Phosphorylated Pyk2 Inducing MCU-Dependent Mitochondrial Iron Overload Mediates ELABELA-Induced Thrombosis

Aim:ELABELA is an endogenous ligand for APJ receptor,which has been detected in many area,such as human embryos as well as the cardiovascular system.Several studies have found links between the ELABELA/APJ system and a variety of cardiovascular diseases,including eclampsia,hypertension,heart failure,etc.Moreover,preliminary experiments of the research group found that the ELABELA/APJ system is also closely related to platelet aggregation and thrombosis:ELABELA concentration-dependently promotes the occurrence of platelet aggregation and the formation of thrombus in vitro,while the APJ receptor inhibitor F13A and Gq protein inhibitor YM-254890 can significantly inhibit ELABELA’s promotion of platelet aggregation and in vitro thrombosis.This project intends to further explore the molecular mechanism that phosphorylated Pyk2 induces MCU-dependent mitochondrial iron overload,promotes the production of mt ROS and then mediates ELABELA-induced thrombosis.This subject provides the experimental and theoretical basis for APJ receptor which is expected to become a new target for the treatment of thrombotic diseases.Methods:1.Multi-ring thrombus detector detects the formation of external thrombus in New Zealand rabbits.2.Western Blot detects the protein expression in platelets of New Zealand rabbits（Pyk2,phosphorylated Pyk2,MCU and phosphorylated MCU）.3.Scepter^TM Cell Counter detects the diameter and volume of New Zealand rabbit platelet cells.4.Mito SOX^TM fluorescent probe detects the level of mt ROS in New Zealand rabbit platelets.5.RPA fluorescent probe to detect the level of mitochondrial iron in platelets of New Zealand rabbits.6.JC-1 fluorescent probe detects the changes of mitochondrial membrane potential（MMP）in New Zealand rabbit platelets.7.BCA method to detect the protein concentration in the extracted New Zealand rabbit platelets.Results:1.Pyk2 phosphorylation inhibitor PF-431396,MCU inhibitor Ru360,mitochondrial iron chelator Dexrazoxane and mitochondria-specific superoxide scavenger Mito-TEMPO all antagonize ELABELA induction.2.ELABELA does not affect the protein expression of Pyk2 and MCU,but it promotes the protein expression of phosphorylated Pyk2 and phosphorylated MCU in a concentration-dependent manner;APJ receptor inhibitor F13A and Gq protein inhibitor YM-254890 can both antagonize ELABELA-induced protein expression of phosphorylated Pyk2 and phosphorylated MCU.3.ELABELA promotes the increase in the diameter and volume of New Zealand rabbit platelet cells,but APJ receptor inhibitor F13A,Gq protein inhibitor YM-254890,Pyk2 phosphorylation inhibitor PF-431396,MCU inhibitor Ru360,mitochondrial iron chelator Dexrazoxane and Mito-TEMPO,a mitochondria-specific superoxide scavenger,all can antagonize the increase in diameter and volume of New Zealand rabbit platelet cells induced by ELABELA.4.ELABELA promotes the increase of mitochondrial iron levels in New Zealand rabbit platelets,but APJ receptor inhibitor F13A,Gq protein inhibitor YM-254890,Pyk2 phosphorylation inhibitor PF-431396and MCU inhibitor Ru360 all antagonize ELABELA-induced New Zealand Increased mitochondrial iron level in rabbit platelets.5.ELABELA promotes the increase of mitochondrial reactive oxygen species in New Zealand rabbit platelets,but APJ receptor inhibitor F13A,Gq protein inhibitor YM-254890,Pyk2 phosphorylation inhibitor PF-431396,MCU inhibitor Ru360 mitochondrial iron chelator Dexrazoxane are all antagonize ELABELA-induced increase of mt ROS in platelets.6.ELABELA reduces the New Zealand rabbit platelets’MMP,but APJ receptor inhibitor F13A,Gq protein inhibitor YM-254890,Pyk2phosphorylation inhibitor PF-431396,MCU inhibitor Ru360,mitochondrial iron chelator Dexrazoxane and mitochondria-specific superoxide scavenger Mito-TEMPO all antagonized the changes of MMP in New Zealand rabbit platelets induced by ELABELA.Conclusion:Phosphorylated Pyk2 inducing MCU-dependent mitochondrial iron overload mediates ELABELA-induced thrombosis.