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

Changes in the concentrations of protein-mixed disulfides (XS-SP) of glutat hione (GSH), cysteine (CSH), and cysteinylglycine (CGSH) were studied in hu man platelets treated with diamide and t-BOOH in timecourse experiments (ti me 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 thi ol composition of XS-SP (threshold dose: diamide, 0.03 mM; t-BOOH, 0.5 mM) and caused reversible XS-SP peaks whose magnitude was related to the concen tration 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 great er 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 glutathi one reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PDH) for t-BO OH, The addition of cysteine (0.3 mM, at 4 min) after treatment of platelet s with 0.4 mM diamide increased the rate of reversal of GS-SP peaks to norm al values, but also caused a relevant change in CGS-SP with respect to that of platelets treated with diamide alone. An increased gamma-glutamyltransp eptidase activity was found in platelets treated with diamide, Moreover, un treated platelets were found to release and hydrolyze GSH to CGSH and CSH, Ratios of thiols/disulfides (XSH/XSSX) 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. (C) 2000 Academic Press.