| Backgroud and objectiveAcute myocardial infarction(AMI)is the most serious type of coronary heart disease,and it is also the leading cause of death from cardiovascular disease.It is very important to clarify the diagnosis of AMI as soon as possible,to recover the blocked coronary blood flow and to save the ischemic myocardium.In recent years,long-term antiplatelet therapy has become one of the main treatment methods of AMI after percutaneous coronary intervention.Clopidogrel and aspirin are commonly used antiplatelet drugs.However,more and more clinical studies showed that using the routine antiplatelet drugs,or even increasing the dose of medication or replacing new antiplatelet drug ticagrelor,there was still a part of patients with clopidogrel resistance or high residual platelet reactivity.There was also a significant increase in the risk of adverse cardiovascular events.Therefore,it is urgent to find the key regulatory molecules and signaling pathways involved in the aggregation and activation of residual platelets.Chemokine C-C motif 2(CCL2),also known as monocyte chemotactic protein 1,is a promoter of inflammatory response,which can induce the production and release of other inflammatory mediators by positive feedback,and accelerate the development of atherosclerosis(AS).Studies have shown that the risk of cardiovascular events in acute coronary syndrome patients with high expression of CCL2 in plasma was significantly higher than that in patients with low expression of CCL2.Our previous studies showed that the expression of CCL2 in plasma and platelets in patients with ST segment elevation myocardial infarction(STEMI)was significantly higher than that in the control group.CCR2 is a specific receptor of CCL2,which is a G protein coupled receptor with 7 transmembrane domains.CCL2 plays an important role in the development of AS and AMI in several stages,such as the formation of lipid streaks,plaque rupture and thrombosis,by combining with CCR2.However,there are no reports about whether CCL2 participates in the formation of arterial thrombosis by affecting the aggregation and activation of residual platelets,and the regulation mechanism of platelet activation.Based on the above research background,we firstly discuss the difference of plasma CCL2 expression in patients with STEMI after taking ticagrelor,the relationship between the expression of CCL2 in plasma and the residual platelet aggregation rate(RPA),the difference of CCL2 and CCR2 in platelets,and the correlation between plasma CCL2 level and the prognosis of patients.Secondly,the healthy volunteers and mice were selected as the research objects.Protein chip screening and Western Blot were used to verify which signaling pathway in platelets was regulated by CCL2.Finally,the effects of CCL2 on platelet aggregation,particle secretion and arterial thrombosis in mice were investigated.To determine whether CCL2 participates in the activation of residual platelets and specific regulation mechanisms,and to verify whether CCL2 is involved in arterial thrombosis,the findings might provide a new way to research for AMI and thrombotic diseases.Methods and results1.CCL2 was involved in residual platelet activation in patients with STEMI and was independently associated with major adverse cardiac and cerebral events(MACCE)within 1 year.1)In this part,a total of 420 patients with STEMI were enrolled.The baseline clinical datas of 80 STEMI patients taking ticagrelor were collected.The RPA of the patients was detected at different time points(2h,4h,6h)after taking the medicine by light transmission aggregometry,and the patients were divided into two groups according to the results.The baseline clinical datas were compared between the normal platelet reactivity group(normal reactivity group)and high platelet reactivity group(high reactivity group).There was no significant difference of each data between the two groups(P>0.05).The proportion of high residual platelet reactivity of the patients was 43.8 %,28.8 %,17.5 % respectively at different time points(2h,4h,6h)after taking the medicine.Moreover,the P2Y12 reaction unit of the patients was detected at different time points(2h,4h,6h)after taking ticagrelor by Verifynow,and the patients were divided into two groups according to the results.The proportion of high residual platelet reactivity of the patients was 34.8%,24.6%,14.3% respectively at different time points(2h,4h,6h)after taking the medicine.2)The plasma CCL2 concentration in the high reactivity group and normal reactivity group according to the results of light transmission aggregometry was detected by enzyme linked immuno sorbent assay(ELISA).We found that the plasma CCL2 concentration in the high reactivity group(2 h: 209.72±84.09 pg/ml;4 h: 195.54±63.66 pg/ml;6 h: 184.29±56.74 pg/ml)was higher than that in the normal reactivity group(2 h: 167.52±36.92 pg/ml;4 h: 160.63±42.31 pg/ml;6 h: 152.43±42.48 pg/ml)at different time points after taking ticagrelor(P<0.01 or P<0.05).The correlation of the RPA and CCL2 concentration in plasma at different time points was analyzed by Pearson correlation.We found that there was a linear correlation between the RPA and the CCL2 concentration in plasma(2 h: r=0.474;4 h: r=0.622;6 h: r=0.425,P<0.01).3)Five patients were selected in each group of the high reactivity group and normal reactivity group at 2 h after taking ticagrelor according to the results of light transmission aggregometry.The expression of CCL2 and CCR2 in platelets of the high reactivity group and normal reactivity group was detected using Western Blot.We found that the expression of CCL2 and CCR2 in platelets of the high reactivity group was higher than that in normal reactivity group(P<0.01).4)All 420 patients with STEMI were followed up for 1 year.ELISA was used to detect the concentration of CCL2 in plasma of patients with STEMI with or without MACCE.The MACCE incidence was 8.8 %(37/420)within 1 year.The plasma CCL2 concentration was markedly higher in STEMI patients with MACCE(total: 244.56±105.85 pg/ml;all-cause death: 237.39±107.79 pg/ml;heart failure: 267.39±160.56 pg/ml;cerebral infarction: 254.90±91.40 pg/ml;recurrent myocardial infarction: 240.38±90.13 pg/ml)than that in patients without MACCE(190.65±50.72 pg/ml,P<0.01 or P<0.05);the plasma CCL2 concentration was markedly higher in patients with MACCE within 24 h and 24 h-90 d(269.83±115.27 pg/ml;251.25±132.74 pg/ml)than that in patients without MACCE(P<0.01).5)All 420 patients with STEMI were divided into two groups according to the median concentration of CCL2 in plasma,then the baseline clinical datas and the MACCE incidence were compared between the two groups.Kaplan-Meier method was used to analyze survival curve and Cox regression model was used to analyze the correlation between the plasma CCL2 concentration and MACCE.The median concentration of CCL2 in plasma was 179.89 pg/ml.The age,MACCE incidence,and cerebral infarction incidence of the patients whose CCL2 concentration in plasma>179.89 pg/ml was higher than those of the patients whose CCL2 concentration in plasma≤179.89 pg/ml(P=0.000,P=0.009,P=0.030).The plasma CCL2 concentration of STEMI patients was independently associated with MACCE within 1 year(HR=1.008,P=0.000).2.CCL2 can regulate platelet activation through P38 mitogen-activated protein kinase(P38MAPK)-heat shock protein 27(HSP27)signaling pathway.1)In this part,20 healthy volunteers were enrolled.The platelets of 8 healthy volunteers were isolated,CCL2(1000 ng/ml)was used to stimulate the isolated platelets of 4 volunteers,and the platelets of the other 4 volunteers were used as controls.The expression of the phosphorylated kinases in platelets was detected by the protein chip.Next,the platelets of all the 20 healthy volunteers were isolated.Similarly,the expression of the phosphorylated kinases which were screened preciously by protein chip was examined by Western Blot after stimulation with CCL2(1000 ng/ml)in the presence or absence of CCL2 neutralizing antibody(50 μg/ml),CCR2 antagonist(RS 201895,10 μM)or P38 MAPK inhibitor(SB 203580,10 μM).The present study showed the expression of 12 phosphorylated kinases were elevated by the protein chip screening(P<0.05),and the phosphorylation of P38MAPK(T180/Y182)and HSP27(S78/S82)was significantly increased by Western Blot after stimulation with CCL2,which could be prevented by preincubation with CCL2 neutralizing antibody,RS 102895,or SB 203580(P<0.01).2)The platelets of 10 wild type(WT)mice and 10 CCL2-/-mice were isolated.Western blot analysis of the phosphorylation of P38MAPK(T180/Y182)and HSP27(S78/S82)was done in WT mouse and CCL2-/-mouse platelets after stimulation with adenosine diphosphate(ADP,10 μM)or collagen(5 μg/ml).The present study showed the phosphorylation of P38MAPK(T180/Y182)and HSP27(S78/S82)was significantly reduced in the absence of CCL2(P<0.05).3.CCL2 participates in platelet aggregation,platelet secretion and arterial thrombosis.1)The platelets of 10 WT mice and 10 CCL2-/-mice were isolated.Platelet aggregation was measured by light transmission aggregometry after stimulation with ADP or collagen.Defective platelet aggregation in the absence of CCL2 was observed upon stimulation with different doses of ADP(10 μM,20 μM,P<0.05)or collagen(2 μg/ml,5 μg/ml,10 μg/ml,P<0.05).2)The platelets of 10 WT mice and 10 CCL2-/-mice were isolated.CD40 L secreted from platelet granules was measured by ELISA after stimulation with ADP or collagen.Impaired CD40 L secretion in the absence of CCL2 was observed upon stimulation with different doses of ADP or collagen(P<0.05).3)Similarly,PF4 secreted from platelet granules was measured by ELISA after stimulation with ADP or collagen.Impaired PF4 secretion in the absence of CCL2 was observed upon stimulation with different doses of ADP or collagen(P<0.05).4)Platelets and WBCs were counted using a hematology analyzer.Tails of anesthetized mice were severed at 2 mm from the tip with a scalpel and the time to the cessation of blood flow was recorded.No significant differences in the number of circulating platelets or WBCs were found between CCL2-/-mice and WT mice(P>0.05).The average tail bleeding time was markedly prolonged in CCL2-/-mice(9.55±3.36 min)compared with WT mice(7.27±2.67 min,P<0.05).5)Fibrin clot formation was initiated by adding thrombin(1 U/ml)to mouse platelet-rich plasma(PRP)and observed the clot retraction.Thrombus formation was induced by applying a piece of filter paper saturated with 10 % FeCl3 solution on the adventitial surface of the artery for 1 min.Carotid blood flow was continuously monitored for 15 min or until complete occlusion occurred.The clot volume of CCL2-/-mice was larger than that of WT mice at different time points(P<0.05).The average occlusion time was significantly prolonged in CCL2-/-mice(13.01±6.21 min)compared with that in WT mice(9.20±3.32 min,P<0.05).ConclusionThis paper preliminarily revealed CCL2 particapates in the aggregation and activation of residual platelets,and the regulation of platelet activation through P38MAPK-HSP27 signaling pathway.Besides,this paper described CCL2 participates in platelet aggregation,platelet secretion and arterial thrombosis.The findings not only provided the intervention target for residual platelet activation and aggregation of AMI patients,but also provided new strategies and methods for the prevention and treatment of AMI and thrombotic diseases. |
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