title: A novel mutation in the P2Y12 receptor and a function-reducing polymorphism in protease-activated receptor 1 in a patient with chronic bleeding.
abstract: 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 P2RY(12) 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.
  
  
  OBJECTIVE
  To determine the functional consequences of the R122C substitution for P2Y(12) function.
  
  
  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 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.
authors.affiliation: Department of Cardiology, Cardiovascular Division, King's College London, The Rayne Institute, St Thomas' Hospital, London, UK.
authors.forename: Y M
authors.initials: YM
authors.lastname: Patel
authors.forename: M
authors.initials: M
authors.lastname: Lordkipanidzé
authors.forename: G C
authors.initials: GC
authors.lastname: Lowe
authors.forename: S P
authors.initials: SP
authors.lastname: Nisar
authors.forename: K
authors.initials: K
authors.lastname: Garner
authors.forename: J
authors.initials: J
authors.lastname: Stockley
authors.forename: M E
authors.initials: ME
authors.lastname: Daly
authors.forename: M
authors.initials: M
authors.lastname: Mitchell
authors.forename: S P
authors.initials: SP
authors.lastname: Watson
authors.forename: S K
authors.initials: SK
authors.lastname: Austin
authors.forename: S J
authors.initials: SJ
authors.lastname: Mundell
mesh_terms: D020816:Amino Acid Motifs; D001792:Blood Platelets / Q000166:cytology*; D045744:Cell Line, Tumor; D002908:Chronic Disease; D005260:Female; D006470:Hemorrhage / Q000201:enzymology*; D006720:Homozygote; D006801:Humans; D008297:Male; D009154:Mutation; D010641:Phenotype; D015539:Platelet Activation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D017354:Point Mutation; D011110:Polymorphism, Genetic; D011487:Protein Conformation; D044463:Receptor, PAR-1 / Q000235:genetics* / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D058925:Receptors, Purinergic P2Y12 / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: C548576:P2RY12 protein, human; D044463:Receptor, PAR-1; D043562:Receptors, G-Protein-Coupled; D058925:Receptors, Purinergic P2Y12
keywords: P2Y12 purinoceptor; bleeding disorder due to P2RY12 defect; blood platelets; point mutation; receptors, G-protein-coupled