Diffusion of peroxynitrite into the human platelet inhibits cyclooxygenase via nitration of tyrosine residues.

Peroxynitrite (ONOO(-)), a reactive oxidant produced by the reaction between nitric oxide and superoxide, was found to diffuse into the platelet cytosol and inhibit arachidonic acid-induced platelet aggregations with IC(50) value of 5.8 +/- 1.2 microM. A fluorescence assay established that ONOO(-) diffused into the platelet cytosol in a manner that was inhibited (50-70%) by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, an inhibitor of HCO(3)(-)/Cl(-) anion exchanger. Treatment of platelets with (-)-epigallocatechin gallate (2 microM), a tea polyphenol and inhibitor of tyrosine nitration, abolished the inhibitory effect of ONOO(-) on arachidonate-induced aggregations by 88%. ONOO(-) (50-300 microM), added to platelets 1 min before arachidonic acid, inhibited (20-100%) formation of platelet cyclooxygenase (COX) products thromboxane A(2) and 12-hydroxyheptadecatrienoic acid. Interestingly, simultaneous addition of ONOO(-) and arachidonic acid stimulated eicosanoid production by 20 to 60%. The inhibition of thromboxane A(2) generation correlated with the 5- to 10-fold increase in the 3-nitrotyrosine levels of the platelet COX. Experiments with purified COX-1 and COX-2 also showed 9-fold increase of 3-nitrotyrosine levels, which correlated with decreased (93-98%) production of prostaglandin H(2) when ONOO(-) (50 microM) was added 1 min before arachidonic acid. However, the addition of ONOO(-) (50-100 microM) simultaneously with arachidonic acid increased prostaglandin H(2) formation by 30 to 60%. Thus, the inhibitory effect of ONOO(-) involved nitration of COX tyrosine residues, whereas the stimulatory effect was likely to be a result of ONOO(-) functioning as a peroxide activator of eicosanoid signaling. Increasing doses of ONOO(-) not only inhibited platelet COX but also induced formation of unique eicosanoids: iso-prostaglandin F(2alpha), epoxyhydroxyeicosatrienoic acid, and trans-arachidonic acids, suggesting that OH and NO(2) radicals were generated from ONOO(-) in platelets. Formation of ONOO(-) from NO and superoxide may function as a platelet hormone-like COX regulatory mechanism in inflammatory processes in which large amounts of these molecules are produced.