Minor thiols cysteine and cysteinylglycine regulate the competition between glutathione and protein SH groups in human platelets subjected to oxidative stress.

Abstract Changes in the concentrations of protein-mixed disulfides (XS-SP) of glutathione (GSH), cysteine (CSH), and cysteinylglycine (CGSH) were studied in human platelets treated with diamide and t -BOOH in timecourse experiments (time range, 1–30 min) in order to understand the contribution of minor thiols CSH and CGSH to the regulation of glutathione–protein mixed disulfides (GS-SP). Diamide was much more potent than t -BOOH in altering the platelet thiol composition of X S-SP (threshold dose: diamide, 0.03 mM; t -BOOH, 0.5 mM) and caused reversible X S-SP peaks whose magnitude was related to the concentration of free thiols in untreated cells. Thus maximum levels of GS-SP (8 min after 0.4 mM diamide) were about 16-fold higher than those of controls (untreated platelets, GS-SP = 0.374 nmol/10 9 platelets), whereas those of CS-SP and CGS-SP were only 4-fold increased (untreated platelets, CS-SP = 0.112 nmol/10 9 platelets; CGS-SP = 0.024 nmol/10 9 platelets). The greater effects of diamide with respect to t -BOOH were explained on the basis of the activities of fast reactive protein SH groups for diamide and glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PDH) for t -BOOH. The addition of cysteine (0.3 mM, at 4 min) after treatment of platelets with 0.4 mM diamide increased the rate of reversal of GS-SP peaks to normal values, but also caused a relevant change in CGS-SP with respect to that of platelets treated with diamide alone. An increased γ-glutamyltranspeptidase activity was found in platelets treated with diamide. Moreover, untreated platelets were found to release and hydrolyze GSH to CGSH and CSH. Ratios of thiols/disulfides ( X SH/ X SS X ) and activities of GR and G-6PDH were also related to a high reducing potential exerted by GSH but not by minor thiols. The lower mass and charge of minor thiols is a likely requisite of the regulation of GS-SP levels in platelets.