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 phos pholipase A(2) activator, on cultured EC ROS generation has been investigat ed. EC were incubated with 0.1-1 unit/ml thrombin and cellular superoxide a nion (O-2(-)) release and hydrogen peroxide (H2O2) production measured. Thr ombin exposure caused an elevation in EC O-2(-) release and H2O2 production . The effects of protein kinase C, arachidonic acid metabolism, NADPH oxida se, and phospholipase Az inhibitors on thrombin-induced EC H2O2 production were examined. EC were exposed to 0.5 unit/ml thrombin and cellular H2O2 pr oduction measured in the presence and absence of the protein kinase C inhib itor, H-7; arachidonic acid metabolism inhibitors, indomethacin, nordihydro guaiaretic acid, and SKF525A; NADPH oxidase inhibitor, apocynin; and phosph olipase A(2) inhibitor, 4-bromophenacyl bromide. All inhibitors, with the e xception of H-7 and indomethacin, suppressed thrombin-induced EC H2O2 produ ction. The pattern of effects of these metabolic antagonists on thrombin-in duced EC ROS production is similar to that previously reported on ROS produ ction in EC exposed to high low-density lipoprotein levels, and in stimulat ed leukocytes. These findings further implicate NADPH oxidase as a major RO S source in EC.