title: Protease-Activated Receptor 4 Variant p.Tyr157Cys Reduces Platelet Functional Responses and Alters Receptor Trafficking.
abstract: Protease-activated receptor 4 (PAR4) is a key regulator of platelet reactivity and is encoded by F2RL3, which has abundant rare missense variants. We aimed to provide proof of principle that rare F2LR3 variants potentially affect platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms.
  
  
  
  We identified 6 rare F2RL3 missense variants in 236 cardiac patients, of which the variant causing a tyrosine 157 to cysteine substitution (Y157C) was predicted computationally to have the greatest effect on PAR4 structure. Y157C platelets from 3 cases showed reduced responses to PAR4-activating peptide and to α-thrombin compared with controls, but no reduction in responses to PAR1-activating peptide. Pretreatment with the PAR1 antagonist vorapaxar caused lower residual α-thrombin responses in Y157C platelets than in controls, indicating greater platelet inhibition. HEK293 cells transfected with a PAR4 Y157C expression construct had reduced PAR4 functional responses, unchanged total PAR4 expression but reduced surface expression. PAR4 Y157C was partially retained in the endoplasmic reticulum and displayed an expression pattern consistent with defective N-glycosylation. Mutagenesis of Y322, which is the putative hydrogen bond partner of Y157, also reduced PAR4 surface expression in HEK293 cells.
  
  
  
  Reduced PAR4 responses associated with Y157C result from aberrant anterograde surface receptor trafficking, in part, because of disrupted intramolecular hydrogen bonding. Characterization of PAR4 Y157C establishes that rare F2RL3 variants have the potential to markedly alter platelet PAR4 reactivity particularly after exposure to therapeutic PAR1 antagonists.
authors.affiliation: From the School of Clinical Sciences (J.E.N., M.E.W., S.K.W., A.D.M.), School of Cellular and Molecular Medicine (M.L.J., A.D.M.), School of Biochemistry (R.B.S.), and School of Physiology and Pharmacology (S.J.M.), University of Bristol, Bristol, United Kingdom; and Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom (M.R.C.).
authors.forename: Jane E
authors.initials: JE
authors.lastname: Norman
authors.affiliation: From the School of Clinical Sciences (J.E.N., M.E.W., S.K.W., A.D.M.), School of Cellular and Molecular Medicine (M.L.J., A.D.M.), School of Biochemistry (R.B.S.), and School of Physiology and Pharmacology (S.J.M.), University of Bristol, Bristol, United Kingdom; and Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom (M.R.C.).
authors.forename: Margaret R
authors.initials: MR
authors.lastname: Cunningham
authors.affiliation: From the School of Clinical Sciences (J.E.N., M.E.W., S.K.W., A.D.M.), School of Cellular and Molecular Medicine (M.L.J., A.D.M.), School of Biochemistry (R.B.S.), and School of Physiology and Pharmacology (S.J.M.), University of Bristol, Bristol, United Kingdom; and Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom (M.R.C.).
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Jones
authors.affiliation: From the School of Clinical Sciences (J.E.N., M.E.W., S.K.W., A.D.M.), School of Cellular and Molecular Medicine (M.L.J., A.D.M.), School of Biochemistry (R.B.S.), and School of Physiology and Pharmacology (S.J.M.), University of Bristol, Bristol, United Kingdom; and Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom (M.R.C.).
authors.forename: Mary E
authors.initials: ME
authors.lastname: Walker
authors.affiliation: From the School of Clinical Sciences (J.E.N., M.E.W., S.K.W., A.D.M.), School of Cellular and Molecular Medicine (M.L.J., A.D.M.), School of Biochemistry (R.B.S.), and School of Physiology and Pharmacology (S.J.M.), University of Bristol, Bristol, United Kingdom; and Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom (M.R.C.).
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Westbury
authors.affiliation: From the School of Clinical Sciences (J.E.N., M.E.W., S.K.W., A.D.M.), School of Cellular and Molecular Medicine (M.L.J., A.D.M.), School of Biochemistry (R.B.S.), and School of Physiology and Pharmacology (S.J.M.), University of Bristol, Bristol, United Kingdom; and Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom (M.R.C.).
authors.forename: Richard B
authors.initials: RB
authors.lastname: Sessions
authors.affiliation: From the School of Clinical Sciences (J.E.N., M.E.W., S.K.W., A.D.M.), School of Cellular and Molecular Medicine (M.L.J., A.D.M.), School of Biochemistry (R.B.S.), and School of Physiology and Pharmacology (S.J.M.), University of Bristol, Bristol, United Kingdom; and Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom (M.R.C.).
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Mundell
authors.affiliation: From the School of Clinical Sciences (J.E.N., M.E.W., S.K.W., A.D.M.), School of Cellular and Molecular Medicine (M.L.J., A.D.M.), School of Biochemistry (R.B.S.), and School of Physiology and Pharmacology (S.J.M.), University of Bristol, Bristol, United Kingdom; and Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom (M.R.C.). a.mumford@bristol.ac.uk.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Mumford
mesh_terms: D000368:Aged; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D016022:Case-Control Studies; D003198:Computer Simulation; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004739:England; D005260:Female; D005838:Genotype; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D006860:Hydrogen Bonding; D007783:Lactones / Q000494:pharmacology; D008297:Male; D008958:Models, Molecular; D020125:Mutation, Missense; D010455:Peptides / Q000494:pharmacology; D010641:Phenotype; D015539:Platelet Activation / Q000187:drug effects; D010974:Platelet Aggregation; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D020641:Polymorphism, Single Nucleotide; D011487:Protein Conformation; D021381:Protein Transport; D011725:Pyridines / Q000494:pharmacology; D044463:Receptor, PAR-1 / Q000187:drug effects / Q000378:metabolism; D018179:Receptors, Thrombin / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection
chemical_list: D007783:Lactones; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D011725:Pyridines; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D013917:Thrombin; C112908:protease-activated receptor 4; C530299:vorapaxar
keywords: HEK293 cells; genotype; platelet activation; platelet activation inhibitors; protease-activated receptor 4