A novel mutation in the p2y12 receptor and a function-reducing polymorphism in par-1 in a patient with chronic bleeding

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 a cysteine (R122C) substitution in the G protein-coupled P2Y12 receptor (P2Y12 R). This mutation is found within the DRY motif, which is a highly conserved region in G protein-coupled receptors (GPCRs) speculated to play a critical role in regulating receptor conformational states.

OBJECTIVES: To determine the functional consequences of the R122C substitution upon P2Y12 R function.

PATIENT / METHODS: We performed a detailed phenotypic analysis of an index case and affected family members. An analysis of the variant R122C P2Y12 receptor stably expressed in cells was also performed RESULTS: ADP-stimulated platelet aggregation was reduced as a result of a significant impairment of P2Y12 R activity in the patient and family members. Cell surface R122C-P2Y12 R expression was reduced both in cell lines and in platelets, which in cell lines was as a consequence of agonist-independent internalization followed by subsequent receptor traffic to lysosomes. Strikingly, members of this family also showed reduced thrombin-induced platelet activation due to an intronic polymorphism in the F2R gene which encodes 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 are a complex trait. This article is protected by copyright. All rights reserved.