A Novel F2RL3 Variant Predicting a Tyr157Cys Substitution in Protease-Activated Receptor 4 Is Associated with Reduced Receptor Function in Platelets
Authors: Norman Jane E , Cunningham Margaret R , Jones Matthew L , Westbury Sarah K , Walker Mary , Mundell Stuart J , Mumford Andrew D 
Published: October 2019

Abstract
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<0.0001) and 10 mU.ml-1 α-thrombin (MA P1 3% vs controls 67%; p<0.0001). Platelets from P1 also showed reduced P-selectin exposure, PAC-1 binding and intracellular Ca2+mobilisation in response to AYPGKF (25-1000 μM) compared to healthy controls. Functional responses of platelets from P1 to the PAR1 agonist SFLLRN (0.25-100μM) were similar to controls suggesting a selective defect in PAR4 function. Platelets from P1 showed a mean number of PAR4 receptors/platelet of 13 by flow cytometry, compared to 295 in controls (SEM 98; n=9) indicating markedly reduced surface expression. However, PAR1 surface expression in platelets from P1 was similar to controls. Immunoblots of platelet protein extracts for PAR4 from P1 and controls were similar, suggesting comparable total PAR4 expression.
The c.471A>G F2RL3 variant was identified in two sons of P1, who also showed reduced platelet functional responses to AYPGKF and PAR4 surface expression that were similar to P1, indicating co-segregation of platelet phenotype and the variant F2LR3 genotype. However, these subjects did not display abnormal bleeding.
Y157C PAR4 in HEK293 cells
Variant PAR4 with a C-terminal CFP tag (Y157C PAR4-CFP) expressed in HEK293 cells showed reduced surface expression by flow cytometry (mean of median fluorescence intensities 28% that of wild-type (WT) PAR4-CFP with SEM 5%; n=3). Consistent with this, fluorescence microscopy revealed predominantly intracellular expression of Y157C PAR4-CFP in the HEK293 cells with limited cell surface expression when compared to WT PAR4-CFP.
Conclusion
The Y157 residue is highly conserved within transmembrane domain 3 of PAR4 that, in other G protein coupled receptors, is crucial for maintaining normal receptor structure. Therefore, the Y157C substitution identified in the index case in PAR4 is likely to adversely affect receptor function. Consistent with this, our data from multiple functional assays show reduced platelet responses to α-thrombin and to a PAR4 specific agonist, but not to a PAR1 specific agonist. This platelet phenotype co-segregated with the heterozygous Y157C substitution in the study kindred suggesting a causal association. However, this was not associated with bleeding in all kindred members. In both platelets and HEK293 cells we demonstrated the Y157C substitution was associated with loss of PAR4 function and reduced surface receptor expression.
This is the first description of a missense PAR4 variant causing loss of receptor function as a heritable trait.

Disclosures
No relevant conflicts of interest to declare.