Competitive mode and site of interaction of ticagrelor at the human platelet P2Y12-receptor

K. Hoffmann, D. A. Lutz, J. Straßburger, Y. Baqi, C. E. Müller, I. von Kügelgen

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Background: The G-protein-coupled P2Y12-receptor plays a crucial role in platelet aggregation. Recently, ticagrelor was licensed as the first perorally active and reversible P2Y12-receptor antagonist. Objective: The present study investigated the site and the antagonistic mode of action of ticagrelor at wild-type or mutant human P2Y12-receptors. Methods: Recombinant wild-type or mutant human P2Y12-receptors were stably expressed in Chinese hamster ovary Flp-In cells. Receptor function was assessed by quantification of ADP- and 2-methylthio-ADP-mediated inhibition of forskolin-induced cellular cAMP production either using a [3H]cAMP-radioaffinity assay or a cAMP response element-driven luciferase reporter gene assay. Results: The natural agonist ADP inhibited forskolin-induced cAMP formation at the wild-type P2Y12-receptor with a lower potency (EC50 209 nm) than the synthetic agonist 2-methylthio-ADP (EC50 1.0 nm). Ticagrelor shifted the concentration-response curves of both agonists in a parallel and surmountable manner to the right. Increasing concentrations of ticagrelor caused increasing shifts. Schild-plot analysis revealed pA2 values of 8.85 for ticagrelor against ADP, and 8.69 against 2-methylthio-ADP, and slopes of the regression lines not different from unity. In cells expressing a recombinant C194A5.43-mutant P2Y12-receptor construct, ticagrelor lost antagonistic potency when tested against ADP or 2-methylthio-ADP. Conclusions: The experiments reveal a surmountable and competitive mode of antagonism of ticagrelor at P2Y12-receptors activated by either the natural agonist ADP or the synthetic agonist 2-methylthio-ADP. Cys1945.43 is likely to be involved in the interaction of ticagrelor with ADP and 2-methylthio-ADP. The data give new insights into the site and mode of action of ticagrelor at the human P2Y12-receptor.

Original languageEnglish
Pages (from-to)1898-1905
Number of pages8
JournalJournal of Thrombosis and Haemostasis
Volume12
Issue number11
DOIs
Publication statusPublished - Nov 1 2014

Fingerprint

Blood Platelets
Adenosine Diphosphate
Colforsin
Purinergic P2Y Receptor Antagonists
Ticagrelor
Response Elements
G-Protein-Coupled Receptors
Cricetulus
Luciferases
Platelet Aggregation
Reporter Genes
methylthio-ADP
Ovary

Keywords

  • Binding site
  • Mode of action
  • Platelets
  • Receptors, purinergic P2Y12

ASJC Scopus subject areas

  • Hematology
  • Medicine(all)

Cite this

Competitive mode and site of interaction of ticagrelor at the human platelet P2Y12-receptor. / Hoffmann, K.; Lutz, D. A.; Straßburger, J.; Baqi, Y.; Müller, C. E.; von Kügelgen, I.

In: Journal of Thrombosis and Haemostasis, Vol. 12, No. 11, 01.11.2014, p. 1898-1905.

Research output: Contribution to journalArticle

Hoffmann, K, Lutz, DA, Straßburger, J, Baqi, Y, Müller, CE & von Kügelgen, I 2014, 'Competitive mode and site of interaction of ticagrelor at the human platelet P2Y12-receptor', Journal of Thrombosis and Haemostasis, vol. 12, no. 11, pp. 1898-1905. https://doi.org/10.1111/jth.12719
Hoffmann, K. ; Lutz, D. A. ; Straßburger, J. ; Baqi, Y. ; Müller, C. E. ; von Kügelgen, I. / Competitive mode and site of interaction of ticagrelor at the human platelet P2Y12-receptor. In: Journal of Thrombosis and Haemostasis. 2014 ; Vol. 12, No. 11. pp. 1898-1905.
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AU - Lutz, D. A.

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AU - Baqi, Y.

AU - Müller, C. E.

AU - von Kügelgen, I.

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N2 - Background: The G-protein-coupled P2Y12-receptor plays a crucial role in platelet aggregation. Recently, ticagrelor was licensed as the first perorally active and reversible P2Y12-receptor antagonist. Objective: The present study investigated the site and the antagonistic mode of action of ticagrelor at wild-type or mutant human P2Y12-receptors. Methods: Recombinant wild-type or mutant human P2Y12-receptors were stably expressed in Chinese hamster ovary Flp-In cells. Receptor function was assessed by quantification of ADP- and 2-methylthio-ADP-mediated inhibition of forskolin-induced cellular cAMP production either using a [3H]cAMP-radioaffinity assay or a cAMP response element-driven luciferase reporter gene assay. Results: The natural agonist ADP inhibited forskolin-induced cAMP formation at the wild-type P2Y12-receptor with a lower potency (EC50 209 nm) than the synthetic agonist 2-methylthio-ADP (EC50 1.0 nm). Ticagrelor shifted the concentration-response curves of both agonists in a parallel and surmountable manner to the right. Increasing concentrations of ticagrelor caused increasing shifts. Schild-plot analysis revealed pA2 values of 8.85 for ticagrelor against ADP, and 8.69 against 2-methylthio-ADP, and slopes of the regression lines not different from unity. In cells expressing a recombinant C194A5.43-mutant P2Y12-receptor construct, ticagrelor lost antagonistic potency when tested against ADP or 2-methylthio-ADP. Conclusions: The experiments reveal a surmountable and competitive mode of antagonism of ticagrelor at P2Y12-receptors activated by either the natural agonist ADP or the synthetic agonist 2-methylthio-ADP. Cys1945.43 is likely to be involved in the interaction of ticagrelor with ADP and 2-methylthio-ADP. The data give new insights into the site and mode of action of ticagrelor at the human P2Y12-receptor.

AB - Background: The G-protein-coupled P2Y12-receptor plays a crucial role in platelet aggregation. Recently, ticagrelor was licensed as the first perorally active and reversible P2Y12-receptor antagonist. Objective: The present study investigated the site and the antagonistic mode of action of ticagrelor at wild-type or mutant human P2Y12-receptors. Methods: Recombinant wild-type or mutant human P2Y12-receptors were stably expressed in Chinese hamster ovary Flp-In cells. Receptor function was assessed by quantification of ADP- and 2-methylthio-ADP-mediated inhibition of forskolin-induced cellular cAMP production either using a [3H]cAMP-radioaffinity assay or a cAMP response element-driven luciferase reporter gene assay. Results: The natural agonist ADP inhibited forskolin-induced cAMP formation at the wild-type P2Y12-receptor with a lower potency (EC50 209 nm) than the synthetic agonist 2-methylthio-ADP (EC50 1.0 nm). Ticagrelor shifted the concentration-response curves of both agonists in a parallel and surmountable manner to the right. Increasing concentrations of ticagrelor caused increasing shifts. Schild-plot analysis revealed pA2 values of 8.85 for ticagrelor against ADP, and 8.69 against 2-methylthio-ADP, and slopes of the regression lines not different from unity. In cells expressing a recombinant C194A5.43-mutant P2Y12-receptor construct, ticagrelor lost antagonistic potency when tested against ADP or 2-methylthio-ADP. Conclusions: The experiments reveal a surmountable and competitive mode of antagonism of ticagrelor at P2Y12-receptors activated by either the natural agonist ADP or the synthetic agonist 2-methylthio-ADP. Cys1945.43 is likely to be involved in the interaction of ticagrelor with ADP and 2-methylthio-ADP. The data give new insights into the site and mode of action of ticagrelor at the human P2Y12-receptor.

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