Pannexin1-P2X7 And Autophafy Pathway Mediates The Effects Of Different Subtypes Apelin And ELABELA On Rabbits Platelet Aggregation

Aim:Apelin and ELABELA are APJ endogenous ligand. This article studied the effect of apelin subtypes apelin-12、apelin-13、apelin-17、apelin-36 and ELABELA on rabbits platelet aggregation. And investgated whether pannexin1(Panx1)-P2X7 pathway mediated apelin-12 、apelin-17、apelin-36、ELABELA in promoting rabbits platelet aggregation; besides, we also searched whether autophagy pathway mediated the inhibition effect of apelin-13 on platelet aggregation. Overall, thesefindings find new biological function of apelin subtypes and ELABELA. In addition, this study also provided a new theoretical basis and drug screening targets for the mechanism of thrombotic diseases.Methods: 1、Determination of platelet aggregation of rabbit platelet rich plasma in turbidimetry.2、Western blot detected the expression of proteins.Results:1、Apelin-12(1*10-7~1.0μM)induced rabbits platelet aggregation in a dose dependent manner,themaximum aggregation ratiois51.5±6.6(%),the median effective concertration(EC50) is 0.03981*10-3μM and the time to peak is 36.3 ±15.6(s), the peak rate is 3.9 ±1.4(%/s).2、Apelin-17( 1*10-9~0.01 μ M) induced rabbits platelet aggregation in a dose dependent manner, themaximum aggregation ratiois50.08 ±4.468(%),the EC50 is 0.0014*10-3μM, and the time to peak is(26.1 ±7.1),the peak rate is 2.9 ±0.9(%/s);3、Apelin-36(1*10-7~1.0μM)induced rabbits platelet aggregation in a dose dependent manner,themaximum aggregation ratiois45.8 ±2.7(%),the EC50 is 0.18*10-3μMand the time to peak is22.9 ±6.8(s),the peak rate is 3.1 ±0.8(%/s);4、ELABELA( 0.001~1.0 μ M) induced rabbits platelet aggregation in a dose dependent manner,themaximum aggregation ratiois46.7 ±0.9(%),the EC50 is 0.028μM and the time to peak is 31.7 ±13.8(s),the peak rate is 3.6 ±1.1(%/s);5、Apelin-13( 0.001~1.0 μ M) inhibited rabbits platelet aggregationinducedby ADP,the inhibiton effective concertration(IC50) is 0.0219μM;However, apelin-13 have no effect on collagen、thrombin and AA induced rabbits platelet aggregation;6、Apelin-12 promoted ADP and thrombin induced rabbits platelet aggregation,however have no effect on collagen and AA induced rabbit platelet aggregation.7、Apelin-17 did not influence rabbits platelet aggregation induced by AA、ADPand collagen, however increased thrombin induced rabbits platelet aggregation8、Apelin-36 have no effect on ADP 、collagen、thrombin、AA and induced rabbit platelet aggregation.9、ELABELA promoted ADP、AA and collagen induced rabbits platelet aggregation, but have no effect on thrombin induced rabbits platelet aggregation.10、APJ blocker F13 A blocked apelin-12, apelin-17, apelin-36. ELABELA induced rabbits platelet aggregation and reversed the inhibited effect of apelin-13 on ADP induced rabbits platelet aggregation.11、Pannexin1 antagonists probenecid and carbenoxolone significantly inhibited apelin-12、apelin-17、apelin-36 and ELABELA induced rabbits platelet aggregation.12、P2X7 antagonists Brilliant blue G inhibited apelin-12、apelin-17、apelin-36 and ELABELA induced rabbits platelet aggregation.13、Autophagy agonists rapamycin blocked the inhibition effect of apelin-13 on ADP induced rabbits platelet aggregation and 3MA enhanced the inhibition effect.14、Apelin-13 had no effect on apelin-12、apelin-17、apelin-36 alone or their mixture induced rabbits platelet aggregation.Conclusion:1、Apelin subtypesapelin-12, apelin-17, apelin-36 and ELABELAinduced rabbits platelet aggregation; Apelin-13 inhibited rabbits platelet aggregation in dose dependent manner.2、Pannexin1-P2X7 pathway mediated apelin-12, apelin-17, apelin-36 and ELABELA induced platelet aggregation; Autophagy pathway regulated inhibition effect of apelin-13 on ADP induced rabbits platelet aggregation.