Vacuolar Protein Sorting 34 (VPS34) Is Involved In Platelet Activation And Thrombosis Formation

Circulating platelets is the key regulatory cells for physiological hemostasis.Excessive activation of platelets may cause pathological thrombosis and lead to life-threatening diseases such as myocardial infarction and stroke.On activation,platelets undergo cytoskeletal reorganization,calcium mobilization,granule content release,adhesion,and aggregation through integrin ??b?3 activation and eventually form thrombi.A wide array of these platelet activation events are subject to the regulation of phosphoinositide 3-kinases(PI3Ks),which are responsible for producing the second messengers D3-phosphorylated phosphoinositides.Up to now,there are eight mammalian PI3K isoforms separated into three subtypes according to their substrate specificity,structure and functional homologies.Class I PI(3)Ks possess a well-defined role in platelets activation by producing PtdIns(3,4,5)P3 from Ptdlns(4,5)P2;Class ?PI(3)Ks convert PtdIns(4)P or PtdIns to PtdIns(3,4)P2 or PtdIns(3)P.Platelets expressed PI3KC2? and PI3KC2?,and PI3KC2a is reported to regulate platelets membrane structure,especially normal open canalicular system formation;Vacuolar protein sorting(VPS)34 is the only member of class? PI3K,which phosphorylate Ptdlns to PtdIns(3)P.We reported the role of VPS34 in hemostasis and thrombosis for the first time.VPS34 in platelets is important for thrombosis but dispensable for hemostasis,indicating VPS34 may have the potential to become a promising target to improve antithrombotic strategy.We have found VPS34 positively regulate platelets function byNADPH oxidase directed ROS generation.However VPS34 also participate in mTOR signaling activation,membrane trafficking and autophagy.Whether these biological process is responsible for reduced platelets activity in VPS34 deficient platelets remain obscure.And how traditional signal pathway in platelets is linked to VPS34 is also unknown.In this study,we investigate how the upstream and downstream of VPS34 cooperate to regulate platelets activation and thrombosis formation.Elucidating the functions of Class ? PI3Ks in platelets may provide essential information for the regulation mechanisms of thrombosis and hemostasis.We identified our previous finding:platelets specific VPS34 deletion mice showed prolonged arterial thrombosis formation,but normal tail bleeding time.VPS34-/-platelets displayed an impaired aggregation and dense granule secretion in response to low doses of collagen or thrombin,higher dose of agonist could abolish the aggregation difference between VPS34 deficient and WT platelets.VPS34 deficient platelet showed delayed clot retraction but normal spreading area on immobilized fibrinogen.VPS34 deficiency in platelets hampered NADPH Oxidase(NOX)assembly during platelet activation.NOX-dependent reactive oxygen species(ROS)generation was identified as the major downstream executor of VPS34 to mediate platelet activation.We focus on VPS 3 4 regulated mTOR signaling and platelet autophagy in the subsequent study.We 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.Characterization of signaling events revealed VPS34 deficiency altered basal level of autophagy in resting platelets.mTOR(mammalian target of rapamycin)is serine/threonine protein kinase,and is a member phosphatide inositol related kinases.It exerts extracellular energy and nutrient signal to the execution of cell growth and division,owing to the ability of TOR protein kinase to sense energy,nutrients and stress.mTOR signaling is impaired in VPS34-/-platelets,however rapamycin and PP242 do not inhibit platelets aggregation,nor NADPH oxidase assemble.It indicates VPS34 regulates platelets activation in an mTOR independent manner.In addition,basal autophagy flux is inhibited by VPS34 deficiency in platelets,however autophagy is not induced upon platelets activation.Atg7 or Beclinl(key regulator of autophagy)deficient mice platelets showed significantly reduced platelets aggregation,indicating autophagy may regulate platelets activation in an unknown manner.Rubicon is a negative regulator for VPS34 activation.In order to further study VPS34 associated network of thrombosis and hemostasis,platelets-specific Rubicon deficiency mice were generated and characterized.Rubicon-/-platelets showed enhanced reactivity in response to collagen,while reduced thrombosis stability under shear with normal ROS production.These data indicate VPS34 may regulate thrombosis stability by interacting with Rubicon.In summary,VPS34 is a pivotal positive regulator of platelet activation and thrombosis formation.And VPS34 regulates mTOR signaling and basal level autophagy in platelets,which is not responsible for platelets activation.Enhancing VPS34 activity by Rubicon knock out leads to platelet hyper-reactivity and unstable thrombosis.