Superoxide anion and hydroxyl radical release by collagen-induced plateletaggregation - Role of arachidonic acid metabolism

Previous study demonstrated that platelets undergoing anoxia-reoxygenation generate superoxide anion (O-2(-)) and hydroxyl radical (OH degrees) which in turn contribute to activate arachidonic acid (AA) metabolism. However it has not been clarified if oxygen free radicals (OFRs) are also generated w hen platelets are aggregated by common agonists. We used two probes, i.e. l ucigenin and salicylic acid (SA), to measure platelet release of O-2(-) and OH degrees, respectively. Among the agonists used, such as ADP, thrombin a nd collagen, the release of O-2(-) and OH degrees was observed mainly when platelets were stimulated with collagen. Such release was inhibited in plat elets pre-treated by aspirin suggesting that AA metabolism was the main sou rce of O-2(-) and OH degrees formation. To further analyze this relationshi p, O-2(-) and OH degrees formation was measured during AA-stimulated platel et aggregation (PA); we observed that O-2(-) and OH degrees release were de pendent upon AA concentration. Furthermore, we found that the incubation of platelets with AACOCF(3), a potent inhibitor of cytosolic phospholipase A( 2), inhibited collagen-induced platelet O-2(-) and OH degrees release. The incubation of platelets with salicylic acid or ascorbic acid, which blunt O H degrees and O-2(-) respectively, inhibited both collagen-induced platelet aggregation and AA-release. This study demonstrated that collagen-induced platelet aggregation is associated with O-2(-) and OH degrees formation, wh ich is dependent upon AA release and metabolism.