COLLAGEN-BINDING INDUCES CHANGES IN ITS PLATELET INTEGRIN RECEPTOR ALPHA(2)BETA(1)

Integrins can signal upon binding of their ligand, presumably because of conformational changes induced by ligand-binding. It has been postu lated that ligand binding causes changes in the affinity of the integr in to its ligand. In order to test for ligand-induced change in the af finity of platelet alpha(2)beta(1) to collagen, labelled viable platel ets were passaged through a column of fibrillar collagen and stringent lysis conditions were used to remove all low-affinity receptors, A hi gh-affinity fraction left on the collagen could be eluted with dithiot hreitol (DTT) and 2% Sodium dodecyl sulfate (SDS), Antibodies raised a gainst this fraction, identified alpha(2)beta(1) by Western-blotting. Functional tests performed with the antibodies confirmed the involveme nt of the high-affinity proteins in platelet-collagen interactions att ributed to alpha(2)beta(1): inhibition of collagen-specific platelet a dhesion and aggregation. EDTA, chaotropic agents or low pH did not elu te the high affinity fraction of alpha(2)beta(1). However, DTT followe d by acetic acid did, which indicates that the steps necessary to disr upt the high-affinity collagen-alpha(2)beta(1) bond are reduction of d isulfide bond(s) followed by disruption of electrostatic interactions. Our data suggest that (i) ligand binding induces the formation of a n ew disulfide bond in a fraction of alpha(2)beta(1), (ii) that this bon d is an intrareceptor, and (iii) that this change increases the affini ty of the receptor to its ligand.