Multiple binding sites in fibrinogen for integrin α_Mβ ₂ (Mac-1)

The leukocyte integrin α_Mβ₂ (Mac-1) is a multiligand receptor that mediates a range of adhesive reactions of leukocytes during the inflammatory response. This integrin binds the coagulation protein fibrinogea providing a key link between thrombosis and inflammation. However, the mechanism by which α_Mβ₂ binds fibrinogen remains unknown. Previous studies indicated that a model in which two fibrinogen γC domain sequences, P1 (γ190-202) and P2 (γ377-395), serve as the α_Mβ₂ binding sites cannot fully account for recognition of fibrinogen by integrin. Here, using surface plasmon resonance, we examined the interaction of the ligand binding α_MI-do-main of α_Mβ₂ with the D fragment of fibrinogen and showed that this ligand is capable of associating with several α_MI-domain molecules. To localize the alternative α_MI-domain binding sites, we screened peptide libraries covering the complete sequences of the γC and βC domains, comprising the majority of the D fragment structure, for α_MI-domain binding. In addition to the P2 and P1 peptides, the α_MI-domain bound to many other sequences in the γC and βC scans. Similar to P1 and P2, synthetic peptides derived from γC and βC were efficient inhibitors of α_Mβ₂-mediated cell adhesion and were able to directly support adhesion suggesting that they contain identical recognition information. Analyses of recognition specificity using substitutional peptide libraries demonstrated that the α_MI- domain binding depends on basic and hydrophobic residues. These findings establish a new model of α_Mβ₂ binding in which the α_MI-domain interacts with multiple sites in fibrinogen and has the potential to recognize numerous sequences. This paradigm may have implications for mechanisms of promiscuity in ligand binding exhibited by integrin α_Mβ₂.