Multiple electrode aggregometry and P2Y12 antagonists

Thromb Haemost 2008; 99: 1127–1129 Dear Sir, In a recent paper, Sibbing et al. used multiple electrode aggregometry (MEA) to quantitate the effects of clopidogrel on ADP-induced platelet aggregation following administration of the drug (1). This is the most recent of several publications on the use of MEA to assess platelet aggregation (2–5) and highlights the potential value of the approach as a means of monitoring the effects of P2Y12 antagonists on platelet function. MEA provides a measurement of platelet aggregation in whole blood by monitoring changes in electrical impedance via disposable test cells each containing two pairs of electrodes (6), and is a development of earlier forms of impedance aggregometry that are also currently being used to monitor the effects of clopidogrel and other P2Y12 antagonists (7–10). The manufacturers of the Multiplate® instrument (Dynabyte GmbH, Munich, Germany) suggest two possible approaches to monitor the effects of P2Y12 antagonists: the ADPtest and the high-sensitivity ADPtest (ADPtest HS) (6). The ADPtest is used to determine platelet aggregation in response to ADP in whole blood; the ADPtest HS is used to determine platelet aggregation in response to ADP in whole blood in the presence of a low concentration of PGE1 which is known to potentiate the ability of P2Y12 antagonists to inhibit platelet aggregation (11). In both cases the current recommendation is that the tests are performed using blood containing hirudin as the anticoagulant. The use of hirudin is suggested rather than citrate (the conventional anticoagulant used for studies of platelet aggregation) because of its ability to prevent blood coagulation without reducing the concentrations of divalent cations to very low levels. Against this background we used MEA to compare the ADPtest and the ADPtest HS in blood anticoagulated using hirudin (using tubes supplied by the manufacturers of the Multiplate instrument) or citrate (trisodium citrate dihydrate at a final concentration in blood of 0.313% w/v), and to measure the degree of inhibition achieved using a range of concentrations of three different P2Y12 antagonists. One was AR-C69931 (now known as cangrelor) which is a direct-acting agent that is currently undergoing late-stage clinical evaluation for intravenous use during coronary procedures (12). The second was the active metabolite of clopidogrel (C-AM) which mediates the inhibitory effects of clopidogrel following administration to patients with cardiovascular disease (13, 14). The third was the active metabolite of prasugrel (P-AM) a new P2Y12 antagonist that is currently undergoing clinical evaluation. Apparently, an advantage of prasugrel over clopidogrel is that P-AM is produced more rapidly and in larger quantities than C-AM following oral administration of the parent drug (15). C-AM and P-AM were kindly provided by Daiichi Sankyo Co. Ltd. (Tokyo, Japan) and Eli Lilly and Company (Indianapolis, IN, USA). At the same time we looked at the effects of the agents on platelet aggregation induced by TRAP6-using the TRAPtest. In performing the experiments the P2Y12 antagonist (in 0.3ml isotonic saline) was added to whole blood (0.3 ml) in a Multiplate test cell which was then warmed for 3 min to bring the temperature up to 37oC. The ADP, ADP/PGE1 or TRAP-6 was then added and aggregation was measured over 6 min. The conditions of the experiments were exactly as recommended by the manufacturers of the Multiplate instrument (6) and platelet aggregation was expressed as area under the curve (AUC). For each of the P2Y12 antagonists all experiments were performed using blood from six normal healthy volunteers. The results are shown in Figure 1. In all tests aggregation induced by ADP or TRAP-6 was enhanced in blood containing hirudin rather than citrate. AR-C69931 markedly inhibited platelet aggregation both in the ADPtest and in the ADPtest HS. It also reduced TRAP-induced aggregation although to a much smaller extent. C-AM produced concentration-dependent inhibition of ADP-induced aggregation in the ADPtest, and in the presence of PGE1 (the ADPtest HS) it appeared to produce stronger inhibition. In both tests C-AM produced stronger inhibition in blood containing hirudin. Like AR-C69931 it produced a small inhibition of platelet aggregation induced by TRAP-6. Results obtained using P-AM were very similar to those obtained using C-AM. Again inhibition was strongest using the ADPtestHS and in blood containing hirudin as the anticoagulant. Another recently published paper provides interesting reading in relation to our results with C-AM and P-AM. Frelinger et al. (16) studied the inhibitory effects of the active metabolite of prasugrel on ADP-induced aggregation in whole blood, also using impedance aggregometry. These authors demonstrated that P-AM brings about more rapid inhibition of ADP-induced aggregation in blood anticoagulated with the thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) compared with citrate. Use of PPACK, like hirudin, also achieves anticoagulation without any reduction in the concentration of divalent cations in plasma. Clearly the nature of the anticoagulant has an influence on the extent of the aggregation induced by ADP (and also by TRAP-6) as measured by MEA and influences the degree of inhibition of ADP-induced aggregation brought about by a P2Y12 Letters to the Editor