Object-Platelet isoprostane 8-ISO-prostaglandin F2␣ (8-iso-PGF2␣), a proaggregating molecule, is believed to derive from nonenzymatic oxidation of arachidonic acid. We hypothesized that NADPH is implicated in isoprostane formation and platelet activation. Methods and Results-We studied 8-iso-PGF2␣ in platelets from 8 male patients with hereditary deficiency of gp91 phox , the catalytic subunit of NADPH oxidase, and 8 male controls. On stimulation, platelets from controls produced 8-iso-PGF2␣, which was inhibited Ϫ8% by aspirin and Ϫ58% by a specific inhibitor of gp91 phox . Platelets from patients with gp91phox hereditary deficiency had normal thromboxane A 2 formation but marked 8-iso-PGF2␣ reduction compared with controls. In normal platelets incubated with a gp91 phox inhibitor or with SQ29548, a thromboxane A 2 /isoprostane receptor inhibitor, platelet recruitment, an in vitro model of thrombus growth, was reduced by 44% and 64%, respectively; a lower effect (Ϫ17%) was seen with aspirin. Moreover, thrombus formation under shear stress (blood perfusion at the wall shear rate of 1500 s Ϫ1 ) was reduced in samples in which isoprostane formation was inhibited by NADPH oxidase inhibitors. In gp91 phox -deficient patients, agonist-induced platelet aggregation was within the normal range, whereas platelet recruitment was reduced compared with controls. Incubation of platelets from gp91 phox -deficient patients with 8-iso-PGF2␣ dose-dependently (1 to 100 pmol/L) increased platelet recruitment by mobilizing platelet Ca 2ϩ and activating gpIIb/IIIa; a further increase in platelet recruitment was detected by platelet coincubation with L-NAME, an inhibitor of NO synthase. Conclusion-This
Background-CD40 ligand (CD40L) expression on platelets is mediated by agonists, but the underlying mechanism is still unclear. Methods and Results-CD40L expression was measured in platelets from healthy subjects both with and without the addition of antioxidants or a phospholipase A2 (PLA2) inhibitor and in platelets from 2 patients with an inherited deficiency of gp91phox. Immunoprecipitation analysis was also performed to determine whether normal platelets showed gp91phox expression. Unlike catalase and mannitol, superoxide dismutase inhibited agonist-induced platelet CD40L expression in healthy subjects. Immunoprecipitation analysis also showed that platelets from healthy subjects expressed gp91phox. In 2 male patients with inherited gp91phox deficiency, collagen-, thrombin-, and arachidonic acid-stimulated platelets showed an almost complete absence of superoxide anion (O 2 Ϫ ) and CD40L expression. Incubation of platelets from healthy subjects with a PLA2 inhibitor almost completely prevented agonist-induced O 2
Objective-The inhibition of oxidative stress is among the most relevant pleiotropic effects of statins. The mechanism by which statins exert their antioxidant effect in vivo is still undefined. NADPH oxidase is among the most important sources of reactive oxygen species involved in atherosclerotic disease. Methods/Results-We developed an ELISA to evaluate serum levels of soluble-gp91 phox , the catalytic core of phagocyte NADPH oxidase. In a cross-sectional study performed in 30 hypercholesterolemic patients and in 20 controls, serum soluble-gp91 phox and urinary isoprostane, a marker of oxidative stress, were measured. The 2 variables were also measured in hypercholesterolemic patients, randomized to diet (nϭ15), or diet plus atorvastatin (10 mg daily, nϭ15) and followed for 30 days. Compared to controls, hypercholesterolemic patients had higher and significantly correlated (Rϭ0.71; PϽ0.001) serum soluble-gp91 phox (PϽ0.001) and urinary isoprostanes (PϽ0.001). After follow-up, the statin-allocated group showed a significant reduction of soluble-gp91 phox (Ϫ33%, PϽ0.01), that paralleled that of isoprostanes (Ϫ37%, PϽ0.01) and cholesterol (Ϫ25%, PϽ0.01). The diet-allocated group showed only a weak reduction of cholesterol. Key Words: gp91 phox Ⅲ oxidative stress Ⅲ hypercholesterolemia Ⅲ NADPH oxidase Ⅲ statins P rimary and secondary prevention trials with statins clearly demonstrated that this drug category is able to reduce cardiovascular events. 1,2 Even if the principal mechanism of action of statins is to lower cholesterol, other effects, the so-called pleiotropic effects, have been considered as adjunctive properties potentially accounting for the antiatherosclerotic effect of statins. 3 Inhibition of oxidative stress may be considered an intriguing pleiotropic effect in view of the fact that oxidative stress is thought to be a key event in the initiation and progression of atherosclerotic disease. 4 Reduction of several markers of oxidative stress including isoprostanes, 8-hydroxydeoxyguanosine (8-OHdG), and nitrotyrosine have been observed after statin treatment but the underlying mechanism is still unclear. [5][6][7][8][9] In a pilot study performed in patients with chronic granulomatous disease (X-CGD), a very rare life-threatening disease secondary to hereditary deficiency of gp91 phox (the catalytic subunit of phagocyte NADPH oxidase), we found a significant reduction of urinary isoprostanes. 10 Also, in children with hypercholesterolemia, we observed a significant correlation between platelet gp91 phox expression and urinary isoprostanes. 11 These previous observations suggest a role for the phagocyte NADPH oxidase, one of the most important cellular producers of superoxide anion (O 2 . ), 12 in the formation of this marker of oxidative stress. Because previous studies provided in vitro evidence that statins inhibit the expression and activation of NADPH oxidase, 6,13 we sought to analyze whether this occurs in vivo and ultimately contributes to the reduction of oxidative stress. Thus, we developed ...
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