Superoxide anion and hydroxyl radical release by collagen-induced platelet aggregation -: 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 when platelets are aggregated by common agonists. We used two probes, i.e. lucigenin and salicylic acid (SA), to measure platelet release of O-2(-) and OH degrees, respectively. Among the agonists used, such as ADP, thrombin and collagen, the release of O-2(-) and OH degrees was observed mainly when platelets were stimulated with collagen. Such release was inhibited in platelets pre-treated by aspirin suggesting that AA metabolism was the main source of O-2(-) and OH degrees formation. To further analyze this relationship, O-2(-) and OH degrees formation was measured during AA-stimulated platelet aggregation (PA); we observed that O-2(-) and OH degrees release were dependent 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 OH 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, which is dependent upon AA release and metabolism.