Stimulation of platelet P2Y₁ receptors by different endogenous nucleotides leads to functional selectivity via biased signalling

Abstract

Background and PurposePlatelet function during inflammation is dependent on activation by endogenous nucleotides. Non‐canonical signalling via the P2Y1 receptor is important for these non‐thrombotic functions of platelets. However, apart from ADP, the role of other endogenous nucleotides acting as agonists at P2Y1 receptors is unknown. This study compared the effects of ADP, Ap3A, NAD+, ADP‐ribose, and Up4A on platelet functions contributing to inflammation or haemostasis.Experimental ApproachPlatelets obtained from healthy human volunteers were incubated with ADP, Ap3A, NAD+, ADP‐ribose, or Up4A, with aggregation and fibrinogen binding measured (examples of function during haemostasis) or before exposure to fMLP to measure platelet chemotaxis (an inflammatory function). In silico molecular docking of these nucleotides to the binding pocket of P2Y1 receptors was then assessed.Key ResultsPlatelet aggregation and binding to fibrinogen induced by ADP was not mimicked by NAD+, ADP‐ribose, and Up4A. However, these endogenous nucleotides induced P2Y1‐dependent platelet chemotaxis, an effect that required RhoA and Rac‐1 activity, but not canonical PLC activity. Analysis of molecular docking of the P2Y1 receptor revealed distinct differences of amino acid interactions and depth of fit within the binding pocket for Ap3A, NAD+, ADP‐ribose, or Up4A compared with ADP.Conclusion and ImplicationsPlatelet function (aggregation vs motility) can be differentially modulated by biased‐agonist activation of P2Y1 receptors. This may be due to the character of the ligand‐binding pocket interaction. This has implications for future therapeutic strategies aimed to suppress platelet activation during inflammation without affecting haemostasis as is the requirement of current ant‐platelet drugs.

Investigation Into P2y Receptor Function in Platelets From Patients With Sepsis

“…In the HC, a trend of ADP-induced chemotaxis was observed, peaking at 100 nM (mean CI = 2.4 ± 0.2). Although this data set did not reach statistical significance ( P = 0.08 compared with negative control, n = 3), it does provide a comparison of the amplitude of the signal with platelets isolated and prepared under the conditions experienced with the constraints of this clinical trial setting, with that of our previously published data (20,23) (Fig. 5).…”

“…The role of P2Y receptors expressed on platelets leading to platelet aggregation is well established (18). However, we have reported an additional requirement for platelet P2Y 1 receptor activation for platelet-dependent pulmonary leukocyte recruitment in the absence of changes in hemostasis (19–21), and in the case of P2Y 1 , requiring distinct signaling pathways of platelet activation (RhoA, Rac-1) leading to functional motility (chemotaxis) compared with the canonical phospholipase C pathway implicated in aggregation (20,22,23).…”

Exploring bias in platelet P2Y1 signalling: Host defence versus haemostasis

“…The advent of immunohistochemistry, however, allowed researchers to identify and quantify platelets in extravascular tissue compartments in response to infection or subjected to inflammatory stimuli. Thus, a growing number of groups have now reported the ability of platelets to undergo chemotaxis towards a range of chemokines and chemoattractants, using different in vitro methodologies, including trans‐migration across endothelial monolayers (Amison, Jamshidi, et al, 2018 ; Arkless et al, 2023 ; Czapiga et al, 2005 ; Fan et al, 2018 ; Gaertner et al, 2017 ; Kraemer et al, 2010 , 2011 ; Miao et al, 2020 ; Nicolai et al, 2020 ; Palankar et al, 2022 ; Petito et al, 2018 ; Pitchford et al, 2008 ; Schmidt et al, 2011 , 2012 ; Seifert et al, 2022 ; Shah et al, 2021 ; Valone et al, 1974 ; Witte et al, 2021 ). Fundamentally, this characteristic is by necessity distinct from the action of platelets during haemostasis that induce irreversible adherence to extracellular matrix (e.g.…”

“…While platelet P‐selectin expression by ADP requires P2Y 1 stimulation (Anderson et al, 2020 ), this can be mediated by either PLC or RhoA and Rac1 (Akbar et al, 2007 , 2016 ; Anderson et al, 2020 ). Other studies of non‐thrombotic functions of platelets that are P2Y 1 dependent include platelet killing of parasites (McMorran et al, 2009 ), chemokine CXCL16 ‐mediated platelet adhesion to inflamed endothelium (Borst et al, 2012 ) and platelet chemotaxis (Amison, Jamshidi, et al, 2018 ; Arkless et al, 2023 ). Consequently, the activation of platelets by P2Y 1 in non‐thrombotic diseases may lead to diverse functional outputs and is worthy of exploration in other inflammatory settings (Figure 2 ).…”

Platelet purinergic receptors and non‐thrombotic diseases

“…The potential control of platelet activation in the context of adenosine suppression of inflammation in thus introduced, including approaches to creating more stable analogues of adenosine (Boncler et al, 2024). Lastly, the discovery that activation of P2Y 12 receptors by various endogenous extracellular nucleotides can lead to selective platelet inflammatory functions and not aggregation (Arkless et al, 2024), leads to a discussion about the selective control of platelet activation by P2Y 1 receptors during inflammation compared to haemostasis, and the potential for safe anti‐platelet drugs that suppress inflammation while preserving haemostasis (Pan et al, 2024).…”