Anti-platelet drugs are used to prevent intra-arterial thrombus formation after rupture of atherosclerotic plaques. Until now, the inhibitory effect of present and future anti-platelet drugs such as aspirin, ADP receptor P2Y(1)/P2Y(12) antagonists and glycoprotein (GP) Ibalpha inhibitors on the interaction of platelets with human plaques is not known. To study those effects we obtained human atherosclerotic plaques by surgical endarterectomy. Plaques induced maximal platelet aggregation in hirudinized platelet-rich plasma (PRP) and blood that was effectively inhibited by aspirin, the P2Y(1) antagonist MRS2179 and the P2Y(12) antagonist AR-C69931MX, but not by GPIbalpha blockade with the mAB 6B4. Inhibition of platelet aggregation by MRS2179 was 74 +/- 37% and 68 +/- 20%, by AR-C69931MX 94 +/- 7% and 80 +/- 6%, and by aspirin 88 +/- 19% and 64 +/- 28%, in PRP and blood, respectively (mean +/- SD; n = 6-12 with plaques from 6 patients). The combination of both ADP receptor antagonists completely inhibited plaque-induced platelet aggregation in hirudinized PRP and blood. Under arterial flow conditions (1,500s(-1)), blockade of platelet GPIbalpha resulted in a strong decrease of plaque-stimulated platelet adhesion/aggregate formation of 77 +/- 5% (mean +/- SD; n = 4). Furthermore, MRS2179, AR-C69931MX and their combination reduced plaque-dependent platelet aggregate formation by 35 +/- 14%, 32 +/- 13% and 58 +/- 12% (mean +/- SD; n = 5), respectively. Aspirin was without significant effect. In conclusion, a GPIbalpha-blocking antibody, as well as P2Y(1) and P2Y(12) receptor antagonists, alone or in combination, reduce in contrast to aspirin human plaque-induced platelet thrombus formation under arterial flow. Although these new anti-platelet agents inhibit platelet thrombus formation after plaque rupture, more efficient platelet blockers are required.

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SummarySeveral methods are used to analyse platelet function in whole blood. A new device to measure whole blood platelet aggregation has been developed, called multiple electrode platelet aggregometry (MEA). Our aim was to evaluate MEA in comparison with the single platelet counting (SPC) method for the measurement of platelet aggregation and platelet inhibition by aspirin or apyrase in diluted whole blood. Platelet aggregation induced by different concentrations of ADP, collagen and TRAP-6 and platelet inhibition by apyrase or aspirin were determined in citrateor hirudin-anticoagulated blood by MEA and SPC. MEA indicated that spontaneous platelet aggregation was lower, and stimulated platelet aggregation was higher in hirudin- than citrate-anticoagulated blood. In hirudin-anticoagulated, but not citrate-anticoagulated blood, spontaneous platelet aggregation measured by MEA was inhibited by apyrase. For MEA compared with SPC the dose response-curves of agonist-induced platelet aggregation in citrate- and hirudin-blood showed similar EC50 values for TRAP, and higher EC50 values for ADP (non-significant) and collagen (p<0.05). MEA and the SPC method gave similar results concerning platelet-inhibition by apyrase and aspirin. MEA was more sensitive than SPC to the inhibitory effect of aspirin in collagen-induced aggregation. In conclusion, MEA is an easy, reproducible and sensitive method for measuring spontaneous and stimulated platelet aggregation, and evaluating antiplatelet drugs in diluted whole blood. The use of hirudin as an anticoagulant is preferable to the use of citrate. MEA is a promising technique for experimental and clinical applications.

BackgroundPoor platelet inhibition by aspirin or clopidogrel has been associated with adverse outcomes in patients with cardiovascular diseases. A reliable and facile assay to measure platelet inhibition after treatment with aspirin and a P2Y12 antagonist is lacking. Multiple electrode aggregometry (MEA), which is being increasingly used in clinical studies, is sensitive to platelet inhibition by aspirin and clopidogrel, but a critical evaluation of MEA monitoring of dual anti-platelet therapy with aspirin and P2Y12 antagonists is missing.Design and MethodsBy performing in vitro and ex vivo experiments, we evaluated in healthy subjects the feasibility of using MEA to monitor platelet inhibition of P2Y12 antagonists (clopidogrel in vivo, cangrelor in vitro) and aspirin (100 mg per day in vivo, and 1 mM or 5.4 mM in vitro) alone, and in combination. Statistical analyses were performed by the Mann-Whitney rank sum test, student' t-test, analysis of variance followed by the Holm-Sidak test, where appropriate.ResultsADP-induced platelet aggregation in hirudin-anticoagulated blood was inhibited by 99.3 ± 1.4% by in vitro addition of cangrelor (100 nM; p < 0.001) and by 64 ± 35% by oral clopidogrel (600 mg) intake (p < 0.05; values are means ± SD). Pre-incubation of blood with aspirin (1 mM) or oral aspirin intake (100 mg/day for 1 week) inhibited arachidonic acid (AA)-stimulated aggregation >95% and 100 ± 3.2%, respectively (p < 0.01). Aspirin did not influence ADP-induced platelet aggregation, either in vitro or ex vivo. Oral intake of clopidogrel did not significantly reduce AA-induced aggregation, but P2Y12 blockade by cangrelor (100 nM) in vitro diminished AA-stimulated aggregation by 53 ± 26% (p < 0.01). A feasibility study in healthy volunteers showed that dual anti-platelet drug intake (aspirin and clopidogrel) could be selectively monitored by MEA.ConclusionsSelective platelet inhibition by aspirin and P2Y12 antagonists alone and in combination can be rapidly measured by MEA. We suggest that dual anti-platelet therapy with these two types of anti-platelet drugs can be optimized individually by measuring platelet responsiveness to ADP and AA with MEA before and after drug intake.