A nitric oxide-donor pravastatin hybrid drug exerts antiplatelet and antiatherogenic activity in mice

Aim of the present study was to compare the lipid-lowering, antithrombotic and antiatherogenic properties of NCX-6550, nitropravastatin, a nitric-oxide donating derivative of pravastatin, with those of pravastatin in hypercholesterolemic mice. LDL receptor-deficient mice (LDLR–/–) on a normal diet (ND) showed enhanced cholesterol levels as compared to wild type (WT) mice (6.8±1.2 mmol/L and 2.8±0.82 mmol/L, respectively). High fat diet (HFD) induced a large enhancement of cholesterolemia in LDLR–/– mice (23.7±5.7 mmol/L, p<0.0001 vs LDLR–/– ND and WT mice. Treatment with NCX 6550 (48 mg/kg), but not with equimolar pravastatin, reduced cholesterol in LDLR–/–HFD. Platelet adhesion to collagen under high shear rate (3000 sec–1) was significantly higher in LDLR–/– than in normal mice, and further enhanced in LDLR–/–HFD (-27%, p<0.0001 vs untreated). NCX 6550 (48 mg/kg), but not pravastatin, reduced platelet adhesion, especially in LDLR–/–HFD. U46619-induced platelet aggregation ex vivo was also inhibited by NCX 6550 (48 mg/kg) but not by the parent compound. Finally, photochemically-induced acute (1 hr) femoral artery thrombosis and delayed (21 days) intimal thickening was assessed. Thrombus size was larger in LDLR–/– on HFD than in normocholesterolemic mice (0.46±0.04 vs 0.18±0.08 mg) and it was reduced by NCX 6550 (48 mg/kg) (0.08±0.02 mg, p<0.0001), but not by pravastatin (0.4±0.01 mg p=NS). Intimal thickening was greater in hypercholesterolemic than in normal mice (I/M normal=0.53±0.16, LDLR–/–=1.1±0.15, LDLR–/–HFD=1.75 ±0.25). Both NCX 6550 and pravastatin reduced intimal thickening in normal (-95% and -74.5%, respectively) and LDLR–/– mice (-98% and -91%), while in strongly hyperlipidemic animals (LDLR–/–HFD) NCX 6550 was more effective than pravastatin (-98% vs -65%, p<0.0001). NCX 6550 shows greater antithrombotic and antiatherogenic activity than pravastatin in highly hypercholesterolemic mice

Loss of matrix metalloproteinase 2 in platelets reduces arterial thrombosis in vivo

Platelet activation at a site of vascular injury is essential for the arrest of bleeding; however, excessive platelet activation at a site of arterial damage can result in the unwarranted formation of arterial thrombi, precipitating acute myocardial infarction, or ischemic stroke. Activation of platelets beyond the purpose of hemostasis may occur when substances facilitating thrombus growth and stability accumulate. Human platelets contain matrix metalloproteinase 2 (MMP-2) and release it upon activation. Active MMP-2 amplifies the platelet aggregation response to several agonists by potentiating phosphatidylinositol 3-kinase activation. Using several in vivo thrombosis models, we show that the inactivation of the MMP-2 gene prevented thrombosis induced by weak, but not strong, stimuli in mice but produced only a moderate prolongation of the bleeding time. Moreover, using cross-transfusion experiments and wild-type/MMP-2−/− chimeric mice, we show that it is platelet-derived MMP-2 that facilitates thrombus formation. Finally, we show that platelets activated by a mild vascular damage induce thrombus formation at a downstream arterial injury site by releasing MMP-2. Thus, platelet-derived MMP-2 plays a crucial role in thrombus formation by amplifying the response of platelets to weak activating stimuli. These findings open new possibilities for the prevention of thrombosis by the development of MMP-2 inhibitors

Stimulation of Platelet Nitric Oxide Production by Nebivolol Prevents Thrombosis

Objective— dl -Nebivolol, a selective β1-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 β−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