Monitoring the effects of platelet glycoprotein IIb/IIIa antagonists with a microtiter plate method for detection of platelet aggregation

Abstract

Measurement of platelet aggregation in platelet-rich plasma (PRP) is a fundamental tool in platelet studies, despite the fact that the technique required for this is time-consuming, may need large volumes of blood, and require particular skill and special equipment. The use of a microplate reader seems useful to perform platelet aggregation more rapidly and with less material. So, the aim of the present study was to validate a simple and rapid method which enables performance of kinetic measurements of platelet aggregation directly in a microtiter plate reader. Platelet aggregation was carried out in 96-well, flat-bottomed microtiter plates. Samples of PRP (140 w l/well) were placed in a microtiter plate. Agonists (10 w l/well) were added using an electronic multichannel dispenser directly before the reading was started. Measurements of the optical density were performed at 650 nm using a THERMOmax™ microplate reader (Molecular Devices, Sunnyvale, USA). During the run time the plate was incubated at 37°C and was mixed with the automix function of the reader. The technique was verified by comparing dose-response curves of platelet agonists and glycoprotein IIb/IIIa antagonists, obtained with the standard aggregometer and with the microtiter plate reader. Platelet aggregation in microtiter plates is simple and rapid. It offers the advantages of lowering the test volumes and the possibility to perform about 90 tests simultaneously. The method was successfully applied to measure platelet inhibition by glycoprotein IIb/IIIa antagonists.