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

The firm adhesion and transplatelet migration of leukocytes on vascular thrombus are dependent on the interaction of the leukocyte integrin Mac-1 ( M 2 , CD11b/CD18) and the platelet counter receptor glycoprotein (GP) Ib . Previous studies have established a central role for the I domain, a stretch of 200 amino acids within the M subunit, in the binding of GP Ib . This study was undertaken to establish the molecular basis of GP Ib recognition by M 2 . The P 201 –K 217 sequence, which spans an exposed loop and amphipathic 4 helix in the threedimensional structure of the M I domain, was identified as the binding site for GP Ib . Mutant cell lines in which the M I domain segments P 201 –G 207 and R 208 –K 217 were switched to the homologous, but non-GP Ib binding, L domain segments failed to support adhesion to GP Ib . 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 M I domains to GP Ib . Synthetic peptides duplicating the P 201 –K 217 , but not scrambled versions, directly bound GP Ib and inhibited M 2 -dependent adhesion to GP Ib and adherent platelets. Finally, grafting critical amino acids within the P 201 –K 217 sequence onto L , converted L 2 into a GP Ib binding integrin. Thus, the P 201 –K 217 sequence within the M I domain is necessary and sufficient for GP Ib binding. These observations provide a molecular target for disrupting leukocyte–platelet complexes that promote vascular inflammation in thrombosis, atherosclerosis, and angioplastyrelated restenosis.