α‐Tocopherol and protein kinase C inhibition enhance platelet‐derived nitric oxide release

Platelet activation is tightly regulated by products of the endothelium and platelets including nitric oxide (NO). Excess vascular oxidative stress has been associated with impaired NO release, and antioxidant status has been shown to alter endothelium‐derived NO bioactivity. Although physiological levels of α‐tocopherol are known to inhibit platelet function, the effect of α‐tocopherol on platelet NO release is unknown. Loading platelets with physiologic levels of α‐tocopherol increased platelet NO production ~1.5‐fold (P<0.01) and reduced extent of aggregation from 84 to 58% (P<0.01). In normal subjects supplemented with 400 IU/day α‐tocopherol, platelet NO release increased 50% (P<0.05; n=5). α‐Tocopherol‐loaded platelets also produced 74% less superoxide as compared with control (P<0.05); however, this did not completely explain its effect on NO production, because platelets aggregated in the presence of superoxide dismutase produced no detectable superoxide, and NO production only increased 72% (P<0.01). Protein kinase C inhibition with chelerythrine also increased NO production by 90% compared with control (P<0.01), and incubation of platelets with α‐tocopherol inhibited PKC‐dependent eNOS phosphorylation as determined by immunoprecipitation. Lastly, platelets isolated from NOS3‐deficient mice released 80% less superoxide as compared with control animals (P=0.011), and incubation of NOS III‐deficient platelets with 500 pM α‐tocopherol only caused a modest additional decrease in platelet superoxide release (NS). Thus, α‐tocopherol appears to enhance platelet NO release both in vitro and in vivo through antioxidant‐ and PKC‐dependent mechanisms.