Platelet Surface Receptor CD40 Regulates Platelet Activation Through CD40-TRAF6 Signaling

【Background】Platelets play a key role in the pathophysiological mechanisms of atherogenesis in the circulation as key cells in inflammatory and immune processes.Activation of CD40 receptors expressed on the plasma membrane surface can interact with endothelial cells,macrophages,leukocytes,neutrophils and other key cells involved in the formation of atherosclerosis and promote atherosclerosis and thrombosis.Our group’s previous study found that the Chinese herbal medicine Allium sativum saponin could play a superior role in inhibiting platelet activation and expression of inflammatory mediators by blocking CD40 receptors,and could inhibit the expression of inflammatory mediators in endothelial cells.Thus,CD40 has an important role in platelet activation and expression of inflammatory mediators by a mechanism that has not been fully defined.In recent years,the invention of small molecule inhibitors has opened a new chapter in anti-atherosclerosis,and small molecule inhibition of CD40-TRAF6 pathway signaling is effective in reducing atherosclerosis in mice,and there is increasing evidence that the CD40-TRAF6 pathway is a key factor in the pathogenesis of atherosclerosis.The critical role of the CD40-TRAF6 pathway in disease is evident,while its regulation of platelets has not been investigated.The aim of this study was to investigate the regulatory relationship between CD40-TRAF6 signaling and its activation and function within platelets,with the aim of finding new targets for antiplatelet therapy.【Methods】1.The cell precipitates obtained by gradient centrifugation were labeled with membrane proteins using flow antibody CD41a-PECy7 and identified by flow cytometry to ensure the purity of the obtained platelet precipitates and facilitate subsequent experiments.2.Using sCD40 L to induce platelet activation for modeling,platelets were incubated with concentration gradient and time gradient and the protein expression of TRAF6 and CD62 P was detected by Western blot technique to explore the relationship between the changes of TRAF6 induced by sCD40 L activation of platelet membrane receptor CD40 and its activation index CD62 P.3.Based on the sCD40L-induced platelet activation model,the effect of TRAF6 inhibition using different treatments(SMI6877002 inhibited the TRFA6 binding site on CD40,NBP2-26506 inhibited TRAF6 with binding to RANK)was investigated using Western blot to explore the effect of TRAF6 on sCD40 L activation of CD40 receptor The regulatory role of CD62 P induced by sCD40 L.4.Based on the sCD40L-induced platelet activation model,the regulation of TRAF6 on platelet membrane protein CD62 P and CD63 expression in the absence or inhibition of TRAF6 was further investigated using flow cytometry.5.Based on the sCD40L-induced platelet activation model,the regulation of TRAF 6on platelet mitochondrial ATP and ROS production without or with inhibition was investigated using a kit assay.6.Based on the sCD40L-induced platelet activation model,the regulation of TRAF 6on platelet mitochondrial membrane potential without or with inhibition was investigated by flow cytometry.7.Based on the sCD40L-induced platelet activation model,Western blot was used to study the regulation of the PI3K/AKT/eNOS signaling pathway in platelets by TRAF6 without or with inhibition.8.The interaction between TRAF6 and PI3 K,an upstream signaling molecule of PI3K/AKT/eNOS signaling pathway,was investigated by immunoprecipitation.【Results】1.sCD40 L upregulated platelet CD62 P and TRAF6 in a concentration gradient and temporal gradient after activation of platelet CD40 receptors.2.Inhibition of TRAF6 reverses the up regulation of CD62 P and CD63 expression in platelets induced by activation of CD40 receptors.3.Inhibition of TRAF6 inhibits the activation of CD40 receptor-induced mitochondrial ATP and ROS production in platelets.4.Inhibition of TRAF6 inhibited the activation of CD40 receptor-induced elevation of mitochondrial membrane potential in platelets.5.Inhibition of TRAF6 inhibits activation of CD40 receptor-induced up regulation of PI3K/AKT/eNOS signaling pathway protein expression in platelets.6.TRAF6 interacts with PI3 K,and thus inhibition of TRAF6 inhibits activation of PI3K/AKT/eNOS signaling pathway in platelets induced by activated CD40 receptors,thereby regulating platelet activation and mitochondrial function.【Conclusion】TRAF6 can regulate platelet activation and mitochondrial function induced by CD40 receptor activation through PI3K/AKT/eNOS signaling pathway.