Protein Disulfide Isomerase and Sulfhydryl-Dependent Pathways in Platelet Activation

The inhibition of blood platelet aggregation and secretion was studied using covalent thiol reagents, maleimides, or mercuribenzoates, or using inhibitors of protein disulfide isomerase (PDI), bacitracin or antibodies to PDI. As expected, both types of inhibitors were effective against stimulation by normal physiologic stimuli. On the other hand, when stimulation was initiated with the peptide LSARLAF, that specifically activates the integrin αIIbβ3 (the fibrinogen receptor), the PDI inhibitors were without effect. LSARLAF-induced aggregation was, however, inhibited by the sulfhydryl reagents. To further investigate the role of sulfhydryl-containing proteins and αIIbβ3, platelets were labeled with membrane-impermeant sulfhydryl reagents. Nine bands were found labeled on gel electrophoresis. Two of the labeled bands were identified as αIIb and β3. The conclusions are that while PDI is required for platelet aggregation and secretion, an additional sulfhydryl-dependent step or protein is also required. This latter reaction occurs at the level of αIIbβ3. In distinction to most literature reports, at least a subpopulation of αIIbβ3 contains free sulfhydryl groups, consistent with the possibility that it is a substrate for PDI or part of the sulfhydryl-dependent response.