The efficacy of P2Y12 receptor antagonist therapy is strongly moderated by the endothelial derived mediators prostacyclin and nitric oxide

R.B Knowles1, M.V Chan1, P.C.J Armstrong1, C.C Shih3,1, M.A Hayman1, I Vojnovic1, A.T Tucker1, A.D Timmis2, T.D Warner1. 1William Harvey Research Institute, London, UK, 2Department of Cardiology, Barts and the London NHS Trust, London, UK, 3Department of Pharmacology, National Defense Medical, Taipei, Taiwan

Circulating platelets are constantly exposed to the endogenous, endothelial mediators, prostacyclin (PGI2) and nitric oxide (NO), which synergise powerfully to keep the platelet in an inhibited state. When stimulated, platelets override these negative influences to produce secondary mediators, ADP and thromboxane (TX) A2, which drive thrombus formation. Consequently, at risk patients are prescribed dual anti-platelet therapy (DAPT), consisting of a P2Y12 receptor antagonist, such as prasugrel, and aspirin, which inhibit these pathways. P2Y12 receptor antagonists have recently been shown to also exert part of their powerful anti-thrombotic effects by amplifying the inhibitory potency of NO and PGI2. Thus, the in vivo synergy between P2Y12 inhibition, NO and PGI2 suggests that the therapeutic potential of P2Y12 inhibitors is strongly determined by the level of endothelial function in individual patients. Our aim, therefore, was to characterise the interplay between these factors.

Three groups of 8 healthy male volunteers received either prasugrel (10mg), aspirin (75mg) or DAPT (prasugrel + aspirin) once daily for 7 days. Platelet-rich plasma (PRP) was obtained by centrifugation before and after treatment. Platelet responses to TRAP-6 amide (25μM) in the presence of PGI2 (1nM) and/or the NO donor, DEA/NONOate (100nM) or vehicle, were assessed by light transmission aggregometry. In parallel in vitro studies, PRP prepared from drug-free volunteers was incubated with aspirin (30μM) and/or prasugrel-active metabolite (PAM; 1.5, 3, 6μM) to represent partial and maximal P2Y12 receptor blockade. Data was analysed by two-way ANOVA with Tukey post-hoc analysis and p<0.05 was taken as significant.

Ex-vivo, there was little effect of PGI2 and/or DEA/NONOate on platelet aggregation prior to anti-platelet therapy. Aspirin alone produced moderate inhibitory effects, though these were significant (p<0.05) in the presence of PGI2 + DEA/NONOate (58±8% to 28±9%). Following prasugrel therapy, inhibitory effects were observed in all conditions, but were significantly (p<0.05) enhanced in the presence of PGI2 + DEA/NONOate (63±3% to 7±3%). Furthermore, the same pattern was seen after DAPT in the presence of PGI2 +DEA/NONOate (67±5% to 19±6%, p<0.05).

In vitro, following aspirin and maximum P2Y12 (PAM, 6μM) inhibition, aggregation to TRAP-6 (25μM) was significantly (p<0.05) reduced in all conditions. Following partial P2Y12 (PAM, 1.5μM) inhibition, however, only PGI2+DEA/NONOate significantly (p<0.05) inhibited aggregation (67%±5 to 35±15%).

We demonstrate that PGI2, and NO synergise with P2Y12 receptor antagonists to produce powerful and optimal platelet inhibition and that even with submaximal P2Y12 blockade, the presence of PGI2 and NO greatly enhances platelet inhibition. Our findings highlight the importance of endothelial driven in vivo modulation of P2Y12 inhibition and introduces the concept of refining ex vivo platelet function testing by incorporating an assessment of endothelial function to better predict thrombotic outcomes in individual patients.

1. Kirkby, N.S, et al. Blockade of the purinergic P2Y12 receptor greatly increases the platelet inhibitory actions of nitric oxide. Proc Natl Acad Sci U S A, 110:15782, (2013).

2. Cattaneo, M. & Lecchi, A. Inhibition of the platelet P2Y12 receptor for adenosine diphosphate potentiates the antiplatelet effect of prostacyclin. J Thromb Haemost 5, 577–582 (2007).