NR4A1 Acts as a Novel Regulator of Platelet Activation and Thrombus Formation

BACKGROUND: Mounting evidence indicates that nuclear receptors play a critical regulatory role in platelet pathophysiology and thrombotic disorders. Although NR4A (the nuclear receptor subfamily 4 group A) plays an important role in cardiovascular pathophysiology, the expression profile and biological function of NR4A member 1 (NR4A1) in platelets have never been reported. METHODS: We evaluated the functions and the underlying mechanisms of NR4A1 in platelet activation and thrombus formation using platelet-specific NR4A1-deficient mice and NR4A1-specific agonists. Using a hyperlipidemic mouse model and platelets from patients with hypercholesterolemia, we explored the influence of hypercholesterolemia on NR4A1 expression and the effects of NR4A1-specific agonists on platelet hyperreactivity induced by hypercholesterolemia. RESULTS: NR4A1 was expressed in both human and mouse platelets. Platelet-specific NR4A1 deletion accelerated FeCl 3 -induced carotid arterial occlusive thrombus formation, enhanced collagen/epinephrine-induced pulmonary thromboembolism, and exacerbated microvascular microthrombi obstruction and infarct expansion in an acute myocardial infarction model. NR4A1-deficient platelets exhibited enhanced agonist-induced aggregation responses, integrin α IIb β 3 activation, dense granule release, α-granule release, platelet spreading, and clot retraction. Consistently, pharmacological activation of NR4A1 by specific agonists decreased platelet activation in both mouse and human platelets. Mechanistically, CAP1 (adenylyl cyclase-associated protein 1) was identified as the direct downstream interacting protein of NR4A1. NR4A1 deletion decreased cAMP levels and phosphorylation of VASP (vasodilator-stimulated phosphoprotein), while NR4A1-specific agonists increased cAMP levels and phosphorylation of VASP in platelets. Importantly, NR4A1 expression in platelets was upregulated in the setting of hypercholesterolemia, which was derived from its upregulation in megakaryocytes in a reactive oxygen species-dependent manner. Platelets from hypercholesterolemic patients and mice exhibited hyperreactivity. However, NR4A1-specific agonists significantly inhibited the activation of hypercholesterolemic platelets to the levels of healthy control platelets. CONCLUSIONS: We provide the first evidence that nuclear receptor NR4A1 negatively regulates platelet activation and thrombus formation. NR4A1 may serve as a novel therapeutic target for managing thrombosis-based cardiovascular diseases, especially with hypercholesterolemia.