S-glutathionylation in human platelets by a thiol-disulfide exchange-independent mechanism

Protein-glutathione mixed disulfide formation was investigated in vitro by exposure of human platelets to the thiol-specific oxidant azodicarboxylic acid-bis-dimethylamide (diamide). We found that diamide causes a decrease in the reduced form of glutathione (GSH), paralleled by an increase in protein-GSH mixed disulfides (S-glutathionylated proteins), which was not accompanied by any significant increase in the basal level of glutathione disulfide (GSSG). The increase in the appearance of S-glutathionylated proteins was inversely correlated with ADP-induced platelet aggregation. Platelet cytoskeleton was analyzed by SDS-PAGE followed by Western immunoblotting with anti-GSH antibody. The main S-glutathionylated cytoskeletal protein proved to be actin, which accounts for 35% of the platelet total protein content. Our results suggest that neither GSSG formation nor a consequent thiol-disulfide exchange mechanism is involved in actin S-glutathionylation of human platelets exposed to diamide. Instead, a mechanism involving the initial oxidative activation of actin thiol groups, which then react with GSH to the protein-GSH mixed disulfides, makes it likely that platelet actin is S-glutathionylated without any significant increase in the GSSG content.