Thrombin stimulated reactive oxygen species production in cultured human endothelial cells

In order to study the major cellular source of reactive oxygen species (ROS) in perturbed human endothelial cells (EC), the effect of thrombin, a phospholipase A(2) activator, on cultured EC ROS generation has been investigated. EC were incubated with 0.1-1 unit/ml thrombin and cellular superoxide anion (O-2(-)) release and hydrogen peroxide (H2O2) production measured. Thrombin exposure caused an elevation in EC O-2(-) release and H2O2 production. The effects of protein kinase C, arachidonic acid metabolism, NADPH oxidase, and phospholipase Az inhibitors on thrombin-induced EC H2O2 production were examined. EC were exposed to 0.5 unit/ml thrombin and cellular H2O2 production measured in the presence and absence of the protein kinase C inhibitor, H-7; arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A; NADPH oxidase inhibitor, apocynin; and phospholipase A(2) inhibitor, 4-bromophenacyl bromide. All inhibitors, with the exception of H-7 and indomethacin, suppressed thrombin-induced EC H2O2 production. The pattern of effects of these metabolic antagonists on thrombin-induced EC ROS production is similar to that previously reported on ROS production in EC exposed to high low-density lipoprotein levels, and in stimulated leukocytes. These findings further implicate NADPH oxidase as a major ROS source in EC.