The Inhibitory Effect Of Atorvastatin On Platelet Aggregation Depends On The Concentration Of Platelet Puree

Background: Coronary heart disease (CHD) is a common disease whichseverely impairs humanity health. With the development of people’s livingstandard, the morbidity and mortality of CHD increased and the age of patientssuffering from CHD was trending younger. The activation of platelet plays akey role in the occurrence and development of cardiovascular disease. Atpresent, aspirin in combination with clopidogrel, an inhibitor of PY12ADPplatelet receptors and the neotype antiplatelet drug tirofiban, a kind ofglycoprotein Ⅱ b/Ⅲ a inhibitors, areintegral parts of the therapeutic regimenfor patients suffering from CHD, especially for the patients with acute coronarysyndrome (ACS) and those undergoing percutaneous coronary intervention.Statins are powerful lipid-lowering drugs that inhibit cholesterol biosynthesisvia downregulation of hydroxymethylglutaryl coenzyme-A reductase. Statinscan lower the rate of cardiovascular events such as myocardial infarction,stroke, and cardiovascular death. There is a growing body of evidence thatstatins may exert other effects besides from their cholesterol-lowering actions,including inhibition of the formation of foam cells, improvement of plaqueinstability, anti-inflammatory actions and inhibition of platelet activation. Thesefindings suggest the pleiotropic effects of statins. There is an increasing interestin the study of the pleiotropic effects of statins, among which the antiplateleteffects attracting the attention of more and more scholars. Several large clinicaltrials have demonstrated that statins markedly decrease the risk ofcardiovascular events in hypercholesterolemic subjects as well asnormocholesterolemic subjects. These findings suggest that the favorableeffects of statins on the cardiovascular events may be attributable to pleiotropiceffects of statins instead of their cholesterol-lowering actions. The antiplateleteffect of statins remains to be unambiguously established because these results were obtained from in vivo tests, where confounding factors are difficult to becontrolled.We explored the in vitro effect of atorvastatin calcium salt on the plateletaggregation of different concentration of platelet puree collected from healthysubjects.126cases of platelet puree were diluted to different concentrations.Then the samples were incubated in vitro with different concentrations ofatorvastatin calcium salt, platelet aggregation and curve slope were evaluatedwith whole blood impedance method to provide the objective basis for clinicaluse of atorvastatin in the patients of CAD with or withouthypercholesterolemia.Methods: A total of126cases of fresh platelets puree were collected fromthe Blood Center of Hebei Province, which the platelet concentration isbetween960×109/L and1280×109/L.The inclusion and exclusion criteria arestrictly in accordance with national standards for blood donation law. Theplatelets puree were diluted with saline to different concentrations of100×109/L（39cases）、300×109/L(42cases) and600×109/L(45cases),then wereprepared into saline group(CN), the DMSO group(CD),the final concentration ofatorvastatin were10-7mol/L、10-6mol/L、10-5mol/L in the experimental group(C1-3) respectively. Then the samples were incubated for10minutes at37℃.Aggregation and curve slope were evaluated in2hours with whole bloodimpedance analyzer (Model590), made by Chrono-Log Corporation, whichinduced by ADP (10μmol/L), arachidonic acid(AA0.5mmol/L)collagen(2μg/mL) and epinephrine (EP1mg/mL) respectively. Data wereprocessed with SPSS16.0software. When data are subject to normaldistribution, continuous variables were expressed as the mean value±standarddeviation (SD), the overall comparison in each group was evaluated by singlefactor ANOVA analysis. If not, the variables were expressed as the median andquartile (QR), the overall comparison in each group was evaluated byFriedman’s M test. P value of <0.05was considered statistically significant.Results:1The effect of different concentrations of atorvastatin calcium on the platelet aggregation of platelet puree with concentration of100×109/Lfrom healthy subjects.11The effect of different concentrations of atorvastatin calcium on the plateletaggregation induced by ADP(10μmol/L):the platelet aggregation rates in CN、CD、C1-3were3.50（1）ohms、3.00（1）ohms、3.00（1）ohms、3.00（2）ohms and3.14±1.06ohms respectively. There were no statistical differencesamong these groups(P=0.674).The curve slopes were2.00（1）ohms/min、2.00（2）ohms/min、2.00（1）ohms/min、2.00（1）ohms/min and2.00（1）ohms/minrespectively. There were no statistical differences among these groups(P=0.572).12The effect of different concentrations of atorvastatin calcium on the plateletaggregation induced by AA(0.5mmol/L):the platelet aggregation rates in CN、CD、C1-3were3.56±1.15ohms、3.50（1）ohms、3.00（1）ohms、3.00（2）ohms and3.00（2）ohms respectively. There were no statistical differencesamong these groups(P=0.200).The curve slopes were1.50（1）ohms/min、1.50（1）ohms/min、1.00（1）ohms/min、1.00（1）ohms/min and1.00（1）ohms/minrespectively. There were no statistical differences among these groups(P=0.993).13The effect of different concentrations of atorvastatin calcium on the plateletaggregation induced by collagen(2μg/mL):the platelet aggregation rates in CN、CD、C1-3were9.00（2）ohm、8.50±1.78ohms、8.00（3）ohms、8.00（2）ohms and8.00（3）ohms respectively. There were no statistical differencesamong these groups(P=0.362).The curve slopes were2.00（1）ohms/min、2.00（1）ohms/min、2.00（1）ohms/min、2.00（0）ohms/min and2.00（0）ohms/minrespectively. There were no statistical differences among these groups(P=0.986).14The effect of different concentrations of atorvastatin calcium on the plateletaggregation induced by EP(1mg/mL):the platelet aggregation rates in CN、CD、C1-3were4.00(2)ohms、4.00(1)ohms、4.00（1）ohms、4.00（3）ohms and4.00（2）ohms respectively.There were no statistical differences among thesegroups(P=0.266).The curve slopes were2.00（1）ohms/min、2.00（1）ohms/min、 2.00（1）ohms/min、2.00（1）ohms/min and1.50（1）ohms/min respectively.There were no statistical differences among these groups (P=0.915).2The effect of different concentrations of atorvastatin calcium on the plateletaggregation of platelet puree with concentration of300×109/L from healthysubjects.21The effect of different concentrations of atorvastatin calcium on the plateletaggregation induced by ADP(10μmol/L):the platelet aggregation rates in CN、CD、C1-3were11.00±1.85ohms、10.97±1.71ohms、11.00（2） ohms、10.42±1.91ohms and10.00（3）ohms. There were no statistical differencesamong these groups(P=0.172).The curve slopes were4.00（2）ohms/min、4.00（2）ohms/min、4.00（2）ohms/min、4.00（1）ohms/min and4.00（1）ohms/minrespectively. There were no statistical differences among these groups(P=0.068).2.2The effect of different concentrations of atorvastatin calcium on the plateletaggregation induced by AA(0.5mmol/L):the platelet aggregation rates in CN、CD、C1-3were12.06±1.03ohms、12.00（2）ohms、12.00（2）ohms、12.00（2）ohms and11.21±1.51ohms respectively. There were no statisticaldifferences among these groups(P=0.275).The curve slopes were10.00（1）ohms/min、10.33±1.35ohms/min、9.83±1.27ohms/min、9.72±1.60ohms/min and9.68±1.46ohms/min respectively. There were no statistical differences amongthese groups (P=0.341).2.3The effect of different concentrations of atorvastatin calcium on the plateletaggregation induced by collagen(2μg/mL):the platelet aggregation rates in CN、CD、 C1-3were17.00±2.02ohms、17.14±1.36ohms、16.29±1.68ohms、15.03±1.62ohms and14.36±2.15ohms respectively. There were statisticallydifferences among these groups(p＜0.05).By the method of SNK-q, nostatistical difference was found between the groups of physiological saline andDMSO.There were differences between the control groups and the atorvastatincalcium groups and there were differences between the groups of atorvastatinof high concentration (C3) and atorvastatin of low concentration (C1).But nostatistical differences were found between the atorvastatin calcium of moderate concentration group (C2) and the other two atorvastatin calcium groups.Thecurve slopes were15.64±1.65ohms/min,15.55±1.57ohms/min,15.00±2.02ohms/min,14.23±1.75ohms/min and13.86±1.98ohms/min respectively. Therewere statistically differences (P=0.001).By the method of SNK-q, no statisticaldifference was found between the groups of physiological saline and DMSO.There were differences between the control groups and the atorvastatin calciumgroups, but no differences were found among the groups of atorvastatincalcium of different concentrations.2.4The effect of different concentrations of atorvastatin calcium on the plateletaggregation induced by EP(1mg/mL):the platelet aggregation rates in CN、CD、C1-3were15.97±1.66ohms、15.50±1.48ohms、15.37±2.10ohms、14.32±2.39ohms and13.93±2.82ohms respectively. There were statisticallydifferences among these groups (P=0.002).By the method of SNK-q, nostatistical difference was found between the groups of physiological saline andDMSO. There were differences between the control groups and the atorvastatincalcium groups, but no differences were found among the groups ofatorvastatin calcium of different concentrations.The curve slopes were19.10±2.10ohms/min、17.98±2.81ohms/min、17.44±3.61ohms/min、16.33±3.87ohms/min and15.46±4.06ohms/min respectively.There werestatistically differences (P=0.001).By the method of SNK-q, no statisticaldifference was found between the groups of physiological saline andDMSO.There were differences between the control groups and the atorvastatincalcium groups, but no differences were found among the groups ofatorvastatin calcium of different concentrations.3The effect of different concentrations of atorvastatin calcium on the plateletaggregation of platelet puree with concentration of600×109/L from healthysubjects.3.1The effect of different concentrations of atorvastatin calcium on the plateletaggregation induced by ADP(10μmol/L):the platelet aggregation rates in CN、CD、 C1-3were22.63±2.14ohms、22.58±1.79ohms、22.35±2.70ohms、21.21±2.30ohms and19.59±1.85ohms respectively. There were statistically differences among these groups (P＜0.05).By the method of SNK-q, nostatistical difference was found between the groups of physiological saline andDMSO. There were differences between the control groups and the atorvastatincalcium groups, and differences were found among the groups of atorvastatincalcium of different concentrations. The curve slopes were21.34±2.62ohms/min、20.77±2.36ohms/min、20.09±2.53ohms/min、19.03±1.94ohms/min and18.72±2.20ohms/min respectively. There werestatistically differences(P＜0.05). By the method of SNK-q, no statisticaldifference was found between the groups of physiological saline andDMSO.There were differences between the control groups and the atorvastatincalcium groups and there were differences between the groups of atorvastatinof high concentration (C3) and atorvastatin of low concentration (C1).But nostatistical differences were found between the atorvastatin calcium of moderateconcentration group (C2) and the other two atorvastatin calcium groups.3.2The effect of different concentrations of atorvastatin calcium on the plateletaggregation induced by AA(0.5mmol/L):the platelet aggregation rates in CN、CD、C1-3were16.60±2.77ohms、16.33±2.66ohms、15.50（4） ohms、15.06±2.84ohms and14.00(3)ohms respectively. There were statisticallydifferences among these groups (P=0.03). By the method of Mann-WhitneyU(α adjusted to0.005)，no statistical difference was found between the groupsof physiological saline and DMSO. There were differences between the controlgroups and the atorvastatin calcium groups, but no differences were foundamong the groups of atorvastatin calcium of different concentrations.The curveslopes were21.27±2.02ohms/min、21.07±2.28ohms/min、20.71±2.37ohms/min、19.41±1.47ohms/min and18.00(2)ohms/min respectively. Therewere statistically differences among these groups(P＜0.05). By the method ofMann-Whitney U(α adjusted to0.005)，no statistical difference was foundbetween the groups of physiological saline and DMSO. There were differencesbetween the control groups and the atorvastatin calcium groups and there weredifferences between the groups of atorvastatin of high concentration (C3) andatorvastatin of low concentration (C1).But no statistical differences were found between the atorvastatin calcium of moderate concentration group (C2) and theother two atorvastatin calcium groups.3.3The effect of different concentrations of atorvastatin calcium on the plateletaggregation induced by collagen(2μg/mL):the platelet aggregation rates in CN、CD、 C1-3were23.53±1.98ohms、22.43±1.75ohms、22.06±2.19ohms、21.39±2.57ohms and20.30±1.94ohms respectively. There were statisticallydifferences among these groups(P＜0.05). By the method of SNK-q, nostatistical difference was found between the groups of physiological saline andDMSO. There were differences between the control groups and the atorvastatincalcium groups and there were differences between the groups of atorvastatinof high concentration (C3) and atorvastatin of low concentration (C1).But nostatistical differences were found between the atorvastatin calcium of moderateconcentration group (C2) and the other two atorvastatin calcium groups. Thecurve slopes were26.00(2)ohms/min、26.00±1.55ohms/min、25.00(3)ohms/min、24.45±1.95ohms/min and22.00（4）ohms/min respectively.There were statistically differences among these groups(P＜0.05). By themethod of Mann-Whitney U(α adjusted to0.005)，no statistical difference wasfound between the groups of physiological saline and DMSO There weredifferences between the control groups and the atorvastatin calcium groups andthere were differences between the groups of atorvastatin of high concentration(C3) and atorvastatin of low concentration (C1).But no statistical differenceswere found between the atorvastatin calcium of moderate concentration group(C2) and the other two atorvastatin calcium groups.3.4The effect of different concentrations of atorvastatin calcium on the plateletaggregation induced by EP(1mg/mL): the platelet aggregation rates in CN、CDC1-3were20.68±2.30ohms、20.63±1.86ohms、19.60±2.16ohms、18.97±1.83ohms and17.76±1.66ohms respectively. There were statisticallydifferences among these groups (P＜0.05). By the method of SNK-q, nostatistical difference was found between the groups of physiological saline andDMSO. There were differences between the control groups and the atorvastatincalcium groups and there were differences between the groups of atorvastatin of high concentration (C3) and atorvastatin of low concentration (C1).But nostatistical differences were found between the atorvastatin calcium of moderateconcentration group (C2) and the other two atorvastatin calcium groups. Thecurve slopes were24.76±2.01ohms/min、24.63±1.77ohms/min、22.93±2.68ohms/min、21.94±1.92ohms/min and20.90±3.08ohms/minrespectively. There were statistically differences(P＜0.05). By the method ofSNK-q, no statistical difference was found between the groups of physiologicalsaline and DMSO.There were differences between the control groups and theatorvastatin calcium groups and there were differences between the groups ofatorvastatin of high concentration (C3) and atorvastatin of low concentration(C1).But no statistical differences were found between the atorvastatin calciumof moderate concentration group (C2) and the other two atorvastatin calciumgroups.Conclusions:The in vitro effect of atorvastatin calcium salt on the platelet aggregationof platelet puree collected from healthy subjects depends on the concentrationof platelet puree. Atorvastatin calcium salt can inhibit the platelet aggregationof platelet puree with concentration of300×109/L and concentration of600×109/L,but the inhibitory effect of atorvastatin calcium salt on the plateletaggregation of platelet puree with low concentration(100×109/L) was notobserved. We observed that the inhibitory effect of atorvastatin calcium salt onthe platelet aggregation induced by collagen and EP were more significantcompared with that induced by the other two agonists. The results indicate thatthe reactivity of platelet aggregation to atorvastatin may vary with the differentpathways of platelet activation. In addition, between the concentrations of10-7mol/L and10-5mol/L,atorvastatin dose not dose-dependently inhibit theplatelet aggregation of platelet puree.