Utility of 96-well plate aggregometry and measurement of thrombi adhesion to determine aspirin and clopidogrel effectiveness

Abstract

SummaryAspirin and clopidogrel are key anti-thrombotic therapies. Results from platelet reactivity testing during therapy, have been shown to correlate with future events and would allow for the optimisation of therapy. However, there is little agreement among current tests and there remains a clear clinical need for a universal standardised test. It was the objective of this study to explore the potential of 96-well plate aggregometry as a definitive clinical test of platelet reactivity with respect to aspirin and clopidogrel.A small non-blinded trial of 16 healthy male volunteers assigned to seven days of aspirin (75mg/day) or clopidogrel (75mg/day) therapy. Blood was collected before and on day 7 of treatment. Platelet aggregation was measured using a 96-well plate based aggregation method, and thrombi adhesion measured by colourimetric assay. Platelet agonists used were ADP (0.1–30µM), arachidonic acid (0.03–1.3mM), collagen (0.1–30µg/ml), adrenaline (0.001–100µM), ristocetin (0.2–3mg/ ml),TRAP6 amide (0.130µM) and U46619 (0.130µM). Concentration response curves were constructed to each agonist under the various conditions and used to extract data such as log EC50, Hill slope, and area under the curve. These demonstrated low intraand inter-assay variability and strong discrimination of drug effects.This study demonstrates the ability of the 96-well plate based aggregation and adhesion method to detect and differentiate between stable aspirin and clopidogrel treatment in healthy volunteers.Moreover,this assay marries the ability to test subjects or patients using a range of platelet agonists with more rapidity and ease than the current gold standard platelet assay, traditional light transmission aggregometry, making it a serious alternative assay for use in clinical settings.