Fluvastatin alters platelet aggregability in patients with hypercholesterolemia: possible improvement of intraplatelet redox imbalance via HMG-CoA reductase.

BACKGROUND: Hypercholesterolemia enhances platelet aggregability. Statins have beneficial effects on cardiovascular events. The purpose of this study is to investigate whether statins inhibit platelet aggregation and, if so, the mechanisms. METHODS AND RESULTS: Twelve patients with hypercholesterolemia were prospectively randomized in a crossover design to receive either fluvastatin (20 mg/d) or colestimide (3000 mg/d) for 12 weeks. The subjects were switched to the opposite arm for additional 12 weeks. Before and after first and second treatments, experiments were performed. Eleven age-matched volunteers with normal lipid profiles served as controls. ADP-induced platelet aggregation, platelet-derive nitric oxide (PDNO) release, intraplatelet levels of GSH and GSSG, and intraplatelet nitrotyrosine production during platelet aggregation were measured. Fluvastatin and colestimide equally lowered total and low density lipoprotein cholesterol levels in hypercholesterolemia. Platelet aggregation was greater in hypercholesterolemia than in normocholesterolemia before treatment and was altered by fluvastatin. PDNO release, intraplatelet glutathione level, and GSH/GSSG ratio were lower in hypercholesterolemia than in normocholesterolemia before treatment and were increased by fluvastatin. Intraplatelet nitrotyrosine formation was greater in hypercholesterolemia than in normocholesterolemia, and decreased by fluvastatin. Colestimide did not have such effects. In vitro application of fluvastatin dose-dependently inhibited platelet aggregation. Furthermore, in vitro application of fluvastatin dose-dependently inhibited platelet nitrotyrosine expressions and the inhibitory effects by fluvastatin were reversed by preincubation with geranylgeranylpyrophosphate. CONCLUSIONS: Fluvastatin altered platelet aggregability in hypercholesterolemic patients in a cholesterol-lowering independent manner, which was partly mediated by the improvement of intraplatelet redox imbalance.

Platelet P-selectin plays an important role in arterial thrombogenesis by forming large stable platelet-leukocyte aggregates.

OBJECTIVES: We investigated the role of P-selectin in arterial thrombogenesis by forming large stable platelet-leukocyte aggregates. BACKGROUND: Plaque rupture followed by thrombus formation is a fundamental pathophysiology of acute coronary syndromes. Although the adhesive interaction between platelets and leukocytes via P-selectin is known to mediate platelet-rich thrombi, the true function of P-selectin in thrombus formation in vivo is unknown. METHODS: In wild-type (P(+/+)) and P-selectin-deficient (P(-/-)) mice with ferric chloride (FeCl(3))-induced carotid arterial thrombosis model, we measured in vivo platelet P-selectin expression and adenosine diphosphate (ADP)-induced ex vivo platelet aggregation. We also measured ex vivo ADP-induced whole blood aggregations and their size distribution by flow cytometry. RESULTS: Time to thrombotic occlusion was longer in P(-/-) mice than in P(+/+) mice. Spontaneous reflow after total thrombotic occlusion was observed in 8 of 10 P(-/-) mice but not in any P(+/+) mice. ADP-induced ex vivo platelet aggregation was not different between the two groups. However, ADP-induced ex vivo whole blood aggregation was inhibited in P(-/-) mice compared to P(+/+) mice. FeCl(3) application increased in vivo expressions of platelet P-selectin in P(+/+) mice but not in P(-/-) mice. The number of leukocytes within thrombi was less in P(-/-) mice than in P(+/+) mice. In flow cytometric analysis of size distribution of ADP-induced whole blood aggregates, the number of large aggregates was less in P(-/-) mice than in P(+/+) mice. Using platelet and leukocyte fluorescence makers, the large aggregates were confirmed as platelet-leukocyte aggregates. CONCLUSIONS: Platelet P-selectin plays an important role in arterial thrombogenesis by forming large stable platelet-leukocyte aggregates.

Polymorphonuclear leukocytes may impair endothelial function: results of crossover randomized study of lipid-lowering therapies.

OBJECTIVES: To examine whether polymorphonuclear leukocytes (PMNs) in hypercholesterolemia (HC) are activated to generate large amount of superoxide in vivo and hence impair endothelial function and, if so, whether statins, which possess anti-inflammatory properties, may restore PMN-mediated endothelial dysfunction. METHODS AND RESULTS: At baseline, subjects with HC showed impaired endothelial function (P<0.001), estimated by flow-mediated vasodilation of the brachial artery, and increased susceptibility of low-density lipoprotein (LDL) to oxidation (P<0.0001) compared with control subjects. PMNs obtained from HC produced greater amount of superoxide (P<0.0001), showed higher adhesiveness to cultured endothelial cells (HUVECs) (P<0.0001), and impaired endothelial nitric oxide synthase (eNOS) Ser1177 phosphorylation of HUVECs compared with controls (P<0.001). Crossover administration of fluvastatin or colestimide for 3 months lowered LDL to the same levels (P<0.001 for both). Endothelial function was restored (P<0.0001). LDL oxidation (P<0.0001) and superoxide release from PMNs (P<0.0001) were diminished only in fluvastatin but not in colestimide arm. Fluvastatin attenuated PMN adhesion to HUVECs (P<0.0001) and restored eNOS Ser1177 phosphorylation of HUVECs (P<0.001). CONCLUSIONS: Statins may improve endothelial function at least in part by inactivating neutrophils independently of LDL reduction. Our results raise a novel concept that polymorphonuclear leukocytes may attack endothelia and play a pivotal role in the pathogenesis of atherosclerosis.

HMG-CoA reductase inhibitor protects against in vivo arterial thrombosis by augmenting platelet-derived nitric oxide release in rats.

Acute coronary syndromes are caused by platelet-mediated thrombosis following rupture of a plaque. HMG-CoA reductase inhibitors (statins) reduce the incidence of events early after acute coronary syndromes, which are independent of its cholesterol-lowering effect. Accordingly, we investigated whether statins inhibit platelet-mediated arterial thrombus formation in vivo and, if so, the underlying mechanisms. Rats were divided into 4 groups. Group 1 was treated with the vehicle, whereas groups 2, 3, and 4 were treated with cerivastatin for 7 days (1, 2, and 5 mg/kg i.p., respectively). Cerivatatin did not change serum cholesterol levels. Carotid arterial thrombosis was created by perivascular FeCl3 delivery. Cerivastatin significantly prolonged the time to thrombotic occlusion of carotid artery. Cerivastatin significantly dose-dependently inhibited both ex vivo platelet P-selectin expression, a marker of platelet activation, and platelet aggregation. Cerivastatin significantly augmented platelet-derived nitric oxide (NO) release, and up-regulated platelet and endothelial nitric oxide synthase (NOS) mRNA expressions. N-nitro-L-arginine methylester abolished the effects of cerivastatin. This study demonstrates that in vivo administration of statin protects against platelet-mediated arterial thrombosis, possibly by augmenting platelet- and endothelium-derived NO releases via up-regulation of platelet and endothelial NOS.

Only two-week smoking cessation improves platelet aggregability and intraplatelet redox imbalance of long-term smokers.

OBJECTIVES: We investigated whether and how soon smoking cessation ameliorates the smoking-induced intracellular oxidative stress and platelet aggregability in long-term smokers. BACKGROUND: Smoking is a major risk factor of atherothrombosis. Smoking cessation reduces cardiac events. However, the underlying mechanisms of the beneficial effects remain to be elucidated. METHODS: Twenty-seven male long-term smokers were divided into two groups. Group A (n = 14) quit smoking for four weeks whereas group B (n = 13) resumed smoking two weeks after quitting. Smoking status was monitored by measurement of urinary cotinine. Using gel-filtered platelets, agonist (adenosine diphosphate and collagen)-induced platelet aggregation, platelet-derived nitric oxide (PDNO), intraplatelet nitrotyrosine production, intraplatelet levels of the reduced form of glutathione (GSH) and its oxidized form (GSSG), and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and urinary 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)), as markers of systemic oxidative stress, were measured. The baseline measurements were similar between the two groups. RESULTS: Smoking cessation quickly reduced agonist-induced platelet aggregations, intraplatelet nitrotyrosine level, and urinary productions of 8-OHdG and 8-iso-PGF(2alpha) by two weeks in both groups. In group A, they were maintained at the low levels until four weeks, whereas they were reversed by resmoking in group B; PDNO release and intraplatelet GSH/GSSG ratio were time-dependently increased by smoking cessation but reversed by resmoking. CONCLUSIONS: The present findings are the first demonstration that only two weeks of smoking cessation can ameliorate the enhanced platelet aggregability and intraplatelet redox imbalance in long-term smokers, possibly by decreasing oxidative stress. Our findings may strengthen the motivation for smokers to quit smoking.

Inhibition of platelet adherence to mononuclear cells by alpha-tocopherol: role of P-selectin.

BACKGROUND: Platelet-leukocyte interaction is an early important event for thrombogenesis, and this process is mainly mediated by P-selectin on platelets. Although alpha-tocopherol has been shown to inhibit thrombotic disorders, the effect of alpha-tocopherol on platelet P-selectin expression and platelet-leukocyte interaction is little known. METHODS AND RESULTS: We examined whether alpha-tocopherol inhibited human platelet P-selectin expression and platelet-leukocyte interaction. Alpha-tocopherol (50 to 500 microg/mL) inhibited thrombin-induced or phorbol 12-myristate 13-acetate (PMA)-induced P-selectin expression on platelets. alpha-Tocopherol suppressed platelet-mononuclear cell (MNC) interaction, platelet aggregation, and platelet protein kinase C (PKC) activity stimulated with either PMA (100 nmol/L) or thrombin. Inhibitory actions of alpha-tocopherol against the platelet functions were mimicked by staurosporine, a selective PKC inhibitor. After oral supplementation of alpha-tocopherol (300 mg/d for 3 weeks) in healthy subjects, thrombin-mediated or PMA-mediated P-selectin expression, platelet-MNC interaction, and platelet aggregation ex vivo were suppressed. CONCLUSIONS: alpha-Tocopherol inhibited P-selectin expression on human platelets and thereby attenuated platelet-MNC interactions, which were mediated at least in part by the inhibition of intraplatelet PKC activity. These actions of alpha-tocopherol on platelet functions provide new insights into the antithromboatherogenic properties of alpha-tocopherol.

Vitamin E inhibits lysophosphatidylcholine-induced endothelial dysfunction and platelet activation.

Lysophosphatidylcholine (LPC), a lysolipid contained in oxidized low-density lipoprotein, is an atherogenic molecule that induces endothelial dysfunction and platelet activation and inhibits angiogenesis. Although studies showed that vitamin E has antiatherogenic properties, the effects of vitamin E on LPC-induced endothelial dysfunction and platelet activation are little known. We examined whether vitamin E has protecting actions against LPC-induced alterations of endothelial and platelet functions. Incubation of cultured bovine aortic endothelial cells (BAECs) with LPC (10 microM) significantly inhibited bradykinin (1 microM)-stimulated nitric oxide release, which was prevented by cotreatment with vitamin E (50, 100, and 500 microg/ml) in a concentration-dependent manner. In isolated human platelets, LPC stimulated P-selectin expression and induced leukocyte-platelet interaction, which functionally depends on P-selectin expressed on the platelet surface. Vitamin E treatment significantly prevented the LPC-induced platelet P-selectin expression and leukocyte-platelet interaction. As LPC-induced endothelial dysfunction and platelet activation have been shown to involve the protein kinase C (PKC)-dependent signal transduction pathway, we examined the effects of vitamin E on LPC-induced PKC activation in human platelets and BAECs. Vitamin E significantly inhibited LPC (10 microM)-stimulated PKC activation in a concentration-dependent manner. It is concluded that (a) Vitamin E prevented LPC-induced endothelial dysfunction and preserved endothelial nitric oxide release, (b) vitamin E inhibited LPC-induced platelet activation (P-selectin expression) and leukocyte-platelet interaction, and (c) these mechanisms appeared to be at least partly mediated by suppression of the PKC in endothelial cells and platelets. The present findings may provide new insights into antiatherogenic mechanisms of vitamin E.

Coexistence of impairment of endothelium-derived nitric oxide and platelet-derived nitric oxide in patients with coronary risk factors.

Impairment of endothelium-derived nitric oxide (EDNO) has been demonstrated in patients with coronary risk factors in some studies, as well as impaired platelet-derived nitric oxide (PDNO) in other studies. However, no study has examined whether these impairments coexist. In 24 patients with coronary risk factors, femoral vascular endothelial function was assessed with acetylcholine (ACh: 50, 100, 200 and 400 microg/min) and endothelium-independent vascular function with nitroglycerin (NTG; 50, 100, 200 microg/min) using a Doppler flow-wire technique, as well as ADP (5 micromol/L)-induced PDNO release with an NO-specific electrode. The ACh-mediated percent change in femoral vascular resistance index (% change of FVRI) and PDNO release had a significant correlation with the number of risk factors. The ACh-mediated % change of FVRI, but not that with NTG, significantly correlated with the PDNO release. Both EDNO and PDNO bioactivities are impaired in patients with coronary risk factors and there is a common mechanism.