Detection of P2Y14 protein in platelets and investigation of the role of P2Y14 in platelet function in comparison with the EP3 receptor

Abstract

SummarymRNA encoding the recently discovered P2Y14 receptor has been reported in platelets, but the presence of P2Y14 receptor protein and its functionality have not been studied. If P2Y14 is expressed along with P2Y1 and P2Y12 receptors it may have a role in haemostasis. It was the objective of this study to investigate the presence of the P2Y14 receptor in platelets and its role in platelet function. The effects of the agonist UDP-glucose were compared with those of sulprostone, a selective EP3 receptor agonist. Expression of P2Y14 receptor was investigated by immunoblotting and confocal microscopy. Platelet aggregation in platelet-rich plasma (PRP) and whole blood was measured using light absorbance and platelet counting. VASP phosphorylation was investigated using flow cytometry. Immunoblotting provided evidence for P2Y14 receptor protein and microscopy confirmed its presence on platelets. Despite this, UDP-glucose (up to 100 µM) did not induce platelet aggregation in either PRP or whole blood, and did not potentiate aggregation induced by other agonists. P2Y14 did not substitute for P2Y12 in experiments using the P2Y12 antagonist AR-C69931. No effect of UDP-glucose was seen on adenylate cyclase activity as measured by VASP phosphorylation. In contrast, sulprostone acting via the EP3 receptor promoted platelet aggregation with effects on adenylate cyclase activity. EP3 also partially substituted for P2Y12 receptor. We have demonstrated the presence of P2Y14 receptor protein in platelets, but no contribution of this receptor to several measures of platelet function has been observed. Further studies are necessary to determine whether the P2Y14 receptor in platelets has any functionality.

Lipopolysaccharide (LPS) induced pulmonary neutrophil recruitment and platelet activation is mediated via the P2Y1 and P2Y14 receptors in mice

“…Mice were administered a second P2Y12 antagonist (Ticagrelor). However this did not lead to a suppression of pulmonary neutrophil recruitment induced by LPS at 3mg/kg or 10mg/kg Given that P2Y1, P2Y12, P2Y14, and P2X1 receptors are all expressed on platelets, and platelet activation is necessary for pulmonary leukocyte recruitment in this model of inflammation [12,22,27,38], we further examined the effects of these receptors on platelet dynamics (in vivo) and platelet-neutrophil interactions (in vitro).…”

“…Using a murine model of allergic airway inflammation we have previously demonstrated that pulmonary leukocyte recruitment was mediated via RhoA signalling downstream of the platelet P2Y1 receptor, whilst the P2Y12 and P2X1 receptors showed no involvement in this process [26]. More recently, platelets have been shown to express the P2Y14 receptor on their surface [27]. However, the P2Y14 receptor appears to be redundant in platelet aggregation, whilst demonstrating an ability to mediate cell migration in other cell types [27][28][29][30].…”

“…More recently, platelets have been shown to express the P2Y14 receptor on their surface [27]. However, the P2Y14 receptor appears to be redundant in platelet aggregation, whilst demonstrating an ability to mediate cell migration in other cell types [27][28][29][30]. A physiological role for platelet P2Y14 receptor activation therefore remains unknown in the context of host defence and inflammation.…”

Exploring bias in platelet P2Y1 signalling: Host defence versus haemostasis

“…Platelets express four P2 receptors (ATP‐gated P2X 1 ion channel and the G protein‐coupled receptors [GPCRs] P2Y 1 and P2Y 12 —both activated by ADP, P2Y 14 —activated by UDP‐glucose) and two P1 receptors:‐ the adenosine activated GPCRs A 2A and A 2B receptors (Gachet, 2006 ; Jacobson et al, 2011 ; Wolska & Rozalski, 2019 ). The relevance of P2Y 14 activation of platelets has not been extensively described, but P2Y 14 appears to lack an involvement in platelet aggregation, while preliminary data suggest P2Y 14 might be involved in platelet‐associated neutrophil migration (Amison et al, 2017 ; Dovlatova et al, 2008 ). Elsewhere, a direct role for P2Y 14 on leukocyte activation has been reported (Sesma et al, 2012 , 2016 ).…”