Cellular and Molecular Life Sciences

, Volume 76, Issue 3, pp 561–576 | Cite as

Deciphering biased inverse agonism of cangrelor and ticagrelor at P2Y12 receptor

  • Cédric Garcia
  • Agnès Maurel-Ribes
  • Michel Nauze
  • Du N’Guyen
  • Laurent O. Martinez
  • Bernard Payrastre
  • Jean-Michel Sénard
  • Céline Galés
  • Véronique PonsEmail author
Original Article


P2Y12 receptor (P2Y12-R) is one of the major targets for drug inhibiting platelet aggregation in the treatment/prevention of arterial thrombosis. However, the clinical use of P2Y12-R antagonists faces some limitations, such as a delayed onset of action (clopidogrel) or adverse effect profile (ticagrelor, cangrelor), justifying the development of a new generation of P2Y12-R antagonists with a better clinical benefit–risk balance. Although the recent concept of biased agonism offers the possibility to alleviate undesirable adverse effects while preserving therapeutic outcomes, it has never been explored at P2Y12-R. For the first time, using highly sensitive BRET2-based probes, we accurately delineated biased ligand efficacy at P2Y12-R in living HEK293T cells on G protein activation and downstream effectors. We demonstrated that P2Y12-R displayed constitutive Gi/o-dependent signaling that is impaired by the R122C mutation, previously associated with a bleeding disorder. More importantly, we reported the biased inverse agonist efficacy of cangrelor and ticagrelor that could underlie their clinical efficacy. Our study points out that constitutive P2Y12-R signaling is a normal feature of the receptor that might be essential for platelets to respond faster to a vessel injury. From a therapeutic standpoint, our data suggest that the beneficial advantages of antiplatelet drugs might be more related to inverse agonism at P2Y12-R than to antagonism of ADP-mediated signaling. In the future, deciphering P2Y12-R constitutive activity should allow the discovery of more selective biased P2Y12-R blockers demonstrating therapeutic advantages over classical antiplatelet drugs by improving therapeutic outcomes and concomitantly relieving undesirable adverse effects.


GPCR Constitutive activity Platelet Aggregation 



We thank Lauriane Onfroy for critical reading of the manuscript. We thank the imaging facility (INSERM UMR1048, Toulouse) and the DNA sequencing platform (Plateau de génomique GeT-Purpan de la plateforme Génome et Transcriptome de la Génopole de Toulouse Midi-Pyrénées). BP, JMS, C Galés and VP are supported by the Institut National de la santé et de la Recherche Médicale (INSERM). C Galés is also supported by the Fondation Bettencourt-Schueller (C Galés) and BP by the Fondation pour la Recherche Médicale (FRM) and the Institut Universitaire de France (IUF).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

18_2018_2960_MOESM1_ESM.tif (4.8 mb)
Supplementary Figure S1:Relative expression of Gα protein and CAMYEL probes. a-b. Relative expression of RLuc8-Gα protein probes (a) or CAMYEL sensor (b) was assessed by luminescence measurement in each experiment. Data represent the mean ± SEM of at least three independent experiments. (TIFF 4930 kb)
18_2018_2960_MOESM2_ESM.tif (4.1 mb)
Supplementary Figure S2:Constitutive activation of Gai2 protein depends on P2Y12-R expression. a. Basal BRET signal was measured in HEK293T/17 cells co-expressing Gαi2-RLuc8, GFP2-Gγ2, Gβ1 in the absence (0µg) or in the presence of increasing amounts of WT P2Y12-R (ranging from 0,01 to 4 µg/dish). b. Relative expression of RLuc8-Gα protein probes was assessed by luminescence measurement in each condition. a-b. Data represent the mean ± SEM of at least six independent experiments and statistical significance between cells expressing increasing amounts of WT P2Y12-R or not (0 µg) was assessed using one-way ANOVA followed by a Dunnett’s multiple comparison test (** P < 0.01; *** P < 0.001; ns, not statistically significant). (TIFF 4176 kb)
18_2018_2960_MOESM3_ESM.tif (22.5 mb)
Supplementary Figure S3:WT and mutant P2Y12-R are expressed at the cell surface of HEK293T/17 cells. a. HEK293T/17 cells expressing N-terminally Myc-tagged WT or mutant P2Y12-R were processed for immunofluorescence using an anti-Myc antibody. Nuclei were stained with 5 μg/mL DAPI. Scale bar indicates 10 μm. b. HEK293T/17 cells were transfected with increasing amounts of vectors encoding N-terminally Myc-tagged WT or mutant P2Y12-R (ranging from 15 to 500 ng/well). Then, cell surface expression of the different receptors was quantified using an anti-Myc antibody. Data represent the mean ± SEM of five independent experiments. Statistical significance between cell surface expression of WT and mutant P2Y12-R was assessed using two-way ANOVA followed by a Bonferroni post-test (ns, not statistically significant). (TIFF 23037 kb)
18_2018_2960_MOESM4_ESM.tif (4 mb)
Supplementary Figure S4:Inverse agonist efficacy of cangrelor depends on P2Y12-R expression. Gαi2 protein activation was measured in HEK293T/17 cells co-expressing Gαi2-RLuc8, GFP2-Gγ2 and Gβ1 alone (0µg) or with increasing amounts of vectors encoding WT P2Y12-R (ranging from 0,01 to 4 µg/dish), after stimulation or not with cangrelor (10 μM). Results are expressed as the difference in the BRET signal measured in the presence and in the absence of cangrelor. Data represent the mean ± SEM of six independent experiments. Statistical significance between cells expressing the WT P2Y12-R or not was assessed using one-way ANOVA followed by Kruskal-Wallis and Dunn’s multiple comparison post-tests (* P < 0.05; ** P < 0.01; ns indicates not statistically significant). (TIFF 4048 kb)
18_2018_2960_MOESM5_ESM.tif (4.4 mb)
Supplementary Figure S5:Dose–response curves of Gαi/o protein activation mediated by cangrelor or ticagrelor at WT P2Y12-R. Dose–response curves of each Gαi/o protein activation were assessed in HEK293T/17 cells co-expressing Gαi/o-RLuc8, GFP2-Gγ2, Gβ1 and WT P2Y12-R after stimulation or not with increasing concentrations of cangrelor or ticagrelor (values from Fig. 5). Results are expressed as the difference in the BRET signal measured in the presence and the absence of ligand. Data represent the mean ± SEM of at least three independent experiments. Statistical significance between unstimulated cells and cells stimulated with the different concentrations of cangrelor (*) or ticagrelor (#) was assessed using one-way ANOVA followed by a Dunnett’s multiple comparison test (* or # P < 0.05; ** or ## P < 0.01; *** or ### P < 0.001). (TIFF 4529 kb)


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Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • Cédric Garcia
    • 1
  • Agnès Maurel-Ribes
    • 1
  • Michel Nauze
    • 2
  • Du N’Guyen
    • 2
  • Laurent O. Martinez
    • 2
  • Bernard Payrastre
    • 1
    • 2
  • Jean-Michel Sénard
    • 2
    • 3
  • Céline Galés
    • 2
  • Véronique Pons
    • 2
    Email author
  1. 1.Laboratoire d’HématologieCentre Hospitalier Universitaire de ToulouseToulouseFrance
  2. 2.INSERM U1048, Institut des Maladies Métaboliques et Cardiovasculaires, Université de ToulouseToulouse Cedex 04France
  3. 3.Service de Pharmacologie Clinique, Centre Hospitalier Universitaire de ToulouseUniversité de ToulouseToulouseFrance

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