Relationship Between Peripheral Erythrocyte-Derived Microparticles With Severity Of Coronary Artery Lesions

BackgroundAcute coronary syndrome(ACS)is a series of clinical manifestations caused by acute myocardial ischemia.The main pathogenesis is atherosclerotic plaque rupture,damage,and then thrombosis,eventually affecting the blood supply to the heart.According to the report of World Health Organization(WHO),7.4 million deaths were attributed to ischemic heart diseases,and ACS was on the top for WHO's 10th leading causes of death.The vast majority of ACS occur with thrombosis.Vascular endothelial cell damage and hypercoagulable state in vivo play a key role in thrombosis.The existing gold standard examination—coronary angiography,can accurately assess the degree of coronary artery stenosis,but cannot assess the degree of vascular endothelial injury and atherosclerotic plaque stability.However,coronary angiography is invasive and relative lag.Therefore,we need to look for a non-invasive and early biomarker to assess the severity of coronary artery lesions as well as predict the risk of ACS,Microparticles are small membrane vesicles originate from blood cells(such as platelets,white blood cells,red blood cells,etc.)and blood vessel wall cells(such as endothelial cells,smooth muscle cells)upon activation or during apoptosis,the size of which typically ranges from 0.1 ?m to 1 ?m.Erythrocyte-derived microparticles(ErMPs)release with the activation of erythrocyte,which is an integral part of the erythrocyte ageing process.Not only ErMPs can directly remove the endothelial protective factor—nitric oxide(NO),but also reduce the bioavailability of NO and indirectly impair the function of endothelial cells.In addition,the outer membrane of ErMPs contains a large amount of phosphatidylserine,which activates coagulation factors ?,? and ?,causing a coagulation cascade.The study found that ErMPs and thromboembolic diseases are closely related.ErMPs in thromboembolic diseases(such as paroxysmal nocturnal hemoglobinuria,nephrotic syndrome,Crohn's disease)was significantly higher.In FeCl3-mediated thrombosis models,ErMPs can also participate in thrombus formation by mediating platelet adhesion to the intact endothelial surface.These evidences further suggest that ErMPs may be involved in the pathological process of ACS such as endothelial injury and coagulation activation.We speculate that the level of ErMPs significantly increased with the severity of coronary artery lesions.It may be a new biomarker that can evaluate the severity of coronary artery lesions.What's more,the level of ErMPs significantly increased in patients with ACS,suggesting that ErMPs may be a new biomarker predicting ACS risk.Based on the above questions,we designed this research.Objectives1.To study the difference of ErMPs level between ACS patients and control patients;2.To analyze the predictive value of ErMPs in diagnosis and treatment of ACS;3.To explore the correlation between the level of ErMPs and the severity of coronary artery lesions;4.To investigate the value of ErMPs level in predicting the degree of coronary artery stenosis.Methods and materials1.A total of 305 subjects were enrolled,and they are divided into control group and ACS group.The control group consisted of 70 subjects(41 males and 29 females,mean age 59± 10 years):Those subjects had no previous history of coronary artery disease(CAD).Coronary angiography showed no stenosis or the stenosis less than 50 percent.The ACS group:Coronary angiography showed coronary stenosis more than or equal to 50 percent.This group was further divided into two groups:unstable angina(UA)group and acute myocardial infarction(AMI)group,which of them are in accordance with the 2016 Chinese Medical Association diagnostic criteria.(1)The UA group consisted of 159 subjects(104 males and 55 females,mean age 62±8years).(2)The AMI group consisted of 76 subjects(58 males and 18 females,mean age 60±years).2.Blood sampling and information of case files collecting(1)Blood sampling:Prior to coronary angiography,peripheral blood samples were collected and centrifuged to obtain platelet-poor plasma(PPP).Leave for detecting by flow cytometry.(2)Information of case files included baseline clinical characteristics(such as gender,age,heart rate,blood pressure,etc.),laboratory data(blood lipids,blood glucose,serum creatinine,red blood cell counts,etc.),coronary angiography results,and therapy(aspirin,clopidogrel/ticagrelor,statins,etc.).(3)Gensini score:Based on coronary angiography results,Gensini scoring system could evaluate the degree of coronary artery stenosis.The Gensini score was the sum of the products of the vessels weight coefficients and the stenosis weight coefficients,based on the results of coronary angiography.3.Detection and analysis of ErMPsErMPs were detected by the flow cytometry.Before detecting,CD235a-APC and Annexin V-FITC were added.We used FlowJo software to analyze the test results.The concentration of ErMPs and the ratio of ErMPs in plasma were calculated based on the number of collected microparticles and the number of absolute counted fluorescent beads,the absolute known number of fluorescent microspheres,the volume of plasma sample.4.Statistical analysisThe software SPSS19.0(SPSS Inc.,Chicago,Illinois,USA)was used for statistical analysis.The continuous variables were compared by t-test or One-way ANOVA,and LSD-t test was used to compare every pair;Non-parametric test was applied in continuous data without normal distribution and homogeneity of variance;Frequency and non-grade parameter data were compared by Chi-square test;Multiple linear regression was used to screening the influencing factors of ErMPs concentration and ErMPs percentage;Covariance was used to analysis of differences in ErMPs among three groups;The correlation between ErMPs and risk of ACS was analyzed by logistic regression;The association between ErMPs and severity of coronary artery stenosis was analyzed by multivariate linear regression analysis;The ROC curve was used to evaluate the value of ErMPs concentration and ErMPs percentage in predicting coronary stenosis.Results1.The basic clinical data of three groups:There was no significant difference in sex,age,heart rate,hypertension and alcohol intake among the three groups(P>0.05).Compared with the control group,diabetes mellitus(P<0.001),smoking(P=0.021),pre-MI/pre-PCI(P<0.001),SBP(P<0.001)and DBP(P=0.011)in UA group were significantly increased;diabetes mellitus(P=0.006),family history of CAD(P=0.021),smoking(P=0.004),pre-MI/pre-PCI(P<0.001)in AMI group were significantly increased,SBP in AMI group was significantly lower(P<0.001).There was no significant difference in diabetes mellitus,smoking and pre-MI/pre-PCI between UA group and AMI group.2.The laboratory data of three groups:There was no significant difference in triglyceride,TC,LDL-C,RBC,hemoglobin,RDW among the three groups(P>0.05).Compared with the control group,HDL-C in the UA group was significantly lower(P=0.013)and FBG was significantly higher(P=0.021),HDL-C in the AMI group was significantly lower(P<0.001)and FBG was significantly higher(P<0.001).Compared with the UA group,the HDL-C in the AMI group were significantly lower(P=0.001),and SCr increased significantly(P=0.001).cTnI in the AMI group was significantly higher than that in the other two groups(P<0.001).3.The therapy of three groups:Compared with the control group,the use rates of aspirin,clopidogrel/ticagrelor,nitrates,statins and ACEI/ARB in UA group and AMI group were significantly increased(P<0.001).The utilization rate of low molecular weight heparin in the AMI group was significantly higher than that in the other two groups(P<0.001).There was no significant difference in the utilization rate of calcium channel blockers among three groups(P>0.05).4.The severity of coronary artery lesions among three groups:Compared with the control group,the maximal stenosis degree of coronary,the number of stenotic coronary artery segments,the number of stenotic coronary artery branches and the Gensini score of UA group and AMI group were significantly increased(P<0.001).Compared with the UA group,the maximal stenosis degree of coronary in the AMI group was significantly increased.There was no significant difference in the number of stenotic coronary artery segments,the number of stenotic coronary artery branches and the Gensini score between UA group and AMI group(P>0.05).5.Influencing factors of ErMPs concentration:ErMPs concentration negatively correlated with HDL-C(r=-0.128,P=0.050).Compared with the non-CAD family history group,there was a significant increase in ErMPs concentrations in the CAD family history group(P=0.034).Then multiple linear regression was conducted,we found that HDL-C(P=-0.156,P=0.016)was a protective factor and CAD family history(?=0.152,P=0.019)was a risk factor of ErMPs concentration.6.Influencing factors of ErMPs percentage:ErMPs percentage positively correlated with SCr(r=0.169,P=0.009)and cTnI(r=0.131,P=0.045).Then multiple linear regression was conducted,we found that SCr(?=0.155,P=0.016)was a risk factor and CCB(?=-0.141,P=0.029)was a protective factor of ErMPs percentage.7.The levels of ErMPs in three groups:In the.uncontrolled confounding circumstances,compared with the control group,ErMPs concentration in ACS group increased significantly(P=0.021).Compared with the control group,ErMPs concentrations in UA group(P=0.019)and AMI group(P=0.005)were both significantly increased.There was no significant difference in ErMPs concentrations between UA group and AMI group(P>0.05).Compared with the control group,ErMPs percentage in ACS group increased,but did not reach statistical significance(P>0.05).Compared with the control group,ErMPs percentage of UA group and AMI group increased but did not reach statistical significance(P>0.05).There was no significant difference in ErMPs percentage between UA group and AMI group P>0.05).After controlling the confounding factors(gender,alcohol intake,family history,SCr,HDL-C,RBC,RDW,CCB),the difference in ErMPs concentration among three groups was statistically significant(P=0.040).Compared with the control group,ErMPs concentrations were significantly increased in the UA group(P=0.028)and the AMI group(P=0.022).ErMPs concentrations did not differ between three groups.8.The correlation between ErMPs and ACS:ErMPs concentration(OR=1.009,95%Cl[1.002-1.017],P=0.015),SBP(OR=1.034,95%CI[1.015-1.053,P<0.001],DM(OR=3.048,95%CI[1.368-6.791],P=0.006),cTnI(OR=1.021,95%CI[1.000-1.043],P=0.048)were risk factors of ACS.The ErMPs percentage(OR=1.476,95%CI[0.311-7.008],P=0.624)remained unrelated to ACS.9.The correlation between ErMPs and UA,as well as ErMPs and AMI:The concentration of ErMPs(OR=1.009,95%CI[1.001-1.016],P=0.020)was risk factor for UA,and HDL-C(OR=0.278,95%CI[0.089-0.867],P=0.027)was protective factor for UA.ErMPs concentration(OR=1.010,95%CI[1.002-1.018],P=0.014)was risk factor for AMI.The ErMPs percentage(OR=1.254,95%CI[0.279-5.645],P=0.768)was not associated with UA.The ErMPs percentage(OR=4.319,95%CI[0.769-24.251],P=0.097)also did not correlate with AMI.10.The correlation between ErMPs and the severity of coronary artery lesions:There was a positive correlation between Gensini score and ErMPs concentration(P=0.206,P=0.001),as well as age(?=0.607,P=0.001),FBG(?=0.272,P=0.008),pre-MI/pre-PCI(?=0.154,P<0.001),but negatively correlated with HDL-C(?=0.124,P=0.001).ErMPs percentage(?=0.161,P=0.023),age(?=0.644,P=0.001),FBG(?=0.292,P=0.007),pre-MI/pre-PCI(?=0.161,P=0.001)were positively correlated with Gensini score.HDL-C(?=-0.422,P=0.006)was negatively correlated with Gensini score.11.ErMPs concentration,ErMPs percentage and their combined predictive value of coronary stenosis(>50%):ErMPs concentration:AUC:0.624,95%CI:0.567-0.679;ErMPs percentage:AUC:0.548,95%CI:0.490-0.605;Combined model of ErMPs concentration and ErMPs percentage:AUC:0.628,95%CI:0.552-0.704.12.ErMPs concentration,ErMPs percentage and their combined predictive value of coronary stenosis(?70%):ErMPs concentration:AUC:0.617,95%CI:0.559-0.671;ErMPs percentage:AUC:0.565,95%CI:0.508-0.622;Combined model of ErMPs concentration and ErMPs percentage:AUC:0.615,95%CI:0.549-0.681.Conclusions1.The concentration of ErMPs in ACS patients increased significantly.Furthermore,ErMPs concentration associated with the occurrence of ACS.ErMPs may be a new biomarker of ACS risk.2.The concentration of ErMPs in UA and AMI patients increased significantly.ErMPs concentration was associated with the occurrence of UA and AMI.ErMPs may be a new biomarker of UA and AMI risk.3.There was a positive correlation between ErMPs and the severity of coronary artery lesions.ErMPs may be a new biomarker of coronary artery lesions.4.The predictive value of ErMPs concentration on the degree of coronary artery lesions was better than that of ErMPs percentage.