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

Abstract

Aspirin 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 (0.1-30microM), arachidonic acid (0.03-1.3mM), collagen (0.1-30microg/ml), adrenaline (0.001-100microM), ristocetin (0.2-3mg/ml), TRAP6 amide (0.130microM) and U46619 (0.130microM). Concentration response curves were constructed to each agonist under the various conditions and used to extract data such as log EC(50), Hill slope, and area under the curve. These demonstrated low intra- and 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.

Standardised optical multichannel (optimul) platelet aggregometry using high-speed shaking and fixed time point readings

“…As we have previously reported in standard 96-well plate and optimul kinetic aggregometry [1,[4][5][6], we found that aspirin significantly inhibited ( p < 0.05) aggregation induced by EPI and completely abolished responses to AA and collagen. PAM, however, significantly attenuated ( p < 0.05) aggregation induced by all agonists tested, except for ristocetin, which acts through a process that involves agglutination and not aggregation.…”

96-well plate-based aggregometry

“…A further variation between research groups is the duration of shaking, reports include bursts of intermittent shaking for up to 18 minutes or one continuous shake for 5 minutes (17,25). We have previously published traces of absorbance measured every 15 seconds following shaking after agonist stimulation over 16 minutes (12). Since this is slower than LTA, we have subsequently utilized a stand-alone high-speed shaker that provides uniform mixing through the production of vortices in each well of the plate and generates more rapid platelet responses.…”

Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay

“…Optimization and characterization of the Optimul assay have been previously reported. 13,14,17 An Optimul panel was defined as abnormal if the dose-response curve was below the lower limit of the 95% reference interval for .1 agonist.…”