Ligand recognition specificity of leukocyte integrin αMß2 (Mac-1, CD11b/CD18) and its functional consequences.

The broad recognition specificity exhibited by integrin α(M)ß2 (Mac-1, CD11b/CD18) has allowed this adhesion receptor to play innumerable roles in leukocyte biology, yet we know little about how and why α(M)ß2 binds its multiple ligands. Within α(M)ß2, the α(M)I-domain is responsible for integrin's multiligand binding properties. To identify its recognition motif, we screened peptide libraries spanning sequences of many known protein ligands for α(M)I-domain binding and also selected the α(M)I-domain recognition sequences by phage display. Analyses of >1400 binding and nonbinding peptides derived from peptide libraries showed that a key feature of the α(M)I-domain recognition motif is a small core consisting of basic amino acids flanked by hydrophobic residues. Furthermore, the peptides selected by phage display conformed to a similar pattern. Identification of the recognition motif allowed the construction of an algorithm that reliably predicts the α(M)I-domain binding sites in the α(M)ß2 ligands. The recognition specificity of the α(M)I-domain resembles that of some chaperones, which allows it to bind segments exposed in unfolded proteins. The disclosure of the α(M)ß2 binding preferences allowed the prediction that cationic host defense peptides, which are strikingly enriched in the α(M)I-domain recognition motifs, represent a new class of α(M)ß2 ligands. This prediction has been tested by examining the interaction of α(M)ß2 with the human cathelicidin peptide LL-37. LL-37 induced a potent α(M)ß2-dependent cell migratory response and caused activation of α(M)ß2 on neutrophils. The newly revealed recognition specificity of α(M)ß2 toward unfolded protein segments and cationic proteins and peptides suggests that α(M)ß2 may serve as a previously proposed "alarmin" receptor with important roles in innate host defense.

Control of integrin alphaIIb beta3 outside-in signaling and platelet adhesion by sensing the physical properties of fibrin(ogen) substrates.

The physical properties of substrates are known to control cell adhesion via integrin-mediated signaling. Fibrin and fibrinogen, the principal components of hemostatic and pathological thrombi, may represent biologically relevant substrates whose variable physical properties control adhesion of leukocytes and platelets. In our previous work, we have shown that binding of fibrinogen to the surface of fibrin clot prevents cell adhesion by creating an antiadhesive fibrinogen layer. Furthermore, fibrinogen immobilized on various surfaces at high density supports weak cell adhesion whereas at low density it is highly adhesive. To explore the mechanism underlying differential cell adhesion, we examined the structural and physical properties of surfaces prepared by deposition of various concentrations of fibrinogen using atomic force microscopy and force spectroscopy. Fibrinogen deposition at high density resulted in an aggregated multilayered material characterized by low adhesion forces. In contrast, immobilization of fibrinogen at low density produced a single layer in which molecules were directly attached to the solid surface, resulting in higher adhesion forces. Consistent with their distinct physical properties, low- but not high-density fibrinogen induced strong alpha(IIb)beta(3)-mediated outside-in signaling in platelets, resulting in their spreading. Moreover, while intact fibrin gels induced strong signaling in platelets, deposition of fibrinogen on the surface of fibrin resulted in diminished cell signaling. The data suggest that deposition of a multilayered fibrinogen matrix prevents stable cell adhesion by modifying the physical properties of surfaces, which results in reduced force generation and insufficient signaling. The mechanism whereby circulating fibrinogen alters adhesive properties of fibrin clots may have important implications for control of thrombus formation and thrombogenicity of biomaterials.