(N)-methanocarba-2MeSADP (MRS2365) is a subtype-specific agonist that induces rapid desensitization of the P2Y 1 receptor of human platelets D. M. Bourdon * , S. K. Mahanty † , K. A. Jacobson ‡ , J. L. Boyer † , and T. K. Harden * * Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC † Inspire Pharmaceuticals Inc., Durham, NC ‡ Molecular Recognition Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA Summary Adenosine diphosphate (ADP) initiates and maintains sustained aggregation of platelets through simultaneous activation of both the G q -coupled P2Y 1 receptor and the G i -coupled P2Y 12 receptor. We recently described the synthesis and P2Y 1 receptor-specific agonist activity of (N)-methano- carba-2MeSADP (MRS2365). Consequences of selective activation of the P2Y 1 receptor by MRS2365 have been further examined in human platelets. Whereas MRS2365 alone only induced shape change, addition of MRS2365 following epinephrine treatment, which activates the G i/z - linked,  2A -adrenergic receptor, resulted in sustained aggregation that was indistinguishable from that observed with ADP. Conversely, the platelet shape change promoted by ADP in the presence of the GP IIb / IIIa antagonist eptifibatide was similar to that promoted by MRS2365. Preaddition of the high affinity P2Y 1 receptor antagonist MRS2500 inhibited the effect of MRS2365, whereas addition of MRS2500 subsequent to MRS2365 reversed the MRS2365-induced shape change. Preactivation of the P2Y 1 receptor with MRS2365 for 2 min resulted in marked loss of capacity of ADP to induce aggregation as evidenced by a greater than 20-fold rightward shift in the concentration effect curve of ADP. This inhibitory effect of P2Y 1 receptor activation was dependent on the concentration of MRS2365 (EC 50 = 34 nM). The inhibitory effect of preincubation with MRS2365 was circumvented by activation of the Gq-coupled 5-HT 2a receptor suggesting that MRS2365 induces loss of the ADP response as a consequence of desensitization of the G q -coupled P2Y 1 receptor. The time course of MRS2365-induced loss of aggregation response to epinephrine was similar to that observed with ADP. These results further demonstrate the P2Y 1 receptor selectivity of MRS2365 and illustrate the occurrence of agonist-induced desensitization of the P2Y 1 receptor of human platelets studied in the absence of P2Y 12 receptor activation. Keywords adenosine diphosphate; desensitization; MRS2365; P2Y 1 receptor; P2Y 12 receptor; platelets Introduction Adenosine diphosphate (ADP) was first reported to be a potent activator of platelet aggregation in 1961 [1] and is now known to be the cognate agonist for the P2Y 1 , P2Y 12 , © 2006 International Society on Thrombosis and Haemostasis Correspondence: T. Kendall Harden, Department of Pharmacology, School of Medicine, University of North Carolina, CB# 7365, Chapel Hill, NC 27599-7365, USA. Tel.: +1 919 966 4816; fax: +1 919 966 5640; tkh@med.unc.edu. NIH Public Access Author Manuscript J Thromb Haemost. Author manuscript; available in PMC 2012 August 08. Published in final edited form as: J Thromb Haemost. 2006 April ; 4(4): 861–868. doi:10.1111/j.1538-7836.2006.01866.x. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript