Protein disulfide isomerase mediates integrin-dependent adhesion.

Cell adhesion is mediated by the integrin adhesion receptors. Receptor-ligand interaction involves conformational changes in the receptor, but the underlying mechanism remains unclear. Our earlier work implied a role for sulfhydryls in integrin response to ligand binding in the intact blood platelet. We now show that non-penetrating blockers of free sulfhydryls inhibit beta(1) and beta(3) integrin-mediated platelet adhesion regardless of the affinity state of the integrin. Removal of the inhibitors prior to adhesion fully restores adhesion despite the irreversible nature of inhibitor-thiol interaction, indicating sulfhydryl exposure in response to adhesion. We further show that blocking protein disulfide isomerase (PDI) inhibits adhesion. These data indicate that: (a) ecto-sulfhydryls are necessary for integrin-mediated platelet adhesion; (b) disulfide exchange takes place during this process; (c) surface PDI is involved in integrin-mediated adhesion.

Collagen binding induces changes in its platelet integrin receptor alpha2beta1.

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 alpha2beta1 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 alpha2beta1 by Western-blotting. Functional tests performed with the antibodies confirmed the involvement of the high-affinity proteins in platelet-collagen interactions attributed to alpha2beta1: inhibition of collagen-specific platelet adhesion and aggregation. EDTA, chaotropic agents or low pH did not elute the high affinity fraction of alpha2beta1. However, DTT followed by acetic acid did, which indicates that the steps necessary to disrupt the high-affinity collagen alpha2beta1 bond are reduction of disulfide bond(s) followed by disruption of electrostatic interactions. Our data 2 1 suggest that (i) ligand binding induces the formation of a new disulfide bond in a fraction of alpha2beta1, (ii) that this bond is an intrareceptor, and (iii) that this change increases the affinity of the receptor to its ligand.

Affinity modulation in platelet alpha 2 beta 1 following ligand binding.

In order to test for ligand-induced change in the affinity of platelet alpha 2 beta 1 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 alpha 2 beta 1. Functional tests performed with the antibodies confirmed the involvement of the high affinity proteins in platelet-collagen interactions attributed to alpha 2 beta 1: inhibition of collagen-specific platelet adhesion and aggregation. EDTA, chaotropic agents or low pH did not elute the high affinity fraction of alpha 2 beta 1. 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 alpha 2 beta 1, 2) that this bond is intrareceptor and 3) that this change increases the affinity of the receptor to its ligand.