PI3-kinase Utilizes RhoA-mDia1 Pathway To Mediate The Thrombin-induced Actin Cytoskeleton Remodelling In Platelets

Backgroung and Objectives: Platelets are non-nuclear disc cells. Agonists such as adenosine thrombin can activate GlycoproteinαⅡbβ3 (GPαⅡbβ3) on the surface of platelets("inside-out"Pathway).Activated GpαⅡbβ3 has high-affinity to bind to ligands such as fibrinogen (Fg), and facilitate platelet aggregation or spreading on immobilized Fg. The dynamia of Platelet spreading and aggregation are actin cytoskeleton remodelling. Phosphatidylinositol 3 kinase (PI3K) play important roles in platelet GPαⅡbβ3activation. But the downstream mechanisms mediating their functions in activated GpαⅡbβ3 induced actin cytoskeleton remodelling ("outside-in"pathway)are unclear.mDia1 is a mammalian homologue of Drosophila diaphanous and a member of the forminhomology family of proteins. mDia1 is a cytoskeleton remodeling protein that regulates cellular cytoskeleton remodeling processes such as cytokinesis, polarity, motility, stress fiber formation and neurite outgrowth. mDia1, functioning as downstream of a small GTPase RhoA, regulates actin cytoskeleton remodeling through its interacting with actin polymerizing protin profilin. Given its demonstrated effects on the cytoskeleton in nucleated cells, little is known about their physiological roles in platelets. Therefore, the aim of this study was to determine the expression and subcellular localization of mDia1 in platelet and the role of mDia1 or PI3K in the process of thrombin-induced platelet actin cytoskeleton remodelling such as spreading and aggregation.Methods:①The expression and subcellular localization of mDia1 in platelets, the changes of platelet morphology or actin cytoskeleton were measured by immunofluorescence.②The extent of platelet aggregation was measured in optical density by the platelet aggregation system.③The expression of mDia1 and the relation with F-actin in quiescent, spreading or aggregated platelets was measured by Western blot.④S mall GTPase activation of RhoA,Rac1 and Cdc42 in platelets in different states was measured by GST pull-down assay.⑤T he proteins among RhoA,Rac1 and Cdc42 interacted with mDia1 in platelets were measured by immunoprecipitation.Results:①mDia1 expressed in quiescent or activated platelets, and the expression level of mDia1 in platelets was not different.②mDia1 and F-actin were colocalized in cytosol of spreading platelets.③mDia1 moved from a Triton-X100-soluble cytosolic fraction to insoluble cytoskeleton fraction after platelets aggregation stimulated by thrombin.④Anti-mDia1 antibody inhibited the thrombin induced spreading of platelets seeded on Fg coated coverslips and platelet aggregation.⑤PI3K inhibitor wortmannin inhibited platelet aggregation agitated by thrombin and inhibited the mDia1 translocation process from Triton-X100-soluble cytosolic fraction to insoluble cytoskeleton fraction.⑥PI3K inhibitor wortmannin inhibited platelet spreading agitated by thrombin and inhibited the mDia1 colocalizing with F-actin.⑦Thrombin elevated the activation of RhoA and the binding of RhoA with mDia1 during the progression of thrombin induced platelet aggregation and spreading on Fg coated coverslips, and wortmannin inhibited the rising of RhoA activation and the level of RhoA binding with mDia1 induced by thrombin.⑧Thrombin elevated the activation of Rac1 and Cdc42 during the progression of thrombin induced platelet aggregation, but could not induced Rac1 or Cdc42 binding with mDia1.and wortmannin could not inhibite the rising of Rac1 and Cdc42 activation induced by thrombin.Conclusion:①RhoA-mDia1 pathway mediates the thrombin-induced actin cytoskeleton remodelling in platelets.②PI3-kinase functions as the upstream of RhoA-mDia1 pathway.