A novel mutation in the P2Y12 receptor and a function-reducing polymorphism in protease-activated receptor 1 in a patient with chronic bleeding

Abstract

Summary: Background: The study of patients with bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets. We have identified a patient with a chronic bleeding disorder expressing a homozygous P2RY12 mutation, predicting an arginine to cysteine (R122C) substitution in the G-protein-coupled P2Y12 receptor. This mutation is found within the DRY motif, which is a highly conserved region in G-protein-coupled receptors (GPCRs) that is speculated to play a critical role in regulating receptor conformational states. Objectives: To determine the functional consequences of the R122C substitution for P2Y12 function. Patient/methods: We performed a detailed phenotypic analysis of an index case and affected family members. An analysis of the variant R122C P2Y12 stably expressed in cells was also performed. Results: ADP-stimulated platelet aggregation was reduced as a result of a significant impairment of P2Y12 activity in the patient and family members. Cell surface R122C P2Y12 expression was reduced both in cell lines and in platelets; in cell lines, this was as a consequence of agonist-independent internalization followed by subsequent receptor trafficking to lysosomes. Strikingly, members of this family also showed reduced thrombin-induced platelet activation, owing to an intronic polymorphism in the F2R gene, which encodes protease-activated receptor 1 (PAR-1), that has been shown to be associated with reduced PAR-1 receptor activity. Conclusions: Our study is the first to demonstrate a patient with deficits in two stimulatory GPCR pathways that regulate platelet activity, further indicating that bleeding disorders constitute a complex trait. © 2014 International Society on Thrombosis and Haemostasis.