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.