The broad recognition specificity exhibited by integrin αMβ2 (Mac-1, CD11b/CD18) has allowed this adhesion receptor to play innumerable roles in leukocyte biology, yet we know little about how and why αMβ2 binds its multiple ligands. Within αMβ2, the αMI-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 αMI-domain binding and also selected the αMI-domain recognition sequences by phage display. Analyses of >1400 binding and nonbinding peptides derived from peptide libraries showed that a key feature of the αMI-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 αMI-domain binding sites in the αMβ2 ligands. The recognition specificity of the αMI-domain resembles that of some chaperones, which allows it to bind segments exposed in unfolded proteins. The disclosure of the αMβ2 binding preferences allowed the prediction that cationic host defense peptides, which are strikingly enriched in the αMI-domain recognition motifs, represent a new class of αMβ2 ligands. This prediction has been tested by examining the interaction of αMβ2 with the human cathelicidin peptide LL-37. LL-37 induced a potent αMβ2-dependent cell migratory response and caused activation of αMβ2 on neutrophils. The newly revealed recognition specificity of αMβ2 toward unfolded protein segments and cationic proteins and peptides suggests that αMβ2 may serve as a previously proposed “alarmin” receptor with important roles in innate host defense.

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 αIIbβ3-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.

Recent evidence suggests that substrate rigidity controls cell adhesion through integrin‐mediated signaling. We have found that binding of soluble fibrinogen to the surface of insoluble fibrin matrix inhibits integrin‐mediated cell adhesion by creating an anti‐adhesive fibrinogen layer. To gain understanding of the mechanism underlying the differential ability of cells to adhere to the naked fibrin gel (rigid substrate) and to the loose fibrinogen layer formed on the surface of fibrin (soft substrate), we examined integrin αIIbβ3‐mediated signaling in platelets. Platelet adhesion to fibrin clots induced strong phosphorylation of FAK and Syk kinases as compared to adhesion on soft substrates. Also, the rigid, but not soft substrate induced recruitment of signaling molecules talin and skelemin to integrin‐containing focal adhesions. Consistent with their limited ability to induce sufficient signaling, soft substrates do not support platelet spreading. Differential platelet signaling was also observed when fibrinogen was adsorbed on plastic at low and high densities, a condition which mimics rigid and soft substrates, respectively. These data suggest that soluble fibrinogen prevents stable platelet adhesion to the fibrin clot by modifying its mechanical properties which results in insufficient signaling and reduced force generation.NIH HL 63199

Recent evidence suggests that substrate rigidity controls cell adhesion through integrin‐mediated signaling. We have found that binding of soluble fibrinogen to the surface of insoluble fibrin matrix inhibits integrin‐mediated cell adhesion by creating an anti‐adhesive fibrinogen layer. To gain understanding of the mechanism underlying the differential ability of cells to adhere to the naked fibrin gel (rigid substrate) and to the loose fibrinogen layer formed on the surface of fibrin (soft substrate), we examined integrin αIIbβ3‐mediated signaling in platelets. Platelet adhesion to fibrin clots induced strong phosphorylation of FAK and Syk kinases as compared to adhesion on soft substrates. Also, the rigid, but not soft substrate induced recruitment of signaling molecules talin and skelemin to integrin‐containing focal adhesions. Consistent with their limited ability to induce sufficient signaling, soft substrates do not support platelet spreading. Differential platelet signaling was also observed when fibrinogen was adsorbed on plastic at low and high densities, a condition which mimics rigid and soft substrates, respectively. These data suggest that soluble fibrinogen prevents stable platelet adhesion to the fibrin clot by modifying its mechanical properties which results in insufficient signaling and reduced force generation. NIH HL 63199