Stimulation of Platelet Nitric Oxide Production by Nebivolol Prevents Thrombosis

Abstract

H ypertension is associated with an increased risk of arterial thrombotic events, such as stroke and myocardial infarction, 1 in which platelets are deeply involved. Previous studies have shown that β-adrenoceptor blockers reduce platelet activation and aggregation, 2 but this does not seem to be a class effect because not all β-blockers share this property. 3 dl-Nebivolol (a racemic mixture of d-and l-nebivolol) is a third-generation β 1 adrenoceptor-selective antagonist that, besides its β-blockade-mediated hypotensive effect, possesses direct vasodilatory properties through a mechanism involving the l-arginine/nitric oxide (NO) pathway. 4,5 Of the 2 enantiomers forming the racemic mixture of dl-nebivolol, d-nebivolol, which has an ≈200-fold greater affinity for β 1 -adrenoceptors than that of l-nebivolol, seems to be mainly responsible of selective β-blockade, 6 whereas the l-form is the main effector of the endothelium-dependent vasorelaxant effect. 7,8 dl-Nebivolol exerts its vasodilatory action by activating endothelial nitric oxide synthase (eNOS) and by preventing eNOS uncoupling, thus enhancing NO production. 9 Endothelium-derived NO is a powerful inhibitor of platelet activation and an antithrombotic agent. 10,11 Besides endothelium, eNOS is present in other cells, including platelets, [12][13][14] and several in vitro and in vivo observations in animals and in humans suggest that platelet-derived NO plays a significant anti-inflammatory and antithrombotic role. [15][16][17][18][19] dl-Nebivolol was previously reported to inhibit ADP-and collagen-induced aggregation of human platelets in vitro, 20 an effect prevented by the previous exposure of platelets to a NOS inhibitor, such as L-NG-nitroarginine methyl ester, and enhanced by preincubation with l-arginine, the substrate of NOS, suggesting that the antiplatelet effect of dl-nebivolol is mediated by an enhanced release of NO from platelets. 4 © 2014 American Heart Association, Inc. 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 ...

Visualization of nitric oxide production by individual platelets during adhesion in flowing blood

“…16 In fact, it has been demonstrated that platelet-derived NO affects platelet adhesion at intermediate and high shear and that the production of NO in platelets is dominated by receptormediated interactions at low-intermediate shear and by mechanotransduction at high shear. 9,38,50 In our experimental conditions, platelet adhesion and thrombus formation onto a collagen surface under flow conditions take place in only a few minutes with the contribution and interplay of different receptors like GPIb-V-IX, GPVI, a 2 b 1 , the purinergic receptors P2Y 1 and P2Y 12 , and a IIb b 3 which, to a different extent, regulate the global increase of intracytoplasmic Ca 11 .…”

“…In agreement with this hypothesis, mice with only circulating platelets deficient of eNOS and with a normal endothelium showed a shortened bleeding time upon endothelial damage. 39 The preeminent role of platelet-instead of endothelium-derived NO in the antithrombotic activity of DL-nebivolol previously reported 12 further suggests that NO generated by platelets upon activation at an arterial damage site is more effective than that generated by endothelium-derived NO in preventing thrombosis, probably because it acts exactly where platelets become activated. 12 These different roles would explain why the endothelial NO response to shear must be an immediate and continuous reaction, whereas platelet NO production would occur only "on demand" and when the initial hemostatic process has already taken place.…”

“…9,10 It has been reported that platelet-derived NO plays a significant role in modulating platelet-thrombus formation in vivo. [11][12][13][14][15] Therefore, the generation of NO by platelets may act as a negative feedback mechanism to limit platelet activation after an aggregating stimulus and to reduce platelet recruitment to a growing thrombus. [11][12][13] However, because of the evanescent nature of NO and the very small amounts produced by platelets, estimated at about 5 3 10 217 mol/platelet (determined by microelectrode in platelet-rich plasma [PRP] stimulated for 2 minutes with 5 mM ADP), 16 several studies have questioned the pathophysiologic role of platelet-derived NO in regulating platelet activation and in vivo thrombus formation, [17][18][19] with a few even negating the presence of eNOS in platelets.…”

“…[11][12][13][14][15] Therefore, the generation of NO by platelets may act as a negative feedback mechanism to limit platelet activation after an aggregating stimulus and to reduce platelet recruitment to a growing thrombus. [11][12][13] However, because of the evanescent nature of NO and the very small amounts produced by platelets, estimated at about 5 3 10 217 mol/platelet (determined by microelectrode in platelet-rich plasma [PRP] stimulated for 2 minutes with 5 mM ADP), 16 several studies have questioned the pathophysiologic role of platelet-derived NO in regulating platelet activation and in vivo thrombus formation, [17][18][19] with a few even negating the presence of eNOS in platelets. 20 NO is a small free radical very much diffusible in the aqueous environment of blood and across cellular membranes, but it is highly reactive and has a very short half-life in the circulation, in the order of a few seconds; therefore it acts mainly as an autoacoid exerting its biological effects on the cells that have produced it or in their immediate proximity.…”

“…By using mouse models of platelet thrombus formation, contradictory data on the role of eNOS in limiting platelet activation were reported; in fact, although some reports did not observe a thrombotic phenotype in eNOS 2/2 mice, others described an enhanced thrombotic response. 12,[17][18][19]39 Recently it was documented that eNOS plays a role in the resolution of a platelet thrombus upon moderate to strong thrombotic stimulation. 53 In our experimental flow conditions on type I collagen, probably mimicking a rather intense thrombotic stimulus, the role of NO in controlling platelet deposition was confirmed by the significant increase of the collagen surface covered by platelets observed with blood from eNOS 2/2 mice compared with blood from wild-type mice.…”