| Aim: Apelin is an endogenous ligand for the G protein coupled receptor APJ.Apelin is widely expressed in the cardiovascular system and plays important roles such as regulation of blood pressure,contraction of the myocardium,proliferation of vascular smooth muscle,and production of blood vessels.Previous studies in the research group also found that the pannexin1-P2X7 pathway mediates apopin-12,-17,and-36 dose-dependent platelet aggregation,while apelin-13 inhibits ADP-induced platelet aggregation and thrombus formation in rabbits.However,studies have shown that apelin-12 synthesizes cyclic peptides and has the opposite effect on apelin itself.After synthesis of cyclic peptides,it can inhibit adenylate cyclase(AC)-regulated cAMP accumulation and Akt and ERK1/2 signaling pathways.It even shows a biological effect against apelin antagonism.Therefore,it is a very important and meaningful topic to study the effects of cyclic peptides of each subtype of apelin on rabbit platelet aggregation and thrombosis in vitro.This study was to investigate the effect of apelin cyclic peptide on rabbit platelet aggregation and thrombosis in vitro,and to investigate whether autophagy pathway mediates 1,12-cyclic apelin-12 inhibition of ADP-induced rabbit platelet aggregation and thrombus formation in vitro.From the perspective of autophagy,the subject reveals the relationship between apelin cyclic peptide and platelet aggregation and thrombosis in vitro,and explores the possible mechanism of action of cyclic peptides.By discovering the new biological effects of apelin’s subtypes of cyclic peptides.Provide new ideas and exploration for the development of new stable and highly effective antiplatelet drugs.Methods: 1.The peptide synthesizer synthesizes the corresponding apelin subtype cyclic peptide,2.Bioinformatics analysis of the properties of synthetic cyclic peptides,3.Platelet aggregation is detected by turbidimetry,4.Thrombosis is observed by rabbit thrombosis detector in vitro,5.Mitochondrial fluorescence probes were used to mark and observe the mitochondria in the platelets,6.Reactive oxygen probes were used to label the reactive oxygen species in platelets of New Zealand rabbits and observed.7.Transmission electron microscopy analysis of mitochondrial autophagy in platelets of New Zealand rabbits.Results: 1.1,12-cyclic apelin12(0.001μmol/L,0.01μmol/L,0.1μmol/L,1.0μmol/L)does not affect platelet aggregation in New Zealand rabbits,but it inhibits endogenous coagulant ADP-induced platelet aggregation in New Zealand rabbits in a dose-dependent manner.2.The newly synthesize apelin cyclic peptides are all strongly alkaline or weakly alkaline,structurally unstable,all of which are hydrophilic proteins.The hydrophilic and hydrophobic residues of cyclic peptides are unevenly distributed,and there is no typical hydrophilic and hydrophobic surface.3.c,c-cApelin12 dose-dependently promotes platelet aggregation in rabbits.4.1,6-cyclic Apelin-12 dose-dependently promotes platelet aggregation in rabbits.5.1,17-cyclic apelin17 promotes platelet aggregation in rabbits.6.1,36-cyclic apelin36 dose-dependently promotes platelet aggregation in rabbits.7.1,12-cyclic apelin12 inhibits endogenous coagulant ADP-induced thrombosis in New Zealand rabbits in a dose-dependent manner.8.1,12-cyclic apelin12 had no effect on rabbit platelet aggregation induced by collagen,thrombin and AA.9.The autophagy inducer rapamycin Rapa attenuate the inhibitory effect of 1,12-cyclic apelin12 on ADP-induced rabbit platelet aggregation,while the autophagy inhibitor 3MA enhanced the inhibitory effect of 1,12-cyclic apelin12.Meanwhile,Rapa It can promote ADP-induced rabbit platelet aggregation,while 3MA can inhibit ADP-induced rabbit platelet aggregation.10.1,12-cyclic apelin12 could not block rabbit platelet aggregation induced by apelin-12,apelin-17 and apelin-36.11.CCCP can attenuate the inhibitory effect of 1,12-cyclic apelin12 on ADP-induced rabbit platelet aggregation.12.Endoplasmic reticulum stress inducer tunicamycin can attenuate the inhibitory effect of 1,12-cyclic apelin12 on ADP-induced rabbit platelet aggregation.13.Mitochondrial ROS scavenger Mito-TEMPO attenuated the inhibitory effect of 1,12-cyclic apelin12 on ADP-induced rabbit platelet aggregation.14.ADP can induce a decrease in the number of mitochondria in rabbit platelets,and 1,12-cyclic apelin12 can reverse the decrease in the number of mitochondria in rabbit platelets induced by ADP.15.ADP can induce increased mitochondrial ROS content in rabbit platelets,while 1,12-cyclic apelin12 decreases ADP-induced increases in mitochondrial ROS content in rabbit platelets.16.ADP can induce mitochondrial autophagy in rabbit platelets,while 1,12-cyclic apelin12 reduces ADP-induced mitochondrial autophagy in rabbit platelets.17.ADP can induce human platelet aggregation,while 1,12-cyclic apelin12 can attenuate ADP-induced human platelet aggregation.Conclusion:1.The 1,12-cyclic apelin12 inhibits ADP-induced platelet aggregation in a dose-dependent,but does not induce platelet aggregation by itself.The apelin subtype cyclic peptide 1,6-cyclic apelin-12,c-c-cyclic apelin12,1,36-cyclic apelin36,1,17-cyclic apelin17 have the same function of inducing platelet aggregation.2.1,12-cyclic apelin12 inhibits ADP-induced rabbit platelet aggregation and thrombus formation in vitro by inhibiting mitochondrial reactive oxygen species and thereby inhibiting mitochondrial autophagy. |
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