Publication Date:
2014-12-06
Description:
Introduction Protease-activated receptor 4 (PAR4) is expressed widely in haemopoietic and vascular tissues and mediates thrombin-induced platelet activation primarily through Gq, protein kinase C and Ca2+ dependent pathways. Platelet PAR4 function may be modified by common non-coding variants near F2RL3 which encodes PAR4, and by epigenetic regulators which affect F2RL3 expression. However, coding sequence variants which affect PAR4 expression or function have not been reported previously. Here we characterise a novel missense variant in F2RL3associated with loss of PAR4 function in platelets from heterozygous subjects and reduced surface expression in platelets and a cell model. Methods The index case (P1) was identified by re-sequencing platelet G-protein coupled receptor genes in sub-groups of a collection of 2,400 adult cardiac surgery patients (GRAPHICS study; UK REC 12/SW/0286). Platelets were studied by light transmission aggregation (LTA) and by measuring P-selectin exposure, PAC-1 binding and intracellular Ca2+mobilisation in response to activating agonists. Platelet PAR4 expression was measured in platelets by flow cytometry and immunoblot and in transiently transfected HEK293 cells by flow cytometry and fluorescence microscopy. Results P1 was a 66 year old male with lifelong mild mucocutaneous bleeding and two gastrointestinal bleeds during aspirin treatment who harboured a heterozygous c.471A〉G transition in F2RL3predicting a p.Tyr157Cys (Y157C) substitution in PAR4. Platelet phenotype Compared to healthy controls, platelets from P1 showed reduced aggregation responses to the PAR4 agonist AYPGKF (60-600 μM; EC50 P1 266 μM vs controls 153 μM (n=11)) and to α-thrombin (5-100 mU.ml-1; EC50 P1 51 mU.ml-1 vs controls 15 mU.ml-1 (n=8)) with the greatest reduction in responses at 150 μM AYPGKF (maximum amplitude (MA) P1 1% vs controls 56%; p
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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