Regulated Unmasking of the Cryptic Binding Site for Integrin α_Mβ₂ in the γC-Domain of Fibrinogen

Abstract

Fibrinogen is a ligand for leukocyte integrin alpha(M)beta2 (CD11b/CD18, Mac-1) and mediates adhesion and migration of leukocytes during the immune-inflammatory responses. The binding site for alpha(M)beta2 resides in gammaC, a constituent subdomain in the D-domain of fibrinogen. The sequence gamma383-395 (P2-C) in gammaC was implicated as the major binding site for alpha(M)beta2. It is unknown why alpha(M)beta2 on leukocytes can bind to immobilized fibrinogen in the presence of high concentrations of soluble fibrinogen in plasma. In this study, we have investigated the accessibility of the binding site in fibrinogen for alpha(M)beta2. We found that the alpha(M)beta2-binding site in gammaC is cryptic and identified the mechanism that regulates its unmasking. Proteolytic removal of the small COOH-terminal segment(s) of gammaC, gamma397/405-411, converted the D100 fragment of fibrinogen, which contains intact gammaC and is not able to inhibit adhesion of the alpha(M)beta2-expressing cells, into the fragment D98, which effectively inhibited cell adhesion. D98, but not D100, bound to the recombinant alpha(M)I-domain, and the alpha(M)I-domain recognition peptide, alpha(M)(Glu253-Arg261). Exposure of the P2-C sequence in fibrinogen, D100, and D98 was probed with a site-specific mAb. P2-C is not accessible in soluble fibrinogen and D100 but becomes exposed in D98. P2-C is also unmasked by immobilization of fibrinogen onto a plastic and by deposition of fibrinogen in the extracellular matrix. Thus, exposure of P2-C by immobilization and by proteolysis correlates with unmasking of the alpha(M)beta2-binding site in the D-domain. These results demonstrate that conformational alterations regulate the alpha(M)beta2-binding site in gammaC and suggest that processes relevant to tissue injury and inflammation are likely to be involved in the activation of the alpha(M)beta2-binding site in fibrinogen.

Exposure of acidic residues as a danger signal for recognition of fibrinogen and other macromolecules by integrin αXβ2

“…These observations agree with mapping of a linear binding epitope for the ␣ X and ␣ M I domain in the C-terminal part of the Fg ␥-chain (27). In transgenic mice, mutation of this segment ablated ␣ M ␤ 2 -mediated binding by primary human neutrophils to murine Fg (28), and unmasking of this site enhanced binding by the ␣ M ␤ 2 integrin to human Fg (29). The specificity of these results for ␣ M and ␣ X is emphasized by comparison to the ␣ L I domain, where unmasking is not required for binding to ICAM-1, and guanidine treatment indeed weakened binding (Fig.…”

The Assembly of Nonadhesive Fibrinogen Matrices Depends on the αC Regions of the Fibrinogen Molecule

“…The nature of these interactions is unknown. Because adsorption of fibrinogen induces not only unfolding of the ␣C regions but also elicits unmasking of other sequences (38,39), it may potentially result in exposure of selfassociation sites in other parts of the molecule. Alternatively, ␥C-␥C and ␥C-␤C bonds similar to those that have been proposed to exist in the fibrin fibers (21) may occur in the multilayer fibrinogen.…”

Multiple Binding Sites in Fibrinogen for Integrin αMβ2 (Mac-1)

“…Immobilization of Fg and the D fragment onto the surfaces was also shown to induce exposure of cryptic binding sites, including those for integrins (25). For example, the C-terminal part of P2, ␥390 -395, is hidden in soluble intact Fg and becomes unmasked as a result of unfolding of protein upon adsorption to microtiter plates (26). Because the coupling of proteins to the dextran matrix of the sensor chip by chemical cross-linking differs from its immobilization onto the plastic in that it preserves a more native-like conformation, it could prevent binding of additional ␣ M I-domains.…”