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

Abstract

Background and Purpose: Platelet 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 Approach: Platelets 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 Results: Platelet 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 by Ap3A, NAD+, ADP-ribose or Up4A compared to ADP. Conclusion and Implications: Platelet 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.