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Monitoring Antiplatelet Aggregation In Vivo and In Vitro by Microtiter Plate Method
Qiu-Ling Wu1,2, Jun Dong3, Hua-Wu Zeng2
1School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, China.
Background:
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.
Objectives:
To evaluate the microtiter plate method by using aspirin and clopidogrel in vivo and in vitro.
Methods:
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.
Results:
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.
Conclusions:
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.
Insights
This study validates a microtiter plate method for evaluating platelet function, showing it effectively measures antiplatelet aggregation with aspirin and clopidogrel in vitro and in vivo.
Area of Science:
- Pharmacology
- Biotechnology
- Hematology
Background:
- Traditional light transmission aggregometry for platelet function is time-consuming and lacks parallelism.
- Existing microtiter plate methods offer high-throughput potential but require further practical validation.
Purpose of the Study:
- To evaluate the efficacy of a microtiter plate method for assessing platelet aggregation.
- To validate the method using aspirin and clopidogrel in both in vitro and in vivo settings.
Main Methods:
- Optimized in vitro assays using varying concentrations of arachidonic acid (AA) and adenosine diphosphate (ADP) agonists with aspirin and clopidogrel.
- Combination index (CI) calculation to assess synergistic effects of aspirin and clopidogrel.
- In vivo study in rabbits involving daily administration of aspirin, clopidogrel, or their combination for seven days, followed by platelet aggregation measurement.
Main Results:
- Established optimal concentrations for AA (50 μM) and ADP (5 μM) to induce concentration-dependent platelet aggregation.
- Demonstrated concentration-dependent antiplatelet effects of aspirin and clopidogrel in vitro, with synergistic inhibition observed.
- Confirmed in vivo inhibition of platelet aggregation by aspirin and clopidogrel, with combined therapy showing significantly enhanced effects.
Conclusions:
- The optimized microtiter plate method, utilizing multiple agonist concentrations, offers a robust approach to platelet function testing.
- This method mitigates variations in agonist-induced platelet response, presenting a promising tool for platelet aggregation detection.
