RNF8 Regulates Platelet Activation Through Mediating WDR1 Ubiquitination

Platelet is an important cell that regulates physiological hemostasis in the body,and abnormal platelet function will lead to corresponding bleeding or thrombotic diseases in the body.Therefore,it is very important to explore the molecular mechanism of platelet activation for the treatment of diseases with abnormal coagulation function.Platelet activation is closely related to cytoskeleton dynamics.Previous studies have suggested that WDR1 is an important regulator of ADF/cofilin depolymerization factor and promotes ADF/cofilin mediated depolymerization of F-actin.F-actin activates platelets,causing them to assemble and activate,leading to the formation of microclots that block blood vessels.It affects the dynamic changes of actin cytoskeleton,plays an important role in inhibiting platelet activity,and participates in the occurrence of platelet mediated abnormal bleeding and coagulation and other related diseases.The post-translational modification of proteins is the precondition for the normal function of proteins.Ubiquitin modification is an important type of post-translational modification to maintain the normal level and activity of proteins.Some of the substrate proteins were degraded by the proteasomal pathway after ubiquitination,while the other proteins were modified by ubiquitination to change their active state.WDR1 can be dephosphorylated by ETA3,resulting in abnormal intracellular actin production,thus affecting cytoskeleton dynamics.However,the ubiquitin modification of WDR1 and its effect on platelet biological function are still unclear.RNF8 is an E3 ubiquitin ligase with RING domain.In this study,we speculated that RNF8 could modify WDR1 through ubiquitination and regulate its degradation,thereby participating in the dynamic change of platelet actin skeleton,and thus affecting platelet function.In this study,the ubiquitin ligase RNF8 gene knockout mice were used to supplement the ubiquitin modification of WDR1 and the effect of RNF8 on platelet activity.The results showed that:(1)The number of platelets in RNF8 knockout mice decreased significantly.(2)Thromboelastography showed that the MA value of platelets in RNF8 knockout mice decreased,suggesting abnormal platelet aggregation function.(3)Transmission electron microscopy of platelets extracted without activation inhibitors showed that the number of dense particles in the platelets of RNF8-/-mice increased,suggesting that the secretion of dense particles decreased,and the activation of platelets may be abnormal.(4)The tail bleeding test showed that the tail bleeding time of RNF8^-/-mice was prolonged and the amount of bleeding was increased.(5)In an in vitro thrombosis experiment using whole blood microperfusion to simulate blood flow in vivo,it was found that the stability of platelet thrombosis on the collagen surface of RNF8^-/-mice was significantly reduced under the simulated constant flow of circulating blood.Meanwhile,the static adhesion test showed that the same number of platelets binding collagen in RNF8^-/-mice reduced the number of platelet adhesion,that is,decreased platelet adhesion ability.(6)The results of HE staining of bone marrow showed that the number of bone marrow megakaryocytes in RNF8^-/-mice was compensatory increased,which was consistent with the results of mass spectrometry.(7)Through phenotypic screening and cross-omics analysis,we found that deletion of RNF8 could lead to up-regulation of WDR1 expression,while down-regulation of ubiquitination.WDR1 protein level was increased due to RNF8deficiency in Western blotting.Molecular docking simulation showed that RNF8 and WDR1 had potential interaction sites.The results of immunoprecipitation showed that there was interaction between RNF8 and WDR1.Immunoprecipitation assay showed a decreased level of total ubiquitination of WDR1 in RNF8^-/-mice.In conclusion,RNF8 is an important regulatory protein involved in platelet activation.The deletion of RNF8 leads to a decrease in the number of platelets in peripheral blood of mice,an increase in tail bleeding time,a decrease in platelet adhesion ability and a decrease in the release of compact particles.The mechanism of action is that RNF8 regulates cofilin and its downstream actin skeleton kinetics by ubiquitination modification of WDR1.Affects platelet activation and function.