Involvement of NADH/NADPH oxidase in human platelet ROS production

Platelets play an important role in atherosclerotic and thromboembolic vasc ular diseases. It has been reported that reactive oxygen species (ROS) coul d modify platelet function, and platelets themselves have the ability to pr oduce ROS. However, the enzymatic sources of ROS in platelets have not been fully determined. The NADH/NADPH oxidase system was originally identified as the major source of ROS in phagocytes. Recently, it has become evident t hat this oxidase is functionally expressed not only in phagocytes but also in various cell types. The present study was undertaken to test the hypothe sis that NADH/NADPH oxidase might be expressed in human platelets. Lucigeni n-enhanced chemiluminescence (L-CL) and electron spin resonance (ESR) metho d demonstrated that human platelets obtained from healthy volunteers releas ed ROS, and the released ROS were increased by stimulation with 12-O-tetrad ecanoylphorbol-13-acetate (TPA) or calcium ionophore. Homogenates of human platelets, as well as MEG01: cells, megakaryocytic cell line, had the enzym atic activity to produce superoxide in NADH/NADPH-dependent manners. This e nzymatic activity was suppressed by diphenylene iodonium (DPI), an inhibito r of NADH/NADPH oxidase. Western blot analysis demonstrated that platelets and MEG01 cells expressed p22(phox) and p67(phox) proteins, components of N ADH/NADPH oxidase. Thus, human platelets have the enzymatic activity of p22 (phox)-based NADH/NADPH oxidase, and this oxidase is likely one of the imp ortant sources of ROS in platelets. (C) 2001 Elsevier Science Ltd. All righ ts reserved.