The Effect Of Salvianolate B On Platelet Aggregation In Healthy Subjects And Patients With Coronary Artery Disease

Background: With the development of our society and people’s livingstandard, coronary heart disease （CAD） morbidity and mortality increased,and showed a trend of younger and a serious threat to the health of people’slives. Platelet activitation, adhesion and aggregation are key factors in theoccurrence and development in atherosclerosis, so antiplatelet therapy iscrucial in conventional cardiovascular disease drug therapy. Detection ofplatelet aggregation is commonly used to observe the therapeutic effect.Aspirin, an irreversible inhibitor of thromboxane A₂production, incombination with clopidogrel, an inhibitor of PY12ADP platelet receptors,represents the current standard-of-care of antiplatelet therapy for patients withacute coronary syndrome and those undergoing percutaneous coronaryintervention. With the development of the equipment and technology, Chineseherbal extracts also played an important role in the treatment of coronary heartdisease.At present, Salvianolate B received the most attention, which is thepurification of the extract of Danshen. Some experiments show that theSalvianolate B is antioxidant, inhibiting the oxidative modification of lowdensity lipoprotein, the intercellular connections and the matrixmetalloproteinase-9expression，even can resist inflammatory. Then it canagainst atherosclerosis, protect the heart and brain blood vessels. Others showthat the Salvianolate B can lower the P-selectin, enhance the activity ofplatelet eNOS, and decrease the platelet aggregation induced by ADP and EP.But up to now, these studies are in vivo tests, where confounding factors aredifficult to be controled, so the inhibition of platelet aggregation and itsmechanism has not been confirmed. In our experiment,26cases of healthysubjects whole blood,8cases of CAD patients whole blood and26cases of platelet puree were sellected to associated with different concentrations ofSalvianolate B incubated in vitro, platelet aggregation and curve slope wereobserved with whole blood impedance method to provide the objective basisfor clinical use of Salvianolate B.Methods: The subjects were divided into three parts.①Healthy wholeblood group. A total of26cases of healthy volunteers （ages range from15～64years, men9, women17） were selected. The inclusion criteria include as thefollowings: Subjects with normal liver and kidney function, normal bloodroutine, female are in non-menstrual period.②CAD patients whole bloodgroup. A total of8cases of volunteers （ages range from51～78years, men5,women3） suffering from acute coronary dyndrome （ACS） including STEMI,NSTEMI and UA were enrolled. The criteria for exclusion include as thefollowings in the first two groups: patients with sorts of hematic desease,hemorrhagic disease and bleeding tendency; patients received the therapy ofaspirin, clopidogrel, heparin and other anti-platelet drugs.③Platelet pureegroup. A total of26cases of fresh platelets puree were collected, which theplatelet concentration is about1280×109/L. The inclusion and exclusioncriteria are strictly in accordance with national standards for blood donationlaw.Blood samples were extracted from the elbow vein and femoral vein inthe first two groups, and then were prepaired into blank control group, thefinal concentration of salvianolate B were1mg/L,10mg/L,100mg/L in theexperimental group (C_0-3) respectively. Aggregation and curve slope wereassessed in1hour with whole blood impedance analyzer （Model590）, madeby Chrono-Log Corporation, which induced by ADP （10μmol/L）, arachidonicacid （AA0.5μmol/L） and epinephrine （EP1mg/mL）. The platelet pureesamples were tested in the same way. Data were processed with SPSS13.0software. When data are subject to normal distribution, continuous variableswere expressed as the mean value±standard deviation （SD）, the overallcomparison in each group was evaluated by single factor ANOVA analysis. Ifnot, the variables were expressed as the median and quartile （QR）, the overall comparison in each group was evaluated by Friedman’s M test. A P value of<0.05was considered statistically significant.Results:1The effect of Salvianolate B on platelet aggregation in healthy subjects.1.1Induced by ADP （10μmol/L）: the platelet aggregation rates in C_0-3were7.00（5.00） ohms,7.04±3.17ohms,7.00±3.73ohms, and7.12±4.71ohmsrespectively. There were no significant difference among these groups（P=0.923）. The curve slopes were4.00（3.00） ohms/min,4.00（4.00） ohms/min,4.00（5.00） ohms/min, and4.00（5.00） ohms/min respectively. There were nosignificant difference among these groups （P=0.252）.1.2Induced by AA （0.5μmol/L）: the platelet aggregation rates in C_0-3were10.65±3.53ohms,11.19±4.20ohms,12.00（4.00） ohms, and12.08±3.70ohms respectively. There were no significant difference among these groups（P=0.096）. The curve slopes were6.50（5.00） ohms/min,6.81±2.93ohms/min,7.42±3.25ohms/min, and7.77±3.87ohms/min respectively. There were nosignificant difference among these groups （P=0.296）.1.3Induced by EP （1mg/ml）: the platelet aggregation rates in C_0-3were6.26±4.45ohms,5.00（8.00） ohms,5.42±3.20ohms, and8.95±5.10ohmsrespectively. There were no significant difference among these groups（P=0.157）. The curve slopes were2.00（1.00） ohms/min,2.00（1.00） ohms/min,2.00（1.00） ohms/min, and3.00（2.00） ohms/min respectively. There were nosignificant difference among these groups （P=0.405）.2The effect of Salvianolate B on platelet aggregation in CAD patients.2.1Induced by ADP （10μmol/L）: the platelet aggregation rates in C_0-3were3.50（8.00） ohms,3.50（5.00） ohms,4.63±3.34ohms，and3.88±3.44ohmsrespectively. There were no significant difference among these groups（P=0.546）. The curve slopes were2.00（3.00） ohms/min,2.00（3.00） ohms/min,3.50±1.31ohms/min, and2.00（2.00） ohms/min respectively. There were nosignificant difference among these groups （P=0.071）.2.2Induced by AA （0.5μmol/L）: the platelet aggregation rates in C_0-3were7.25±4.06ohms,6.88±4.05ohms,8.00（3.00） ohms, and7.75±4.43ohms respectively. There were no significant difference among these groups（P=0.919）. The curve slopes were4.88±2.03ohms/min,4.50±2.33ohms/min,4.00±1.60ohms/min, and4.63±2.07ohms/min respectively. There were nosignificant difference among these groups （P=0.850）.2.3Induced by EP （1mg/ml）: the platelet aggregation rates in C_0-3were3.50（5.00） ohms,3.50（4.00） ohms,1.50（3.00） ohms, and1.50（5.00） ohmsrespectively. There were no significant difference among these groups（P=0.312）. The curve slopes were2.00（1.00） ohms/min,2.00（1.00）ohms/min，2.00（1.00） ohms/min, and2.00（1.00） ohms/min respectively. Therewere no significant difference among these groups （P=0.896）.3The effect of Salvianolate B on platelet aggregation in platelet pureefrom healthy subjects.3.1Induced by ADP （10μmol/L）: the platelet aggregation rates in C_0-3were16.65±2.74ohms,15.50（2.00） ohms,15.65±3.15ohms, and15.50（3.00）ohms respectively. There were no significant difference among these groups（P=0.503）. The curve slopes were31.85±4.84ohms/min,32.00（6.00）ohms/min,30.50（5.00） ohms/min, and31.27±6.11ohms/min respectively.There were no significant difference among these groups （P=0.454）.3.2Induced by AA （0.5μmol/L）: the platelet aggregation rates in C_0-3were16.50±2.96ohms,15.65±2.02ohms,16.58±2.64ohms, and16.96±2.31ohms respectively. There were no significant difference among these groups（P=0.293）. The curve slopes were7.69±5.06ohms/min,39.00±4.99ohms/min,37.42±4.93ohms/min, and40.08±3.65ohms/min respectively. There were nosignificant difference among these groups （P=0.155）.3.3Induced by EP （1mg/ml）: the platelet aggregation rates in C_0-3are19.92±3.54ohms,17.50±2.79ohms,17.08±2.71ohms, and17.12±3.50ohmsrespectively. There was statistically difference （P=0.004）. But by way of themethod of SNK-q, no difference was found among concentrations ofsalvianolate B. The curve slopes were25.15±4.92ohms/min,24.23±5.06ohms/min,24.54±6.38ohms/min, and25.19±4.86ohms/min respectively.There were no significant difference among these groups （P=0.894）. Conclusions: Salvianolate B in concentrations used in this current studycan’t inhibite platelet aggregation induced by ADP, AA and EP in healthysubjects and CAD patients whole blood. Only in platelet puree which theplatelet concentration of approximately1280×10⁹/L, the Salvianolate B caninhibit platelet aggregation induced by EP, but there was no difference amonggroups of different concentrations of Salvianolate B. Moreover, Salvianolate Balso can’t inhibit platelet aggregation induced by ADP and AA in plateletpuree.