Inverse agonism at the P2Y12 receptor and ENT1 transporter blockade contribute to platelet inhibition by ticagrelor.
Ticagrelor is a potent antagonist of the P2Y12receptor (P2Y12R) and consequently an inhibitor of platelet activity effective in the treatment of atherothrombosis. Here, we sought to further characterize its molecular mechanism of action. Initial studies showed that ticagrelor promoted a greater inhibition of adenosine 5'-diphosphate (ADP)-induced Ca2+release in washed platelets vs other P2Y12R antagonists. This additional effect of ticagrelor beyond P2Y12R antagonism was in part as a consequence of ticagrelor inhibiting the equilibrative nucleoside transporter 1 (ENT1) on platelets, leading to accumulation of extracellular adenosine and activation of Gs-coupled adenosine A2Areceptors. This contributed to an increase in basal cyclic adenosine monophosphate (cAMP) and vasodilator-stimulated phosphoprotein phosphorylation (VASP-P). In addition, ticagrelor increased platelet cAMP and VASP-P in the absence of ADP in an adenosine receptor-independent manner. We hypothesized that this increase originated from a direct effect on basal agonist-independent P2Y12R signaling, and this was validated in 1321N1 cells stably transfected with human P2Y12R. In these cells, ticagrelor blocked the constitutive agonist-independent activity of the P2Y12R, limiting basal Gi-coupled signaling and thereby increasing cAMP levels. These data suggest that ticagrelor has the pharmacological profile of an inverse agonist. Based on our results showing insurmountable inhibition of ADP-induced Ca2+release and forskolin-induced cAMP, the mode of antagonism of ticagrelor also appears noncompetitive, at least functionally. In summary, our studies describe 2 novel modes of action of ticagrelor, inhibition of platelet ENT1 and inverse agonism at the P2Y12R that contribute to its effective inhibition of platelet activation.
School of Physiology, Pharmacology and Neuroscience, Faculty of Biomedical Sciences, University of Bristol, Bristol, United Kingdom.
Riyaad
R
Aungraheeta
School of Physiology, Pharmacology and Neuroscience, Faculty of Biomedical Sciences, University of Bristol, Bristol, United Kingdom.
Alexandra
A
Conibear
School of Physiology, Pharmacology and Neuroscience, Faculty of Biomedical Sciences, University of Bristol, Bristol, United Kingdom.
Mark
M
Butler
School of Physiology, Pharmacology and Neuroscience, Faculty of Biomedical Sciences, University of Bristol, Bristol, United Kingdom.
Eamonn
E
Kelly
Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden; and.
Sven
S
Nylander
School of Clinical Sciences, Faculty of Health Sciences, University of Bristol, Bristol, United Kingdom.
Andrew
A
Mumford
School of Physiology, Pharmacology and Neuroscience, Faculty of Biomedical Sciences, University of Bristol, Bristol, United Kingdom.
Stuart J
SJ
Mundell
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D033721:Equilibrative Nucleoside Transporter 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D015539:Platelet Activation / Q000187:drug effects*; D058925:Receptors, Purinergic P2Y12 / Q000378:metabolism*; D000077486:Ticagrelor; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D033721:Equilibrative Nucleoside Transporter 1; D008840:Microfilament Proteins; C548576:P2RY12 protein, human; D010750:Phosphoproteins; D058925:Receptors, Purinergic P2Y12; C438649:SLC29A1 protein, human; D000098662:Vasodilator-Stimulated Phosphoprotein; D005576:Colforsin; D000244:Adenosine Diphosphate; D000242:Cyclic AMP; D000077486:Ticagrelor; D000241:Adenosine; D002118:Calcium
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