Collagen binding induces changes in its platelet integrin receptor α₂β₁

Integrins can signal upon binding of their ligand, presumably because of conformational changes induced by ligand-binding. It has been postulated that ligand binding causes changes in the affinity of the integrin to its ligand. In order to test for ligand-induced change in the affinity of platelet α₂β₁ to collagen, labelled viable platelets were passaged through a column of fibrillar collagen and stringent lysis conditions were used to remove all low-affinity receptors. A high-affinity fraction left on the collagen could be eluted with dithiothreitol (DTT) and 2% Sodium dodecyl sulfate (SDS). Antibodies raised against this fraction, identified α₂β₁ by Western-blotting. Functional tests performed with the antibodies confirmed the involvement of the high-affinity proteins in platelet-collagen interactions attributed to α₂β₁: inhibition of collagen-specific platelet adhesion and aggregation. EDTA, chaotropic agents or low pH did not elute the high affinity fraction of α₂β₁. However, DTT followed by acetic acid did, which indicates that the steps necessary to disrupt the high-affinity collagen-α₂β₁ bond are reduction of disulfide bond(s) followed by disruption of electrostatic interactions. Our data suggest that (i) ligand binding induces the formation of a new disulfide bond in a fraction of α₂β₁, (ii) that this bond is an intrareceptor, and (iii) that this change increases the affinity of the receptor to its ligand.