Studies have suggested a pivotal role for free sulfhydryls in platelet integrin function, and enzyme-mediated reduction of disulfide bonds on platelets has been implicated. The platelet fibrinogen receptor ␣ IIb ␤ 3 is the best-studied platelet integrin and serves as a model system for studying the structure-function relation in this family of adhesion receptors. The demonstration of free sulfhydryls on the exofacial domain of purified ␣ IIb ␤ 3 , specifically in its activated conformation, prompted us to explore the potential for activation-dependent, enzymatically catalyzed thiol expression on intact platelets and the possible role of surface-associated protein disulfide isomerase (PDI) in ␣ IIb ␤ 3 ligation. Using the membrane-impermeant sulfhydryl blocker para-chloromercuriphenyl sulfonate, the inhibitor of disulfide exchange bacitracin, and the monoclonal anti-PDI antibody RL90, we examined fibrinogen binding to ␣ IIb ␤ 3 as well as ligation-induced allosteric changes in the conformation of ␣ IIb ␤ 3 . We sought to distinguish the possible involvement of disulfide exchange in agonist-induced platelet stimulation from its role in integrin ligation. Analysis of the role of free thiols in platelet aggregation suggested a thiolindependent initial ligation followed by a thiol-dependent stabilization of binding. IntroductionThe affinity of integrins for their ligands is tightly regulated, both by cellular events and subsequent to ligand binding. [1][2][3][4][5] Such cellular control over the interaction of extracellular proteins implies the existence of a mechanism to propagate information back and forth between the extracellular head and the cytoplasmic tail of the integrin receptor. In recent years, the posited mechanism has been the allosteric conformational changes occurring in the integrin receptor, both in response to cellular events that switch the receptor from low to high affinity (inside-out signaling) and external events that relay information about occupancy of the receptor (outside-in signaling). [6][7][8][9] Although information about the nature of the changes in conformation is slowly emerging (for review, see Woodside et al 10 ) and consequent changes in intracellular interactions are widely documented, 10-13 the molecular mechanism regulating such changes, particularly on the exofacial domain, has not been elucidated. We previously reported that extracellular sulfhydryls participate in conformational changes triggered by interaction of the platelet integrin ␣ 2 ␤ 1 with its natural ligand collagen. 14 We also found that disulfide exchange is required for platelet adhesion mediated by integrins ␣ 5 ␤ 1 and ␣ IIb ␤ 3 as well as ␣ 2 ␤ 1 and that surface-expressed protein disulfide isomerase (PDI) is involved in this exchange. 15 Furthermore, involvement of disulfide exchange in receptor function is a feature specific to the integrin-receptor family (J. L. et al, submitted manuscript, 2002). We therefore proposed that conformational changes subsequent to ligand interaction with the integrin lead to ...

Integrin alpha2beta1 is the principal adhesive receptor for collagen but platelets also adhere through glycoprotein VI (GPVI). Integrin alphaIIbbeta3 may augment platelet adhesion. We have shown that disulfide exchange is necessary for platelet adhesion to fibrinogen, fibronectin, and collagen. However 2 questions remained: (1) Can activated alphaIIbbeta3 explain the observed role of disulfide exchange in adhesion to collagen, or is this role common to other integrins? (2) Is disulfide dependence specific to the integrin receptors or shared with GPVI? To discriminate adhesive functions of alpha2beta1 from those of alphaIIbbeta3 we used Glanzmann platelets and alphaIIbbeta3-specific antibodies applied to normal platelets. To resolve adhesive events mediated by alpha2beta1 from those of GPVI we used synthetic peptides specific to each receptor. We addressed direct integrin ligation using purified alpha2beta1 and recombinant I domain. We observed the following: adhesion to the alpha2beta1-specific peptide was disulfide-exchange dependent and protein disulfide isomerase (PDI) mediated; membrane-impermeant thiol blockers inhibited alpha2beta1, but not GPVI mediated, adhesion; direct blockade of PDI revealed that it is involved in adhesion through alpha2beta1 but not GPVI; and purified alpha2beta1, but not recombinant I domain, depended on free thiols for ligation. These data suggest that the enzymatically catalyzed adhesion-associated reorganization of disulfide bonds is common to members of the integrin family and specific to this family.

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 L L 1 and L L 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 integrinmediated platelet adhesion; (b) disulfide exchange takes place during this process; (c) surface PDI is involved in integrinmediated adhesion. ß