A B S T R A C T Human platelets contain the cuprozinc (cytoplasmic) and manganese (mitochondrial) forms of superoxide dismutase. Nevertheless, superoxide radicals were detectable in the surrounding medium of metabolically viable platelet suspensions by using two assay systems: cytochrome c and nitroblue tetrazolium. The quantity of superoxide generated by platelets (5 x 105 superoxide radicals/platelet per 10 min) was constant and did not increase after aggregation by agents such as collagen and thrombin. The superoxide-generating system was present in the supernate of both aggregated and resting platelets and therefore was not platelet-bound. Platelet superoxide production was unaffected by prior ingestion of aspirin, indicating that the prostaglandin and thromboxane pathways were not involved. Both resting and aggregated platelets exhibited a reductive capacity toward cytochrome c and nitroblue tetrazolium which was unrelated to superoxide production. Furthermore, the aggregation process always resulted in a marked increase in this reduction. The nonsuperoxide reduction associated with aggregation was found to be membrane bound and to decrease with an apparent first order reaction rate (k1 = 0.067 min-'). In addition, accumulative, time-dependent nonsuperoxide-related cytochrome c reduction was also detected. Since there is no superoxide dismutase in plasma, the presence of superoxide radicals in the surrounding medium of platelets may have in vitro significance for platelet and leukocyte concentration and storage and in vivo significance for hemostasis, coagulation, and thrombosis. The nonsuperoxiderelated reducing activities may represent a biochemical basis for platelet-blood vessel interactions, with particular reference to blood vessel integrity.

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Superoxide radicals are measurable in platelet suspensions by two different assay systems (SOD-inhibitable cytochrome c and nitroblue tetrazolium reduction). The -producing mechanism is detectable in platelet supernatants and the rate of generation is proportional to platelet concentration. Prior ingestion of aspirin does not inhibit production by platelets. Following stimulation by collagen and thrombin platelets develop a marked increase in their capacity for cytochrome c and NBT reduction, but in contrast to leukocytes stimulation does not result in an increase in superoxide production. Sodium fluoride, a powerful stimulus for production in leukocytes, and also a platelet aggregating agent, is under study. A concentration of 20 mM sodium fluoride increases platelet superoxide production approximately twofold over baseline values. This level of fluoride induces 88% 5-HT release in the absence of LDH leakage. The increase in superoxide generation does not correlate directly with 5-HT release, and the fluoride effect on may not be related to its influence on secretion. Electrophoretic studies indicated that fluoride had no effect on platelet superoxide dismutase. Experiments with ionophore A23187 indicated that the action of this agent was not comparable to fluoride but was similar to collagen and thrombin in that production did not exceed baseline levels. In some instances the ionophore appeared to inhibit production. Superoxide radicals may play an important role in platelet-platelet or platelet-blood vessel interactions.

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