Dose-responses to inducers and inhibitors of platelet aggregation analysed via a micro-method.

Abstract

The ex vivo study of platelet function in small experimental animals treated with anti-platelet drugs is restricted by the limited availability of platelet-rich plasma (PRP). To circumvent this obstacle, a micro-method for platelet aggregation was developed that enables ED50 measurements for various inducers of platelet aggregation. Fifty microliters of human, hamster, or rabbit PRP was mixed with agonist in each well of a microtiter plate, and shaken at 900 rpm at 37 degrees C for intervals up to 5 min. After stopping the aggregation with formaldehyde in PBS, light scattering was measured vs platelet-poor plasma (PPP) at 620 nm. Thus, aggregation in human PRP by ADP (t = 2 min) occurred with an ED50 = 1.8 microM, whereas the collagen (t = 3 min; ED50 = 2 micrograms/ml) and AA (t = 1 min; ED50 = 0.3 microM) induced aggregation occurred at those concentrations that induce aggregation in classical aggregometry. Likewise, aggregation was inhibited by the anti-GPIIb/IIIa antibody MA-16N7C2 and by the GPIIb/IIIa antagonists G4120 or MK-852. In comparison with human PRP, hamster (ED50 = 0.8 microM at t = 2 min) and rabbit (ED50 = 5 microM at t = 4 min) platelet aggregation by ADP occurred with comparable sensitivities, whereas the aggregation of rabbit platelets by collagen (ED50 = 15 micrograms/ml at t = 3 min) appeared to be slightly less sensitive and subject to large interindividual variations. The method was applied to measure plasma levels of a GPIIb/IIIa antagonist following injection into hamsters.