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

Summary

mRNA 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.

Keywords

P2Y₁₄ - KIAA0001 - GPR105 - UDP-glucose - platelet aggregation - EP₃ - sulprostone - P2Y₁₂ - P2Y₁

^* These authors contributed equally and substantially to this work.

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