Pharmacological Effects Of TRPM4 Inhibitor 9-phe On Platelet Aggregation And Thrombosis

BackgroundIn recent years,with the development of social economy and the intensification of population aging and urbanization,the prevalence of cardiovascular risk factors in China has become obvious,leading to a continuous increase in the number of cardiovascular diseases.The prevalence of cardiovascular disease in China is on the rise.Among them,the number of coronary heart disease patients exceeds 10 million.The mortality rate of coronary heart disease in urban and rural residents in China continues to increase.The number of deaths due to coronary heart disease in my country exceeds 1 million every year.The socio-economic burden caused by cardiovascular diseases is increasing day by day,so it is urgent to strengthen the prevention and treatment of cardiovascular diseases.On the basis of atherosclerosis,intravascular thrombosis caused by abnormal activation of platelets is an important pathological basis for the development of coronary heart disease.Therefore,inhibiting the activation of platelets is the main treatment for coronary heart disease.According to the different targets,the commonly used clinical antiplatelet drugs are mainly divided into four categories:(1)cyclooxygenase-1(cox-1)inhibitors;(2)adenosine diphosphate(ADP)receptor inhibitors;(3)Platelet glycoprotein GPIIb/IIIa antagonist;(4)Phosphodiesterase inhibitor.Platelet drugs have a positive effect,but there are still many problems in clinical application,such as bleeding risk and drug resistance.The existence of these clinical problems requires us to further explore the possible molecular mechanisms of platelet activation.The transient receptor potential(transient receptor potential,TRP)protein superfamily is divided into seven subfamilies based on the homology of amino acid sequence.TRPM4 is a member of the transient potential receptor M subfamily(TRPM)and is a special protein in the TRP family.TRPM4 is a non-selective cation channel protein activated by Ca2+.It is permeable to monovalent cations,such as Na+and K+.It can be activated by increased intracellular Ca2+concentration and regulated by voltage,protein kinase A,PKC and PIP2.TRPM4 is widely expressed in multiple organs and directly participates in many biological processes,including cell depolarization,cardiac rhythm production and immune response.TRPM4 is highly expressed in Purkinje fiber cells and ventricular myocytes.TRPM4 mutation is involved in causing progressive familial heart block and cardiac insufficiency.9-phenanthrol(9-phe)can inhibit the TRPM4 channel,and 9-phe specifically targets the TRPM4 channel.In kidney HEK-293 cells,cerebral artery smooth muscle cells,cardiac Purkinje cells and detrusor smooth muscle cells,TRPM4-like currents can be inhibited by 9-phe.The use of 9-phenanthrol,a selective TRPM4 inhibitor,can inhibit the arrhythmia in the early and subsequent stages,indicating that TRPM4 is related to arrhythmia.TRPM4 is related to hypertension.TRPM4 knockout mice show hypertension.Inhibition of TRPM4 leads to excessive secretion of catecholamines by chromaffin cells.TRPM4 can affect the functions of a variety of immune cells,such as regulating the Ca2+signal to affect T cell function and the activation of bone marrow-derived mast cells,and participate in the activation process of monocytes and macrophages.These studies suggest that TRPM4 is involved in the occurrence of a variety of cardiovascular diseases.However,whether platelets express TRPM4 and participate in platelet activation,and whether TRPM4 specific inhibitor 9-phe can inhibit platelet activation and thrombosis has not been reported.PurposeThe purpose of this research is to clarify the expression of the transient potential receptor TRPM4 in human platelets,to explore the effect and possible mechanism of the transient potential receptor TRPM4 inhibitor 9-phe on platelet activation and thrombosis.So as to provide new anti-platelet targets for the clinic,and to provide new treatment strategies for the prevention and treatment of coronary heart disease.Method1.Study on the expression of TRPM4 in human and mouse plateletsWe used PCR and Western Blot methods to confirm the expression of TRPM4 in human and mouse platelets.2.Evaluation of the effect of transient potential receptor TRPM4 on platelet function2.1 Platelet aggregationNormal human venous blood was separated into platelet rich plasma(PRP)and washed platelet(WP).After incubating PRP or WP with different concentrations of TRPM4 inhibitor 9-phenanthrol(9-phe)at room temperature,we clarified the effect of 9-phe on the platelet aggregation rate induced by ADP,collagen and thrombin.2.2 Platelet spreadingWP was pretreated with a vehicle solvent and 9-phe(100μM).The effect of 9-phe on the spread of platelets on fibrinogen was tested at different time points.2.3 Platelet retractionPRP pretreated with vehicle solvent and 9-phe(200μM)were added to the aggregation tube separately,and the effect of 9-phe on platelet retraction at different time points was tested.3.Establishment of mesenteric thrombosis model9-phe and platelets from isolated mice were injected intravenously.Ferric chloride injury thrombosis experiments were performed at the mesenteric artery,and the effect of 9-phe on arterial thrombosis was tested.4.Statistical methodsThe data in this paper are all expressed by Mean±SEM,and the software Graphpad Prism 8.0 is used to analyze the data.The difference between the two groups is analyzed by the unpaired t-test.When P<0.05,the difference is considered to be statistically significant.ResultThe results of reverse transcription PCR and western blot experiments showed that TRPM4 was expressed on human and mouse platelets at the mRNA level and protein level.In the range of 0-100 μM,the TRPM4 inhibitor 9-phe concentration-dependently inhibits platelet aggregation caused by ADP,collagen and thrombin.In the platelet spreading experiment,200 μM 9-phe significantly inhibited the spreading of platelets on the surface of fibrinogen.In the retraction experiment,100μM 9-phe obviously inhibited the retraction of PRP.In the mouse mesenteric thrombosis model,the thrombosis of the mice in the 9-phe administration group was significantly reduced,and its anti-thrombotic effect was similar to that of the positive drug cangrelor.Conclusion1.TRPM4 is expressed on platelets.2.TRPM4 inhibitor 9-phe inhibits platelet aggregation in a concentration-dependent manner.3.Intravenous injection of TRPM4 inhibitor 9-phe effectively inhibits mesenteric artery thrombosis in mice.