ALBERT

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

Description: Introduction G-protein coupled receptors (GPCRs) are critical mediators of platelet responses to stimulatory and inhibitory agonists. In rare families with mild bleeding, it is recognised that heterozygous loss of function variations in platelet GPCR genes may diminish platelet agonist responses. However, the population prevalence of loss of function variations in these genes is unknown. We have utilised population databases and next generation sequencing from patients with inherited platelet function disorders (IPFD) to describe the extent of genetic variation in the major platelet GPCRs. We have also used predictive computation and a new consensus structure of GPCRs (Venkatakrishnan AJ et al.Nature 2013; 494) to estimate which variations confer loss of function. Methods We interrogated the ESP and 1000 genomes population datasets for single nucleotide (SNV) and insertion-deletion (indel) variations in the genes encoding 6 stimulatory (ADRA2A, F2R, F2RL3, P2RY1, P2RY12, TBXA2R) and 2 inhibitory (PTGER4, PTGIR) platelet GPCRs. Coding and splice region variations within the relevant Refseq transcripts were functionally annotated using the Polyphen-2, SIFT and FATHMM algorithms. Missense variations within GPCR transmembrane (TM) domains, were annotated manually by expressing the substitutions in Ballesteros-Weinstein nomenclature before comparison with the consensus GPCR structure. Missense variations in the N- and C-terminal regions (NR and CR) and the intra- and extra- cellular loops (ICL and ECL) were annotated by identifying the position of the substituted residue relative to experimentally confirmed or putative functional motifs. An identical analysis was performed using exome data from 31 unrelated patients with IPFD recruited through the UK GAPP study with clinical bleeding and abnormal platelet function by light transmission aggregation. Results In 7745 individuals from the ESP and 1000 genomes cohorts, we identified 332 SNV in the target regions of the 8 GPCR genes (40.5 variations/kb) comprising 183 non-synonymous and 148 synonymous coding variants and 4 variations within intronic splice regions. There were no indel variations. Functional annotation of the non-synonymous SNVs identified 41 that potentially conferred loss of function, distributed in all the target GPCRs but with low population frequency (minor allele frequency range 1-0.008%). Five SNVs affected the NT, including Gly48Asp and Arg47His substitutions at the PAR4 receptor thrombin/trypsin cleavage site. There were 12 SNVs affecting the TM domains, of which 4 were predicted to disrupt GPCR folding, including a TPα receptor Pro305Leu substitution within the structural N/DPXXY motif and the P2Y12 receptor Met108Leu and Thr283Ile substitutions predicted to disrupt non-covalent TM network contacts. There were 14 SNVs affecting the ICL including the P2Y12 receptor Asp121Asn substitution in the E/DRY motif and prostacyclin (IP1) receptor Arg212Cys and Arg215Cys substitutions predicted to disrupt Gs coupling. Ten functional SNVs affected the CT. In 31 IPFD patients with complex laboratory phenotypes that could not be explained by loss of a single GPCR, there were 8 non-synonymous SNVs, of which 5 were predicted to confer loss of function (table). Discussion In unselected populations, heterozygous loss of function GPCR gene variations which potentially affect platelet agonist responses are individually rare, but collectively numerous. Loss of function GPCR variations were also present in patients with underlying IPFD. These data illustrate that variations in platelet regulatory genes may act as modifiers of laboratory phenotype in patients with underlying IPFD and that the net phenotype may be the product of multiple gene defects. Disclosures: No relevant conflicts of interest to declare.