Abstract Number: OC 10.1
Meeting: ISTH 2021 Congress
Background: Pharmacological blockade of platelet G protein-coupled receptors (GPCRs), including the P2Y12 receptor (P2Y12R) forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis. Clarification of the intracellular regulation of these clinically important receptors may provide a sound basis for the development of novel and specific drugs aimed at selected elements of this receptor system.
Aims: The aim of this study is to fully characterize the interplay between two critical platelet signalling systems: the cyclic nucleotide signalling system, a negative regulator of platelet reactivity and the platelet expressed P2Y12R that provides a critical feed-forward signal to amplify platelet reactivity. Further study aims to characterize whether specific clinically used antiplatelet drugs potentially activate the cyclic nucleotide system to dampen P2Y12R activity.
Methods: Cell based studies including BRET and receptor immunoprecipitation were undertaken in HEK293T cells transiently expressing FLAG-tagged P2Y12R to assess changes in receptor function. Further studies including pull-down assays, flow cytometry and GTPase-activity assays were used to examine P2Y12R function.
Results: Initial studies in cell lines and platelets revealed that the P2Y12R is phosphorylated in response to cAMP-elevating agents suggesting that the receptor is a target for cAMP signalling. Platelet P2Y12R co-immunoprecipitated with PKA isoforms suggesting that P2Y12R is a novel PKA type I binding protein in platelets.Receptor surface expression (flow cytometry) and activity assays (BRET and GTPase-Glo) revealed that activation of PKA directly attenuated ADP-mediated P2Y12R expression and responsiveness in both cell lines and platelets. Intriguingly ticagrelor a potent inhibitor of the P2Y12R with efficient antithrombotic activity also decreases receptor activity in a PKA-dependent manner.
Conclusions: Overall these data reveal that cAMP mediated PKA signalling negatively regulates P2Y12R activity. Further we have demonstrated that this negative feedback loop may also play a potential role in the mode of action of specific clinically used P2Y12R antagonists.
To cite this abstract in AMA style:Khalil J, Naseem K, Mundell S. Regulation of P2Y12 Receptor Expression and Activity by cAMP Mediated PKA Signalling in Human Platelets; A Novel Mode of Action of Anti-platelet Drugs? [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/regulation-of-p2y12-receptor-expression-and-activity-by-camp-mediated-pka-signalling-in-human-platelets-a-novel-mode-of-action-of-anti-platelet-drugs/. Accessed May 19, 2022.
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