Abstract-Endothelial nitric oxide synthase (eNOS) has been identified in human platelets. Although platelet-derived nitric oxide (NO) has been shown to inhibit platelet recruitment in vitro, its role in the regulation of the hemostatic response in vivo has not been characterized. To define the role of platelet-derived NO in vivo, we studied mice that lacked a functional eNOS gene (NOSIII). Surface P-selectin expression in platelets from eNOS-deficient mice was not significantly altered; however, bleeding times were markedly decreased in eNOS-deficient versus wild-type mice (77.2Ϯ3 versus 133.4Ϯ3 seconds, PϽ0.00005). To determine the contribution of endothelium-versus platelet-derived NO to the bleeding time, isolated platelets from either eNOS-deficient or wild-type mice were transfused into a thrombocytopenic eNOS-deficient mouse and the bleeding time was measured. The bleeding times in mice transfused with eNOS-deficient platelets were significantly decreased compared with mice transfused with wild-type platelets (⌬bleeding time, Ϫ24.6Ϯ9.1 and Ϫ3.4Ϯ5.3 seconds, respectively; PϽ0.04). Platelet recruitment was studied by measuring serotonin release from a second recruitable population of platelets that were added to stimulated platelets at the peak of NO production. There was 40.3Ϯ3.7% and 52.0Ϯ2.1% serotonin release for platelets added to wild-type or eNOS-deficient platelets, respectively (PϽ0.05). In summary, mice that lacked eNOS had markedly decreased bleeding times even after endothelial NO production was controlled. These data suggest that the lack of platelet-derived NO alters in vivo hemostatic response by increasing platelet recruitment. Thus, these data support a role for platelet-derived NO production in the regulation of hemostasis. (Circ Res. 1999;84:1416-1421.)Key Words: selectin Ⅲ mice Ⅲ platelet Ⅲ nitric oxide synthase N ormal hemostatic balance is maintained by tight regulation of platelet activation and recruitment. Adhesion of platelets to the endothelium is prevented by several mechanisms, including endothelial cell production of nitric oxide (NO). 1,2 NO inhibits platelet adhesion and aggregation 3,4 and prevents thrombosis. 5 Exogenous NO inhibits the normal activation-dependent increase in the expression of platelet surface glycoproteins, including P-selectin and the integrin glycoprotein IIb/IIIa complex. 6 In addition, we have shown that a functional decrease in exogenous NO can lead to a clinical thrombotic disorder. 7,8 In addition to its presence in endothelial cells, constitutive NO synthase (NOS) has been identified in human platelets and megakaryocytic cells. 9 -12 Consistent with these observations, studies report NO release from aggregating platelets. [13][14][15] Platelet aggregation is enhanced by incubation with inhibitors of NOS and inhibited by incubation with the NOS substrate L-arginine. 16 Although platelet-derived NO appears to inhibit the primary aggregation response only modestly, we have recently shown that NO release from activated human platelets markedly inhibits ...