Stimulation of platelet nitric oxide production by nebivolol prevents thrombosis.

OBJECTIVE: dl-Nebivolol, a selective ß1-adrenergic receptor antagonist, besides its hypotensive activity exerts vasodilatory and platelet inhibitory effects in vitro by a mechanism involving nitric oxide (NO). Our aim was to evaluate whether nebivolol exerts in vivo antithrombotic effects, to unravel the mechanism of this action and to clarify the relative roles of its 2 enantiomers: d- and l-nebivolol. METHODS AND RESULTS: In wild-type mice, dl-nebivolol, l-nebivolol, and d-nebivolol, but not bisoprolol, reduced mortality consequent to platelet pulmonary thromboembolism induced by the intravenous injection of collagen plus epinephrine (-44%, -45%, -29%, respectively; P<0.05), whereas in eNOS(-/-) mice only dl-nebivolol and d-nebivolol were effective. dl-Nebivolol, l- and d-nebivolol reduced photochemical damage-induced femoral artery thrombosis in wild-type mice, whereas in eNOS(-/-) mice only dl-nebivolol and d-nebivolol were active. Moreover, dl-nebivolol and l-nebivolol increased plasma, urinary-, and platelet-derived nitrites and nitrates (NOx), NO degradation products, in wild-type but not in eNOS(-/-) mice. In vivo platelet activation, assessed by platelet P-selectin expression, was reduced by dl-nebivolol and l- and d-nebivolol in wild-type mice but only by dl-nebivolol and d-nebivolol in eNOS(-/-) mice. In bone marrow-transplanted, chimeric mice with only blood cells, and not the endothelium, producing NO dl-nebivolol and l-nebivolol maintained their antithrombotic activity, whereas they lose it in chimeras with only endothelium, and not blood cells, producing NO. In vitro, with isolated platelets, dl-nebivolol and l-nebivolol, but not d-nebivolol and bisoprolol, increased platelet cGMP and NOx formation. Treatment with dl-nebivolol and l-nebivolol increased phophorylated eNOS in platelets. CONCLUSIONS: Our data show that dl-nebivolol exerts an antithrombotic activity by stimulating the formation of NO by platelets, and that this effect is generated by its l-enantiomer, whereas the d-enantiomer exerts a weak antiplatelet effect because of ß-adrenergic receptor-independent stimulation of adenyly cyclase. These results confirm that platelet-derived NO plays a role in thrombosis prevention and it may represent a target of pharmacological intervention.

NCX 6560, a nitric oxide-releasing derivative of atorvastatin, inhibits cholesterol biosynthesis and shows anti-inflammatory and anti-thrombotic properties.

We compared the lipid-lowering, vasodilating, anti-thrombotic and anti-inflammatory properties of NCX 6560, a novel NO-releasing derivative of atorvastatin, with those of atorvastatin. NCX 6560 and atorvastatin induced similar inhibition of cholesterol biosynthesis in rat smooth muscle cells (IC(50)=1.9+/-0.4 and 3.9+/-1.0 microM, respectively). However, in hyperlipidemic mice, a 5-week oral treatment with NCX 6560 (46.8 mg/kg/day, p.o.) was more effective than equivalent atorvastatin (40 mg/kg/day, p.o.) at lowering serum cholesterol (NCX 6560: -21% vs controls, P<0.05; atorvastatin: -14% vs control, P=NS). In norepinephrine-precontracted rabbit aortic rings, NCX 6560-induced vasodilation (EC(50)=53.5+/-8.3 microM) and in PC12 cells it stimulated cGMP formation (EC(50)=1.8+/-0.7 microM), while atorvastatin was inactive. In lipopolysaccharide from Escherichia coli (LPS)-treated RAW 264.7 macrophages, NCX 6560 reduced iNOS expression and dimer assembly more efficiently than atorvastatin and inhibited nitrite accumulation (IC(50)=6.7+/-1.6 microM) and TNFalpha release. U46619- or collagen plus epinephrine-induced platelet pulmonary thromboembolism in mice was reduced by NCX 6560 at 46.8 mg/kg p.o. (mortality: -44% and -56% vs vehicle, respectively; P<0.05), but not by atorvastatin 40 mg/kg, p.o. In the U46619-induced mortality model, isosorbide mononitrate (ISMN) (20 mg/kg, p.o.), a pure NO-donor, was also active (mortality: -40%, P<0.05). NCX 6560 significantly reduced ex vivo platelet adhesion to collagen at high shear (-31+/-1.3% vs vehicle), and so did ISMN (-33.3+/-1.7% vs vehicle). Atorvastatin was ineffective. NCX 6560, but not atorvastatin, reduced blood pressure in eNOS knockout mice (-16%, P<0.001 vs vehicle), an effect not observed in wild type mice. On the contrary, ISMN provoked a significant drop of blood pressure both in wild type (-20%, P<0.05 vs vehicle) and in eNOS-/- mice (-21%, P<0.05 vs vehicle). In conclusion, NCX 6560 exerts greater lipid-lowering, anti-thrombotic and anti-inflammatory effects than atorvastatin, due to a large extent to NO release.