Objective-Platelet activation is a feature of cardiovascular disease that is also characterized by endothelial dysfunction.The direct relationship between impaired endothelium-derived NO bioavailability and platelet activation remains unclear. We investigated whether acute inhibition of NO production modulates platelet activation in mice and whether specific rescue of endothelial function in diabetes modifies platelet activation. Methods and Results-Intravenous injection of the NO synthase inhibitor N G -nitro-L-arginine methyl ester in wild-type (WT) mice significantly reduced platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation and increased platelet surface expression of P-selectin, CD40 ligand, and fibrinogen platelet binding, demonstrating that NO production exerts tonic inhibition of platelet activation in mice. Diabetes was induced by streptozotocin injection in WT or endothelial-targeted guanosine 5Ј-triphosphate cyclohydrolase I (GCH)-transgenic (GCH-Tg) mice protected from endothelial dysfunction in diabetes by sustained levels of tetrahydrobiopterin in vascular endothelium. Platelet VASP phosphorylation was significantly reduced in diabetic WT but not in diabetic GCH-Tg mice. P-selectin, CD40 ligand expression, and fibrinogen binding were increased in diabetic WT mice but remained unchanged compared with controls in endothelial-targeted GCH-Tg mice. Key Words: diabetes Ⅲ endothelial nitric oxide synthase Ⅲ platelets Ⅲ endothelial dysfunction T he endothelium plays a crucial role in control of vascular tone by releasing endothelium-derived autocoids, the most important of which is NO, 1 generated by endothelial NO synthase (eNOS). NO also inhibits platelet activation, adhesion and aggregation; reduced NO bioactivity is associated with arterial thrombosis in animal models and in individuals with endothelial dysfunction. 2 The importance of NO in platelet function was shown by in vitro experiments such as inhibition of platelet aggregation by endothelial cells 3 or NO donors. 4,5 NO formation inhibition causes platelet activation, 6 and exogenous NO can even reverse agonist-induced activation of platelet glycoprotein (GP) IIb/IIIa. 7 The finding that platelets adhere to dysfunctional endothelium and that expression of potential adhesion molecules is enhanced under these conditions suggests that NO may modulate platelet activation in vascular disease states. 8 We demonstrated recently that inhibition of systemic NO formation in healthy humans rapidly induces platelet activation, an effect that could be reversed by exogenous NO. 6 Indeed, increased platelet activation is observed in diseases characterized by chronic endothelial dysfunction such as acute coronary syndromes, 9 congestive heart failure, 10,11 diabetes, 12,13 and hypercholesterolemia. 14 In patients with advanced atherosclerosis, impaired endotheliumdependent release of NO leads to reduced platelet cGMP formation. 15 It remains uncertain whether endothelial-derived NO has a more important role in tonic suppression of platelet ...