Monitoring Antiplatelet Aggregation In Vivo and In Vitro by Microtiter Plate Method.

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.