Pasquale Pignatelli

Short-term treatment with atorvastatin reduces platelet CD40 ligand and thrombin generation in hypercholesterolemic patients

BACKGROUND Soluble CD40L (sCD40L), a substance that maximally reflects in vivo platelet activation, is increased in patients with hypercholesterolemia. We investigated the relation between sCD40L and platelet CD4OL in hypercholesterolemic patients before and after a short-term treatment with atorvastatin. METHODS AND RESULTS Collagen-induced platelet CD40L and plasma levels of sCD40L and prothrombin fragment F1+2, a marker of thrombin generation, were investigated in 30 hypercholesterolemic patients and 20 healthy subjects. Hypercholesterolemic patients were then randomized to either diet (n=15; group A) or atorvastatin 10 mg/d (group B); the aforementioned variables were measured at baseline and after 3 days of treatment. Compared with referents, hypercholesterolemic patients showed higher values of platelet CD40L (P<0.005), sCD40L (P<0.005), and F1+2 (P<0.003). Platelet CD40L was significantly correlated with sCD40L (P<0.001), and the latter was significantly correlated with F1+2 (P<0.001). The intervention trial showed no changes in group A but a significant decrease in platelet CD40L (P<0.01), sCD40L (P<0.002), and F1+2 (P<0.03) in group B. In vitro studies demonstrated that cholesterol enhanced platelet CD40L and CD40L-mediated clotting activation by human monocytes; also, atorvastatin dose-dependently inhibited platelet CD40L expression and clotting activation by CD40L-stimulated monocytes. CONCLUSIONS This study shows that, in hypercholesterolemia, platelet overexpression of CD40L may account for enhanced plasma levels of sCD40L and F1+2. Atorvastatin exerts a direct antithrombotic effect via inhibition of platelet CD40L and CD40L-mediated thrombin generation, independently of its cholesterol-lowering effect.

gp91phox-Dependent Expression of Platelet CD40 Ligand

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 (O2−) and CD40L expression. Incubation of platelets from healthy subjects with a PLA2 inhibitor almost completely prevented agonist-induced O2− and CD40L expression. Conclusions—These data provide the first evidence that platelet CD40L expression occurs via arachidonic acid–mediated gp91phox activation.

Platelet Activation by Superoxide Anion and Hydroxyl Radicals Intrinsically Generated by Platelets That Had Undergone Anoxia and Then Reoxygenated

BACKGROUND Platelet activation has been demonstrated in experimental and clinical models of ischemia-reperfusion, but the underlying mechanism is still unclear. We mimicked the ischemia-reperfusion model in vitro by exposing platelets to anoxia-reoxygenation (A-R) and evaluated the role of oxygen free radicals (OFRs), which are usually produced during the reperfusion phase, in inducing platelet activation. METHODS AND RESULTS Human platelets were exposed to 15 and 30 minutes of anoxia and then reoxygenated. Compared with control platelets kept in atmospheric conditions, platelets exposed to A-R showed spontaneous platelet aggregation (SPA), which was maximal after 30 minutes of anoxia. Superoxide dismutase (SOD) (-74%, P < .005), catalase (-67%. P < .005). SOD plus catalase (-82%, P < .005), and the hydroxyl radical (OH0) scavengers mannitol (-66%, P < .005) and deoxyribose (-55%, P < .005) inhibited SPA. Platelets that had undergone A-R released superoxide anion (0-2), as detected by lucigenin chemiluminescence. Also, platelets exposed to A-R and incubated with salicylic acid generated 2.3- and 2,5-dihydroxybenzoates, which derive from salicylic acid reaction with OH0. SPA was significantly inhibited by the cyclooxygenase enzyme inhibitors aspirin and indomethacin: by SQ29548, a thromboxane (Tx) A2 receptor antagonist; by diphenyliodonium an inhibitor of flavoprotein-dependent enzymes: and by arachidonyl trifluoromethyl ketone, a selective inhibitor of cytosolic phospholipase A2. Platelets exposed to A-R markedly generated inositol 1,3,4-trisphosphate and TxA2, which were inhibited by incubation of platelets with SOD plus catalase. CONCLUSIONS This study shows that platelets exposed to A-R intrinsically generated 0-2 and OH0, which in turn activate arachidonic acid metabolism via phospholipases A2 and C, and provides further support for the use of antioxidant agents as inhibitors of platelet function in ischemia-reperfusion models.

Effects of resveratrol and other wine polyphenols on vascular function: An update

Several epidemiologic observations show that moderate wine drinking reduces cardiovascular morbidity and mortality. Wine contains several polyphenols, and among them, resveratrol in particular has been shown to exert a number of important biologic activities on the cardiovascular system that may contribute to the protective effects of wine. The mechanisms through which resveratrol and other wine polyphenols protect from ischemic cardiovascular events are many, but protection from oxidative stress and radical oxygen species production, a facilitating activity on nitric oxide production and activity and the ability to modulate the expression of adhesive molecules by blood cells and the vascular wall seem to be the most important. In this overview, the in vitro and in vivo evidence on the activity of resveratrol on vascular function and circulating blood cells, with a special emphasis on blood platelets, is thoroughly presented.

Hereditary deficiency of gp91(phox) is associated with enhanced arterial dilatation: results of a multicenter study.

Background— NADPH oxidase is believed to modulate arterial tone, but its role in humans is still unclear. The objective of this study was to evaluate whether NADPH oxidase is involved in flow-mediated arterial dilation (FMD). Methods and Results— Twenty-five patients with hereditary deficiency of gp91phox, the catalytic core of NADPH oxidase, (X-CGD), 25 healthy subjects, and 25 obese patients matched for sex and age were recruited. FMD, platelet gp91phox, serum levels of nitrite and nitrate as markers of nitric oxide generation, oxidized low-density lipoprotein, and urinary excretion of isoprostanes as markers of oxidative stress were determined. Platelet gp91phox expression was downregulated in X-CGD patients (1.0±0.8 mean fluorescence; P<0.001) and upregulated in obese patients (4.1±2.2 mean fluorescence; P=0.01) compared with healthy subjects (2.9±1.7 mean fluorescence). Urinary excretion of isoprostanes was reduced in X-CGD patients (41.7±33.3 pg/mg creatinine; P=0.04) and increased in obese patients (154.4±91 pg/mg creatinine; P<0.001) compared with healthy subjects (69.5±52.4 pg/mg creatinine). Obese patients had higher serum oxidized low-density lipoprotein than healthy subjects (35.3±6.7 versus 24.8±9.8 U/L; P<0.001) and X-CGD patients (28.5±7.2 U/L; P<0.001). X-CGD patients had significantly higher FMD (14.7±5.9%) compared with healthy subjects (7.9±2.5%; P<0.001); obese patients had lower FMD (5.3±3.0%; P=0.028) compared with healthy subjects. Serum nitrite and nitrate levels were significantly higher in patients with X-CGD (36.0±10.8 μmol/L; P=0.016) and lower in obese patients (9.3±11.0 μmol/L; P=0.001) compared with healthy subjects (27.1±19.1 μmol/L). Serum nitrite and nitrate levels significantly correlated with FMD (Rs=0.403, P<0.001) and platelet gp91phox (Rs=−0.515, P<0.001). FMD inversely correlated with platelet gp91phox (Rs=−0.502, P<0.001) and isoprostanes (Rs=−0.513, P<0.001). Conclusion— This study provides the first evidence that, in humans, gp91phox is implicated in the modulation of arterial tone.

Polyphenols enhance platelet nitric oxide by inhibiting protein kinase C-dependent NADPH oxidase activation: effect on platelet recruitment

Several studies demonstrated an inverse association between polyphenol intake and cardiovascular events. Platelet recruitment is an important phase of platelet activation at the site of vascular injury, but it has never been investigated whether polyphenols influence platelet recruitment. The aim of the study was to analyze in vitro whether two polyphenols, quercetin and catechin, were able to affect platelet recruitment. Platelet recruitment was reduced by NO donors and by NADPH oxidase inhibitors and was enhanced by L‐NAME, an inhibitor of NO synthase. Quercetin and catechin, but not single polyphenol, significantly inhibited platelet recruitment in a concentration‐dependent fashion. The formation of superoxide anion was significantly inhibited in platelets incubated with quercetin and catechin but was unaffected by a single polyphenol. Incubation of platelets with quercetin and catechin resulted in inhibition of PKC and NADPH oxidase activation. Treatment of platelets with quercetin and catechin resulted in an increase of NO and also down‐regulated the expression of GpIIb/IIIa glycoprotein. This study shows that the polyphenols quercetin and catechin synergistically act in reducing platelet recruitment via inhibition of PKC‐dependent NADPH oxidase activation. This effect, resulting in NO‐mediated platelet glycoprotein GpIIb/IIIa down‐regulation, could provide a novel mechanism through which polyphenols reduce cardiovascular disease.—Pignatelli, P., Di Santo, S., Buchetti, B., Sanguigni, V., Brunelli, A., Violi, F. Polyphenols enhance platelet nitric oxide by inhibiting protein kinase C‐dependent NADPH oxidase activation: effect on platelet recruitment. FASEB J. 20, 1082–1089 (2006)

Inherited Human gp91phox Deficiency Is Associated With Impaired Isoprostane Formation and Platelet Dysfunction

Object—Platelet isoprostane 8-ISO-prostaglandin F2&agr; (8-iso-PGF2&agr;), 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&agr; in platelets from 8 male patients with hereditary deficiency of gp91phox, the catalytic subunit of NADPH oxidase, and 8 male controls. On stimulation, platelets from controls produced 8-iso-PGF2&agr;, which was inhibited −8% by aspirin and −58% by a specific inhibitor of gp91phox. Platelets from patients with gp91phox hereditary deficiency had normal thromboxane A2 formation but marked 8-iso-PGF2&agr; reduction compared with controls. In normal platelets incubated with a gp91phox inhibitor or with SQ29548, a thromboxane A2/isoprostane receptor inhibitor, platelet recruitment, an in vitro model of thrombus growth, was reduced by 44% and 64%, respectively; a lower effect (−17%) was seen with aspirin. Moreover, thrombus formation under shear stress (blood perfusion at the wall shear rate of 1500 s−1) was reduced in samples in which isoprostane formation was inhibited by NADPH oxidase inhibitors. In gp91phox-deficient patients, agonist-induced platelet aggregation was within the normal range, whereas platelet recruitment was reduced compared with controls. Incubation of platelets from gp91phox-deficient patients with 8-iso-PGF2&agr; dose-dependently (1 to 100 pmol/L) increased platelet recruitment by mobilizing platelet Ca2+ 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 study provides the first evidence that platelet 8-iso-PGF2&agr; maximally derives from gp91phox activation and contributes to platelet recruitment via activation of gpIIb/IIIa.

Atorvastatin Inhibits gp91phox Circulating Levels in Patients With Hypercholesterolemia

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-gp91phox, the catalytic core of phagocyte NADPH oxidase. In a cross-sectional study performed in 30 hypercholesterolemic patients and in 20 controls, serum soluble-gp91phox and urinary isoprostane, a marker of oxidative stress, were measured. The 2 variables were also measured in hypercholesterolemic patients, randomized to diet (n=15), or diet plus atorvastatin (10 mg daily, n=15) and followed for 30 days. Compared to controls, hypercholesterolemic patients had higher and significantly correlated (R=0.71; P<0.001) serum soluble-gp91phox (P<0.001) and urinary isoprostanes (P<0.001). After follow-up, the statin-allocated group showed a significant reduction of soluble-gp91phox (−33%, P<0.01), that paralleled that of isoprostanes (−37%, P<0.01) and cholesterol (−25%, P<0.01). The diet-allocated group showed only a weak reduction of cholesterol. Conclusion—Our study demonstrates that statins exert an antioxidant effect via inhibition of soluble gp91phox expression.

Immediate Antioxidant and Antiplatelet Effect of Atorvastatin via Inhibition of Nox2

Background— Statins exert an antithrombotic effect in patients at risk of or with acute thrombosis, but no study has investigated whether this effect is immediate and whether there is an underline mechanism. Methods and Results— Patients with hypercholesterolemia were randomly allocated to a Mediterranean diet with low cholesterol intake (<300 mg/d; n=15) or atorvastatin (40 mg/d; n=15). Oxidative stress, as assessed by serum Nox2 and urinary isoprostanes, and platelet activation, as assessed by platelet recruitment, platelet isoprostanes, and thromboxane A2, platelet Nox2, Rac1, p47phox, protein kinase C, vasodilator-stimulated phosphoprotein, nitric oxide, and phospholipase A2, were determined at baseline and after 2, 24, and 72 hours and 7 days of follow-up. An in vitro study was also performed to see whether atorvastatin affects platelet oxidative stress and activation. The atorvastatin-assigned group showed a significant and progressive reduction of urinary isoprostanes and serum Nox2, along with inhibition of platelet recruitment, platelet isoprostanes, Nox2, Rac1, p47phox, and protein kinase C, starting 2 hours after administration. Platelet phospholipase A2 and thromboxane A2 significantly decreased and vasodilator-stimulated phosphoprotein and nitric oxide increased after 24 hours. Low-density lipoprotein cholesterol decreased significantly after 72 hours and further declined after 7 days. No changes were observed in the Mediterranean diet group. In vitro experiments demonstrated that atorvastatin dose-dependently inhibited platelet Nox2 and phospholipase A2 activation, along with inhibition of platelet recruitment, platelet isoprostanes, and thromboxane A2, and increased vasodilator-stimulated phosphoprotein and nitric oxide. Conclusions— The study provides the first evidence that atorvastatin acutely and simultaneously decreases oxidative stress and platelet activation by directly inhibiting platelet Nox2 and ultimately platelet isoprostanes and thromboxane A2. These findings provide a rationale for the use of statins to prevent or modulate coronary thrombosis. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT01322711.

Statins as Antithrombotic Drugs

Statins are powerful lipid-lowering drugs that inhibit cholesterol biosynthesis via downregulation of hydroxymethylglutaryl coenzyme-A reductase.1 They are largely used in patients with or at risk of cardiovascular disease inasmuch as randomized trials have consistently shown that statins lower the rate of myocardial infarction, stroke, and cardiovascular death.2 The majority of trials with statins investigated patients with stable atherosclerotic disease, in whom the reduction in cardiovascular events appeared to be related to the cholesterol-lowering effect of statins and ultimately to plaque stabilization.3 However, experimental data demonstrated that statins may also exert a direct antithrombotic effect in models of arterial and venous thrombosis via a mechanism unrelated to the cholesterol-lowering activity4,5; this may suggest that statins inhibit several pathways of hemostasis, including platelet activation and coagulation cascade. Consistent with these observations are the potential negative properties that have been observed, including the association between statin therapy and cerebral hemorrhage. In this review, we present experimental data in support of the ability of statins to interfere directly with the clotting system and platelet activation, as well as the clinical settings that suggest that statins exert beneficial effects related to their antithrombotic properties. Colli et al6 were the first to show that statins interfere with activation of the clotting system and the coagulation cascade. Specifically, they demonstrated that fluvastatin dose-dependently inhibits activity of tissue factor (TF), a glycoprotein that converts factor X to factor Xa,7 and suggested that downexpression of geranylgeranylated protein is implicated in TF lowering. These data were confirmed in patients affected by polygenic hypercholesterolemia8 in whom simvastatin 20 mg/d for 8 weeks resulted in downregulation of TF antigen and activity and thrombin generation. Of note, inhibition of clotting activation, as assessed by plasma values of prothrombin fragment F1+2, a marker of …