MODULATION OF PLATELET-FUNCTION BY REACTIVE OXYGEN METABOLITES

Reactive oxygen metabolites have been reported to affect platelet aggr egation. However, this phenomenon is still poorly understood. In the p resent study we investigated the effects of superoxide radical and hyd rogen peroxide (H2O2) on platelet function in vitro and correlated tho se effects to possible changes of platelet concentrations of cyclic nu cleotides and thromboxane, since these systems play a key role in the response of platelets to activating stimuli. Human platelets were expo sed to xanthine-xanthine oxidase (X-XO), a system that generates both superoxide radicals and H2O2. Sixty seconds of incubation with X-XO im paired aggregation in response to ADP (by 48%), collagen (by 71%), or the thromboxane mimetic U-46619 (by 50%). This effect was reversible a nd occurred in the absence of cell damage. Impair ment of aggregation in platelets exposed to X-XO was due to H2O2 formation, since it was p revented by catalase but not by superoxide dismutase. Similarly, incub ation with the pure H2O2 generator glucose-glucose oxidase also marked ly inhibited ADP-induced platelet aggregation in a dose-dependent fash ion. Impaired aggregation by H2O2 was accompanied by a > 10-fold incre ase in platelet concentrations of guanosine 3',5'-cyclic monophosphate (cGMP), whereas adenosine 3',5'-cyclic monophosphate levels remained unchanged. The inhibitory role of increased cGMP formation was confirm ed by the finding that H2O2-induced impairment of platelet aggregation was largely abolished when guanylate cyclase activation was prevented by incubating platelets with the guanylate cyclase inhibitor, LY-8358 3. Different effects were observed when arachidonic acid was used to s timulate platelets. Exposure to a source of H2O2 did not affect aggreg ation to arachidonate. Furthermore, in the absence of exogenous H2O2, incubation with catalase, which had no effects on platelet response to ADP, collagen, or U-46619, virtually abolished platelet aggregation a nd markedly reduced thromboxane B-2 production (to 44% of control) whe n arachidonic acid was used as a stimulus. In conclusion, our data dem onstrate that H2O2 may exert complex effects on platelet function in v itro. Low levels of endogenous H2O2 seem to be required to promote thr omboxane synthesis and aggregation in response to arachidonic acid. In contrast, exposure to larger (but not toxic) concentrations of exogen ous H2O2 may inhibit aggregation to several agonists via stimulation o f guanylate cyclase and increased cGMP formation.