Class III PI3K Positively Regulates Platelet Activation and Thrombosis via PI(3)P-Directed Function of NADPH Oxidase

Abstract

Objective— Class III phosphoinositide 3-kinase, also known as VPS34 (vacuolar protein sorting 34), is a highly conserved enzyme regulating important cellular functions such as NADPH oxidase (NOX) assembly, membrane trafficking, and autophagy. Although VPS34 is expressed in platelets, its involvement in platelet activation remains unclear. Herein, we investigated the role of VPS34 in platelet activation and thrombus formation using VPS34 knockout mice. Approach and Results— Platelet-specific VPS34-deficient mice were generated and characterized. VPS34 deficiency in platelets did not influence tail bleeding time. In a ferric chloride–induced mesenteric arteriolar thrombosis model, VPS34 −/− mice exhibited a prolonged vessel occlusion time compared with wild-type mice (42.05±4.09 versus 18.30±2.47 minutes). In an in vitro microfluidic whole-blood perfusion assay, thrombus formation on collagen under arterial shear was significantly reduced for VPS34 −/− platelets. VPS34 −/− platelets displayed an impaired aggregation and dense granule secretion in response to low doses of collagen or thrombin. VPS34 deficiency delayed clot retraction but did not influence platelet spreading on fibrinogen. We also demonstrated that VPS34 deficiency altered the basal level of autophagy in resting platelets and hampered NOX assembly and mTOR (mammalian target of rapamycin) signaling during platelet activation. Importantly, we identified the NOX-dependent reactive oxygen species generation as the major downstream effector of VPS34, which in turn can mediate platelet activation. In addition, by using a specific inhibitor 3-methyladenine, VPS34 was found to operate through a similar NOX-dependent mechanism to promote human platelet activation. Conclusions— Platelet VPS34 is critical for thrombosis but dispensable for hemostasis. VPS34 regulates platelet activation by influencing NOX assembly.