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

Abstract

SJ. A novel mutation in the P2Y 12 receptor and a function-reducing polymorphism in protease-activated receptor 1 in a patient with chronic bleeding. J Thromb Haemost 2014; 12: 716-25.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 P2Y 12 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 P2Y 12 function. Patient/methods: We performed a detailed phenotypic analysis of an index case and affected family members. An analysis of the variant R122C P2Y 12 stably expressed in cells was also performed. Results: ADP-stimulated platelet aggregation was reduced as a result of a significant impairment of P2Y 12 activity in the patient and family members. Cell surface R122C P2Y 12 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 thrombininduced 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.

Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay

“…This mutation maps to the DRY motif, a highly conserved region in G-coupled receptors, which was shown in expression studies to inhibit platelet activation by ADP. 23,24 Representative Optimul traces from this family are shown in Figure 7B. Impairment in all platelet pathways (except for passive agglutination onto ristocetin) was consistent with a platelet activation defect and was seen in all family members.…”

Sodobni pristop za diagnosticiranje prirojenih motenj delovanja trombocitov

“…V to skupino uvrščamo Glanzmannovo trombastenijo (12,13), nenormalnosti receptorja (P2Y 12 ) za ADP (14,15), aspirinu podobne motnje (angl. Aspirin--like defects) (16), motnje skladiščenja substanc (angl.…”

The DRY motif at work: the P2Y12 receptor case

“…Interestingly, binding studies in platelets from these patients showed a decrease in affinity for the P2Y 12 agonist 2-MeSADP and in surface receptor expression compared with healthy donors (HD) [4]. Accordingly, in vitro studies with cells stably expressing the R122C receptor variant confirmed significantly impairment of receptor activity compared with WT P2Y 12 , when cells with similar levels of receptor expression were skillfully selected, and no increase in basal activity.…”

“…Therefore, even though a careful analysis of the signaling effects of non-conservative substitutions in position D 3.49 will be necessary to fully characterize R122C P2Y 12 as a CIM, it appears to possess many of their characteristics. It is thus conceivable that both P2Y 12 [4] and TP receptors [19][20][21] have developed a large activation energy barrier, consistent with their relevance in the homeostasis of the cardiovascular system [18,22], while other receptors (such as b2-AR) might have a low energy barrier between the R and R* states [14]. It is tempting to speculate here that this behavior, common to many other GPCRs, is the result of evolutionary forces that may have selected regulatory mechanisms against more highly active phenotypes that might not be compatible with a physiological milieu.…”