Platelet Shp2 Negatively Regulates Thrombus Stability Under High Shear Stress

Platelets are important regulatory cells for physiological hemostasis and arterial thrombosis,and the insufficient or excessive activation of platelets can lead to severe bleeding and thrombotic diseases.Platelets activation are balanced by its own positive and negative signal transduction,the exploration of molecular mechanism of platelet activation will further elucidate the mechanisms of bleeding or thrombosis and provide basis for new therapeutic targets.The formation and stability of arterial thrombosis are influenced by many factors such as blood flow,platelet activation and coagulation factor.Platelets are the key cells in arterial thrombosis,leading to the development and stabilization of thrombosis.The adhesion,aggregation and secretion of platelets affect the formation and stability of thrombosis.Previous studies have suggested that platelet adhesion receptors(GPIb-V-IX,GPVI,integrin ?IIb?3)mediate platelets adhered to the damaged vascular endothelial matrix.Activated platelets promote platelet activation by secreting physiological stimulants such as,ADP,thromboxane A2(TXA2),thrombin.Integrin ?IIb?3 has been reported to regulate platelet secretion,aggregation,thrombosis formation.However,there is little knowledge about how ?IIb?3 combine with physiological stimulants dynamicly regulate thrombus stability.Shp2 is a tyrosine phosphatase encoded by PTPN11 gene with two SH2 domains,and plays an important biological role in the response of multiple growth factors,hormones and cytokines.PTPN11 gene mutations lead to Noonan syndrome and Leopard syndrome,and those patients suffered from severe bleeding and bruising.Previous studies suggest that Shp2 combined with the ITIM motif of PEC AM1 regulated GPVI signaling pathways,Shp2 knockout platelet showed enhanced spreading on fibrinogen surface,however,the specific mechanism of Shp2 regulation of platelet activation is unclear,we hypothesized that Shp2 regulate integrin ?IIb?3 mediated platelet secretion,and participate in thrombosis formation and stability.In this study,by using megakaryocyte/platelet Shp2(Shp2-/-)specific deficient mice,we investigate the role of Shp2 in platelet activation and thrombosis stability.Results can be summarized as the following:(1)In a mesenteric arterial thrombosis induced by ferric chloride,the arterial thrombosis obstruction time in Shp2-/-mice was comparable with wild type(WT)mice,but the stability of thrombus significantly increased,the number of unstable emboli shed from thrombosis was reduced.(2)An in vitro microfluidic whole-blood perfusion assay was performed to mimic the complicated shear rate during arterial thrombosis formation,as data shows in a pathological shear rate(3000 s-1),thrombi formed on collagen surface by Shp2-deficient platelets were more stable;In thrombosis stability experiments,Shp2 deficiency enhanced the capacity of platelet to against shear stress,demonstrating that Shp2 negative regulates arterial thrombosis stability especially in pathological shear rate conditions.(3)Shp2 deficiency did not alter the platelet responsiveness towards thrombin-,ADP-,and collagen-stimulation,Shp2-/-platelets displayed enhanced dense granule secretion when stimulated by TXA2 analogue U46619.And the enhanced second-wave secretion was specific to TXA2/TP signaling pathways.The inhibition of P2Y12 receptor and integrin ?IIb?3 eliminated the enhanced dense granules in Shp2-/-platelet induced by U46619 stimulation.(4)Moreover,Shp2-/-platelet showed enhanced spreading on fibrinogen,when ADP was hydrolyzed by apyrase,the spreading difference between Shp2-/-platelet and WT platelet were eliminated.Therefore,Shp2 negatively regulates the function of platelet secretion mediated by integrin outside-in signaling,and the deficiency of Shp2 leads to enhanced ADP secretion and spread over fibrinogen.(5)Shp2 was found operating downstream of integrin ?Ilb?3 outside-in signaling,functioning through inhibiting the phosphorylation of Akt.Calmodulin was identified to bind both Shp2 and Akt and serve as a key molecule linking Shp2 to Akt activation.Calmodulin inhibitor W-7 or Akt inhibitor SH-6 effectively eliminated the secretion differences between Shp2-1-platelet and WT platelet in the stimulation of U46619.Hence,Shp2 through Calmoudlin regulate Akt activation,which is involved in integrin outside-in signaling and platelet secretion.To sum up,platelet Shp2 may not be critically involved in the initiation and propagation of thrombus but specifically regulates the perpetuation of thrombus by integrating and fine-tuning the effects of ?IIb?3 signaling,secondary generated agonists,and rheological change.