The Role Of ERK5 In The Glycoprotein Ib-?-mediated Platelet Activation

Background:Platelets,which are derived from megakaryocytes,circulate in mammalian blood and play essential roles in thrombus,hemostasis,angiogenesis,inflammation,tumor growth and metastasis.Abnormal activation of platelets can lead to thrombotic diseases,including arterial thrombosis(myocardial infarction),venous thrombosis(deep vein thrombosis)and microvascular thrombosis(DIC),which are serious threats to life and health.The glycoprotein(GP)Ib-IX complex,which belongs to LRR family,not only mediates platelet adhesion but also transmits signals leading to platelet aggregation and activation.GPIb-IX signal transduction includes Src,PI3K,MAPKs and multiple amplification mechanisms.Mitogen-activated protein kinases(MAPKs)ERKI/2 and p38 have been shown to be important in GPIb-IX-mediated signaling leading to integrin activation.However,the roles of a MAPK extracellular signal-regulated kinase 5(ERK5)in GPIb-IX-mediated platelet activation remain unknown.Objective:To reveal the function and mechanisms of ERK5 in GPIb-IX-mediated platelet activation.Methods:The functions of ERK5 in GPIb-IX-mediated human platelet activation were assessed using botrocetin/NWF,ristocetin/VWF or platelets adhesion to VWF under shear stress in the presence of ERK5 specific inhibitor.ERK5 associated proteins were pull-down and identified by mass spectrometry from Chinese hamster ovary cell transfected with HA tagged-ERK5 and were confirmed in human platelets.Roles of ERK5 associated proteins in GPIb-IX-mediated platelet activation were clarified using specific inhibitors.Results:The phosphorylation levels of ERK5 were significantly enhanced in human platelets stimulated by botrocetin/VWF or ristocetin/VWF.ERK5 inhibitor XMD8-92 suppressed the second wave of human platelet aggregation induced by botrocetin/VWF or ristocetin/VWF,and inhibited human platelet adhesion on immobilized VWF under shear stress.Casein kinase ?(CKII)was identified as an ERK5-associated protein in human platelets.CKII inhibitor TBB,similar to ERK5 inhibitor XMD8-92,specifically restrained PTEN phosphorylation,therefore suppressed Akt phosphorylation in human platelets treated with botrocetin/VWF.Conclusion:ERK5 associates with CKII to play essential roles in GPIb-IX-mediated platelet activation via PTEN/PI3K/Akt Pathway.Others:ERK5 signaling is an agonist-specific signaling pathway in platelets.We tested the effect of ERK5 inhibitor on platelet activation induced by other agonists,such as ADP,collagen,thrombin,and U46619.Results showed that ERK5 take part in platelet aggregation induced by thrombin and U46619,but not by ADP and collagen.Besides,data showed that ERK5 inhibitor suppressed platelet spreading on immobilized Fg.In future,more studies are needed to clarify mechanisms of ERK5 in other platelet agonists.