Cytoskeletal alterations in human platelets exposed to oxidative stress are mediated by oxidative and Ca²⁺-dependent mechanisms

The metabolism of the redox-active quinone, menadione (2-methyl-1,4-naphthoquinone), in human platelets was associated with superoxide anion production, oxidation and depletion of intracellular glutathione, and modification of protein thiols. The cytoskeletal fraction extracted from menadione-treated platelets exhibited a dose-dependent increase in the amount of cytoskeleton-associated protein and a concomitant loss of protein thiols. These alterations were associated with oxidative modifications of actin, including β-mercaptoethanol-sensitive crosslinking of actin to form dimers, trimers, and high-molecular-weight aggregates which also contained other cytoskeletal proteins, i.e., α-actinin and actin-binding protein. In addition, analysis of the cytoskeletal fraction from platelets treated with high concentrations (≥100 μm) of menadione by polyacrylamide gel electrophoresis under reducing conditions revealed a net decrease in the relative abundance of the individual cytoskeletal polypeptides. Under the same incubation conditions the platelets exhibited a sustained increase in cytosolic Ca²⁺ concentration. The presence of glucose, or the omission of Ca²⁺ from the incubation medium, prevented both the increase in cytosolic Ca²⁺ and the decrease in the relative amounts of cytoskeletal proteins. The latter effect was also largely prevented in platelets loaded with Quin-2 tetraacetoxymethyl ester to buffer the menadione-induced elevation of cytosolic Ca²⁺. Finally, the presence of a protease inhibitor, leupeptin, in the incubation medium prevented the menadione-induced decrease in the amount of actin-binding protein but not the decrease in the other cytoskeletal proteins. Our findings demonstrate that the multiple effects of oxidative stress on the platelet cytoskeleton are mediated by oxidative as well as by Ca²⁺-dependent mechanisms.