Affinity modulation in platelet α2β1 following ligand binding

In order to test for ligand-induced change in the affinity of platelet α2β1 to collagen we passaged labeled viable platelets through a column of fibrillar collagen and used stringent lysis conditions to remove all low-affinity receptors. A high affinity fraction left on the collagen could be eluted with DTT and 2% SDS. Antibodies raised against it Western-blotted α2β1. 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. Our data suggest that 1) ligand binding induces the formation of a new disulfide bond in a fraction of α2β1, 2) that this bond is intrareceptor and 3) that this change increases the affinity of the receptor to its ligand.