰¹–K²¹⁷ sequence onto α_L, converted α_Lβ₂ into a GP Ibα binding integrin. Thus, the P²⁰¹–K²¹⁷ sequence within the α_MI 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.