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.
Dalle-Donne
Isabella
I
Department of Biology, University of Milan, via Celoria 26, I-20133 Milan, Italy. quack@unimi.it
Giustarini
Daniela
D
Colombo
Roberto
R
Milzani
Aldo
A
Rossi
Ranieri
R
D000199:Actins / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003599:Cytoskeleton / Q000378:metabolism; D003958:Diamide / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005609:Free Radicals / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry*; D019803:Glutathione Disulfide / Q000737:chemistry; D006801:Humans; D007118:Immunoassay; D015151:Immunoblotting; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013997:Time Factors
D000199:Actins; D004220:Disulfides; D005609:Free Radicals; D013438:Sulfhydryl Compounds; D003958:Diamide; D005978:Glutathione; D010100:Oxygen; D019803:Glutathione Disulfide