The inhibitory effects of nitric oxide and prostaglandin I2 on platelet aggregation are greatly enhanced by blockade of P2Y12 receptors

R Knowles1, MH Lundberg1, NS Kirkby1,2, PC Armstrong1, AD Timmis3, JA Mitchell2, TD Warner1. 1The William Harvery Research Institute, Barts and the LondonSchool of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, UK, 2National Heart and Lung Institute, Imperial College London, London SW3 6LY, UK, 3Department of Cardiology, Barts Health NHS Trust,, London, UK

P2Y12 receptor antagonists such as clopidogrel and prasugrel that block the pro-aggregatory effects of ADP upon platelets are widely used for anti-thrombotic protection. Activation of P2Y12 receptors by ADP leads to inhibition of platelet adenylyl cyclase and a reduction in intra-platelet levels of cAMP, resulting in an increase in platelet reactivity. Notably, cAMP synergises with cGMP to produce greater platelet inhibition than cAMP alone, and we have recently shown that P2Y12 receptor activation strongly reduces the platelet inhibitory effects of intraplatelet cGMP (1), in addition to its interaction with cAMP (2). In the normal circulation, endothelial cells produce prostaglandin I2 (PGI2) and nitric oxide (NO) which increase intraplatelet levels of cAMP and cGMP and synergise to inhibit platelets. Here we have continued to test our hypothesis that part of the anti-thrombotic effect of P2Y12 receptor antagonists is explained by their ability to potentiate the effects of cAMP and cGMP and so amplify the existing in vivo synergy between PGI2 and NO.

Blood was obtained by venepuncture from 8 healthy volunteers into 0.32% (w/v) tri-sodium citrate and platelet rich plasma (PRP) was produced by centrifugation. PRP was then incubated with the P2Y12 receptor blocker prasugel-active metabolite (PAM, 1.5, 3 and 6μM; or vehicle, 0.5% DMSO). Light transmission aggregometry was then used to determine the aggregation of PAM-treated platelets in response to ADP 20μM, collagen 4μg/ml or TRAP-6 25μM in the presence of PGI2 1nM and/or the NO donor, DEA/NONOate 100nM and/or vehicle (n=4 for all experiments). Data was analysed by 1-way ANOVA with Tukey post-hoc analysis. and p<0.05 was taken as significant.

PGI2 or DEA/NONOate alone in the presence of PAM generally caused only minor reductions in platelet aggregation, whereas the combination of PGI2 and DEA/NONOate together with PAM produced far greater inhibitions of aggregation.. For instance, platelet aggregation in response to TRAP-6 in the presence of PAM 3μM + vehicle was 65±4%, PAM + PGI2 63±1%, PAM + DEA/NONOate 55±3%, and PAM + PGI2 + DEA/NONOate 20±6% (p<0.001, combination vs. vehicle). In the presence of PAM 1.5μM, collagen induced platelet aggregation of 74±3% in the presence of vehicle, PAM + PGI2 60±10%, PAM + DEA/NONOate 58±6%, and PAM + PGI2 + DEA/NONOate 21±10% (p<0.001, combination vs. vehicle). Similarly, in the presence of PAM 1.5μM, ADP induced platelet aggregation of 70±5% in the presence of vehicle, PAM + PGI2 49±13%, PAM + DEA/NONOate 30±7%, PAM + PGI2 + NO 12±2% (p<0.001, combination vs. vehicle).

These studies support our previous finding that blockade of P2Y12 receptors potentiates the anti-aggregatory effects of NO, from the donor DEA/NONOate. Moreover, they demonstrate that even partial blockade of P2Y12 receptors in the presence of low concentrations of NO and PGI2 can produce strong inhibition of platelet aggregation. This may well be relevant to the efficacy of anti-platelet therapies in patients with endothelial dysfunction.

References

1. Kirkby NS et al, Proc Natl Acad Sci U S A 110:15782, 2013

2. Cattaneo M & Lecchi A, J Thromb Haemost 5:577, 2007