School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, China.
Department of Phytochemistry, School of Pharmacy, Second Military Medical University, Shanghai, China.
J Cardiovasc Pharmacol. 2020 Apr;75(4):314-320. doi: 10.1097/FJC.0000000000000801.
The current light transmission aggregation method is a recognized conventional method for platelet function evaluation, but it is time-consuming and poor in parallelism and cannot simultaneously monitor multiple inducers at multiple levels. The microtiter plate method has been established because of the high-throughput characteristic, but it needs more practical applications.
To evaluate the microtiter plate method by using aspirin and clopidogrel in vivo and in vitro.
In vitro, the platelet aggregations inhibited by aspirin (0.3, 1, 3, 10, 30, 90 μM) and clopidogrel (1, 3, 10, 30, 100, 300 μM) were evaluated with the presence of arachidonic acid (AA) and adenosine diphosphate (ADP) agonists. Using the combination index (CI), the effect of the combination of aspirin and clopidogrel on platelet aggregation was evaluated. In vivo, New Zealand rabbits (n = 18) were randomly divided into 3 groups, aspirin group (5 mg/kg, intragastrical gavage [i.g.]), clopidogrel group (14 mg/kg at the first day, followed by 4 mg/kg, i.g.), and the combination of these two drugs, administered (i.g.) continuously for 7 days. Then, the blood was collected to measure platelet aggregation.
Different concentrations of AA (12.5, 25, 50, 100 μM) and ADP (1.25, 2.5, 5, 10 μM) could promote platelet aggregation in concentration-dependent manner, and the most stable induction concentrations of AA and ADP were 50 and 5 μM. In vitro, with the above optimized detection system, aspirin and clopidogrel alone or in combination had concentration-dependent antiplatelet aggregation. The combination of aspirin and clopidogrel also showed synergistic inhibition effect within the concentration range studied. In vivo, aspirin and clopidogrel alone or in combination inhibited platelet aggregation induced by multiple concentrations of AA and ADP agonists, and the combined inhibition was more significant during the administration than aspirin or clopidogrel alone.
The improved microtiter plate method combining the use of multiple levels of multiple agonists avoids the variation of the effective inducer concentrations due to individual different response of platelets to agonists. It may be a potential approach in the detection of platelet aggregation.
目前的光传输聚集方法是一种公认的血小板功能评估常规方法，但耗时且平行性差，无法同时监测多个诱导物在多个水平上的情况。微孔板法由于高通量的特点而被建立，但需要更多的实际应用。
通过体内和体外实验评估阿司匹林和氯吡格雷的微孔板法。
在体外，使用花生四烯酸（AA）和二磷酸腺苷（ADP）激动剂评估阿司匹林（0.3、1、3、10、30、90 μM）和氯吡格雷（1、3、10、30、100、300 μM）抑制的血小板聚集。使用合并指数（CI）评估阿司匹林和氯吡格雷联合对血小板聚集的影响。在体内，将新西兰兔（n = 18）随机分为 3 组，阿司匹林组（5 mg/kg，灌胃[ig. ]）、氯吡格雷组（第 1 天 14 mg/kg，然后 4 mg/kg，ig. ）和两药联合给药（ig. ）连续 7 天。然后采集血液测量血小板聚集。
不同浓度的 AA（12.5、25、50、100 μM）和 ADP（1.25、2.5、5、10 μM）可以浓度依赖性地促进血小板聚集，AA 和 ADP 最稳定的诱导浓度分别为 50 和 5 μM。在体外，使用上述优化的检测系统，阿司匹林和氯吡格雷单独或联合使用具有浓度依赖性的抗血小板聚集作用。在研究浓度范围内，阿司匹林和氯吡格雷联合也表现出协同抑制作用。在体内，阿司匹林和氯吡格雷单独或联合抑制由多种浓度的 AA 和 ADP 激动剂诱导的血小板聚集，并且在给药期间的联合抑制比单独使用阿司匹林或氯吡格雷更为显著。
结合使用多个水平和多种激动剂的改良微孔板法避免了由于血小板对激动剂的个体反应不同而导致有效诱导剂浓度的变化。它可能是检测血小板聚集的一种潜在方法。
