Inverse agonism at the P2Y12 receptor and ENT1 transporter blockade contribute to platelet inhibition by ticagrelor

Abstract

Key Points• Ticagrelor acts as an inverse agonist at the P2Y 12 R, inhibiting basal agonistindependent signaling. • Ticagrelor inhibits the adenosine transporter ENT1 not only on erythrocytes, but on platelets too.Ticagrelor is a potent antagonist of the P2Y 12 receptor (P2Y 12 R) 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 59-diphosphate (ADP)-induced Ca 21 release in washed platelets vs other P2Y 12 R antagonists. This additional effect of ticagrelor beyond P2Y 12 R 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 G s -coupled adenosine A 2A receptors. 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 P2Y 12 R signaling, and this was validated in 1321N1 cells stably transfected with human P2Y 12 R. In these cells, ticagrelor blocked the constitutive agonist-independent activity of the P2Y 12 R, limiting basal G i -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 Ca 21 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 P2Y 12 R that contribute to its effective inhibition of platelet activation. (Blood. 2016;128(23):2717-2728

P2Y12 receptor modulation of ADP‐evoked intracellular Ca2+ signalling in THP‐1 human monocytic cells

“…Inhibition of the ADP response was approximately 80% at the highest concentrations of PSB‐0739 tested. Several studies have suggested that ticagrelor produces cardiovascular benefit through a pleiotropic inhibition of the equilibrative nucleoside transporter 1 (ENT‐1) (Aungraheeta et al ., ), and a consequent elevation in extracellular adenosine. To rule out this mechanism of action in THP‐1 cells, we inhibited ENT‐1 using 3 μM 6‐ S ‐[(4‐nitrophenyl)methyl]‐6‐thioinosine (NBMPR), but observed no effect on ADP‐evoked Ca 2+ responses (data not shown).…”

Restored response to ADP downstream of purinergic P2Y12 receptor in apheresis platelets after pathogen‐reducing xenon flash treatment

“…D and E). The observation that the addition of the P2Y 12 receptor antagonist AR‐C66096 enhanced VASP phosphorylation even in the presence of ADP could be explained by the Gi‐independent cAMP increase or inverse agonism by cangrelor or ticagrelor including AR‐C66096 …”

“…The observation that the addition of the P2Y 12 receptor antagonist AR-C66096 enhanced VASP phosphorylation even in the presence of ADP could be explained by the Giindependent cAMP increase or inverse agonism by cangrelor or ticagrelor including AR-C66096. 15,16 Akt is independently phosphorylated at Ser473 by mammalian target of rapamycin complex-2 and Thr308 by 3-phosphoinositide-dependent protein kinase-1, respectively. 17,18 Each phosphorylation plays a different role in Akt-mediated signal transduction of human platelets: p-Akt Ser473 mediates proline-rich Akt1 substrate of 40 kDa phosphorylation but is not essential for the aggregation response, while p-Akt Thr308 mediates glycogen synthase kinase 3b phosphorylation and plays an important role in the PLT functional response.…”

Update of P2Y receptor pharmacology: IUPHAR Review 27

“…Competitive P2Y 12 receptor antagonists include the nucleotide derivatives cangrelor ( 35 , AR‐C69931MX, N 6 ‐(2‐methylthioethyl)‐2‐(3,3,3‐trifluoropropylthio)‐β,γ‐dichloromethylene‐ATP) and AR‐C67085 (2‐propylthio‐β,γ‐dichloromethylene‐D‐ATP, 28 ; Ingall et al, 1999) as well as nucleoside derivatives such as the orally active ticagrelor ( 36 , AZD6140, Springthorpe et al, 2007; Hoffmann et al, 2009; Hoffmann et al, 2014). Ticagrelor has been reported to act as an inverse agonist under some experimental conditions (Aungraheeta et al, 2016; Garcia et al, 2019). A series of novel analogues of Ap 4 A blocks P2Y 12 receptors at nanomolar concentrations; one of the most potent derivatives was compound 37 (Yanachkov et al, 2016).…”