Protease-activated receptor 4 variant p.Tyr157Cys reduces platelet functional responses and alters receptor trafficking

Jane E. Norman, Margaret R. Cunningham, Matthew L. Jones, Mary E. Walker, Sarah K. Westbury, Richard B. Sessions, Stuart J. Mundell, Andrew D. Mumford

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

OBJECTIVE—: 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 on platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms. APPROACH AND RESULTS—: 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 affect most 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. CONCLUSIONS—: 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.

LanguageEnglish
Pages952-960
Number of pages9
JournalArteriosclerosis, Thrombosis, and Vascular Biology
Volume36
Issue number5
Early online date10 Mar 2016
DOIs
Publication statusPublished - 1 May 2016

Fingerprint

Blood Platelets
HEK293 Cells
Thrombin
protease-activated receptor 4
Peptides
Hydrogen Bonding
Glycosylation
Mutagenesis
Endoplasmic Reticulum
Cysteine
Tyrosine
Hydrogen
Pharmaceutical Preparations

Keywords

  • genotype
  • HEK293 cells
  • platelet activation
  • platelet activation inhibitors
  • protease-activated receptor 4

Cite this

Norman, Jane E. ; Cunningham, Margaret R. ; Jones, Matthew L. ; Walker, Mary E. ; Westbury, Sarah K. ; Sessions, Richard B. ; Mundell, Stuart J. ; Mumford, Andrew D. / Protease-activated receptor 4 variant p.Tyr157Cys reduces platelet functional responses and alters receptor trafficking. In: Arteriosclerosis, Thrombosis, and Vascular Biology. 2016 ; Vol. 36, No. 5. pp. 952-960.
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Protease-activated receptor 4 variant p.Tyr157Cys reduces platelet functional responses and alters receptor trafficking. / Norman, Jane E.; Cunningham, Margaret R.; Jones, Matthew L.; Walker, Mary E.; Westbury, Sarah K.; Sessions, Richard B.; Mundell, Stuart J.; Mumford, Andrew D.

In: Arteriosclerosis, Thrombosis, and Vascular Biology, Vol. 36, No. 5, 01.05.2016, p. 952-960.

Research output: Contribution to journalArticle

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AU - Norman, Jane E.

AU - Cunningham, Margaret R.

AU - Jones, Matthew L.

AU - Walker, Mary E.

AU - Westbury, Sarah K.

AU - Sessions, Richard B.

AU - Mundell, Stuart J.

AU - Mumford, Andrew D.

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N2 - OBJECTIVE—: 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 on platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms. APPROACH AND RESULTS—: 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 affect most 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. CONCLUSIONS—: 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.

AB - OBJECTIVE—: 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 on platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms. APPROACH AND RESULTS—: 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 affect most 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. CONCLUSIONS—: 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.

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KW - protease-activated receptor 4

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