The Performance Of Platelet Microbubbles In Clinical Hypertension With Obesity And Clopidogrel

Objective:The overall objective is to:? establish methods to separate,prepare the platelet microparticles(platelet microparticles whose diameters are between 0.5?m and 0.8?m,and small platelet microparticles whose diameters are less than 0.5?m)in plasma and test the immunphenotypes of them;? detect the quantities and characteristics of microparticles,which derive from platelets with different kinds of immunophenotypes,in the peripheral bloods of hypertensive patients;? analyze the clinical parameters of patients and the data of platelet microparticles with significance analysis,univariate and multivariate correlation analysis,and to explore the role of platelet microparticles play in hypertension and the pathological and physiological effects of platelet activation in cardiovascular diseases.Objective of first part:By detecting the number and percentage of platelet microparticles in peripheral bloods of hypertensive patients with or without obesity,and analyzing the immunophenotypes,associated factors and diagnostic threshold of the particles,to investigate the pathophysiological effects of them and the potential significance that the platelet microparticles can be a new biomarker to link between hypertension and obesity.Objective of second part:By studying the numbers and percentages of platelet microparticles with different sizes in peripheral bloods of hypertensive patients and healthy volunteers,and analyzing the correlation factors and diagnostic threshold,we aim to investigate the pathophysiological effects of platelet microparticles during the development of hypertension,and the possible mechanisms that clopidogrel,an anti-platelet drug,may affect the quantity and distribution of platelet microparticles during the treatment of hypertension.Objective of third part:By studying the concentration' distribution of cells' microparticles of different sizes in the peripheral bloods of spontaneously hypertensive rats which are in different groups(clopidogrel treatment group and control group),we explore the potential pathophysiological effects that different sizes of microparticles may have in the development of hypertension,and the possible mechanism clopidogrel may have in the therapy of hypertension.Methods:The overall methods are using flow cytometry and variety of statistical methods.First and second part:We recruited hypertensive patients without obesity(n=19),hypertensive patients with obesity(n=10),hypertensive patients(n=21)and healthy volunteers(n=19).Blood samples(3ml from each subject)were drawn by venipuncture into blue-top vacutainer tubes containing sodium citrate.The samples were centrifuged to obtain platelet-poor plasma within lhour of blood sampling,and stored at-80? until analysis.When tested the platelet microparticles,the specific monoclonal antibodies and isotypes were added at room temperature.For flow cytometry assay,standard beads(0.8pm,0.46pm;Sigma,St.Louis,MO)were applied for determining the size of microparticles,and the number and percentage of platelet microparticles were tested by flow cytometery.Platelet microparticles were defined as CD31+/42b+.The data of platelet microparticles and clinical parameters were analyzed with t-test or non-parametric statistical analysis to compare the changes in the number of platelet microparticles between different groups;linear regression analysis was used to determine the correlation between platelet microparticles and various clinical parameters.Third part:Ten 13-week-old male spontaneously hypertensive rats(SHR)were randomly divided into two groups:placebo control group(1ml 0.5%sodium carboxymethylcellulose solution,CMC-Na)and clopidogrel treatment group(10mg/kg clopidogrel).Drugs therapy was administered by oral perfusion every day for 4 weeks continuously.Blood samples(1ml)were drawn weekly from tail veins of each rat before and after administration and then were centrifuged to obtain platelet-poor plasma within 1 hour of blood sampling,and stored at-80? until analysis.For flow cytometry,samples were warmed into room temperature and diluted 500 times for testing.A mix beads(Apogee Mix,Apogee,UK)was used to determine the size of microparticles,and the number and percentage of the microparticles were tested by flow cytometery.The obtained data were analyzed by t-test to investigate the changes in levels of cellular microparticles of different sizes among different groups.Results:First part:In the peripheral blood of hypertensive patients with obesity,the concentration of platelet microparticles(154.87 ± 216.21)is significantly higher than in the hypertensive patients without obesity(14.72 ± 17.24,p = 0.003).The number of platelet microparticles has no significant correlation with age,sex and other clinical parameters of patients,including BMI,systolic/diastolic blood pressure,total cholesterol(TC),low density lipoprotein(LDL),triglyceride(TG)and platelets'(PLT)levels,but has significant correlation with high density lipoprotein(HDL)and lipid index such as TC/HDL-C,LDL-C/HDL-C,log(TG/HDL-C).Second part:The number of small platelet microparticles(sPMPs,diameter<0.5?m)in patients with hypertension(1.21 ± 1.24)was significantly lower than the healthy group(3.22± 1.40,p = 0.002),while there is no significant difference(hypertension group:2.82 ±1.73;health group:2.80 ± 1.57;p>0.05)in the number of big platelet microparticles(diameter>0.5pm).There was no correlation between small platelet microparticles and various clinical parameters.We supposed that clopidogrel mainly affect the release of small platelet microparticles rather than the large ones.Third part:The number of microparticles with a size of 200-350nm in peripheral bloods of spontaneously hypertensive rats treated with clopidogrel tended to decline,while others(with a size of 350-500nm and 500-800nm)showed an increasing trend.All the microparticles in control group showed an increasing trend.We also found that more than 50%of microparticles in peripheral bloods are between 200nm and 350nm.Conclusion:Platelet microparticles can be used as a potential marker to link the hypertension and obesity,and also as a marker to predict the risk of coronary heart disease(CHD).Clopidogrel can participate in the treatment of hypertension mainly by impacting the release of small platelet microparticles rather than the large ones in hypertensive patients.This also suggests that small platelet microparticles can be used as a new clinical indicator to judge the therapeutic effect of hypertension,and there is a very important potential value in investigating clopidogrel' s anti-inflammatory effect in the therapy of hypertension.With the treatment of clopidogrel in hypertension,the small cellular microparticles in peripheral blood tend to decrease.On the contrary,the large ones tend to increase gradually.This result supports the conclusion that clopidogrel can affect the release of microparticles during the treatment of hypertension.