title: Thrombin-induced reactive oxygen species generation in platelets: A novel role for protease-activated receptor 4 and GPIbα.
abstract: BACKGROUND
  Platelets are essential for maintaining haemostasis and play a key role in the pathogenesis of cardiovascular disease. Upon ligation of platelet receptors through subendothelial matrix proteins, intracellular reactive oxygen species (ROS) are generated, further amplifying the platelet activation response. Thrombin, a potent platelet activator, can signal through GPIbα and protease-activated receptor (PAR) 1 and PAR4 on human platelets, and recently has been implicated in the generation of ROS. While ROS are known to have key roles in intra-platelet signalling and subsequent platelet activation, the precise receptors and signalling pathways involved in thrombin-induced ROS generation have yet to be fully elucidated.
  
  
  OBJECTIVE
  To investigate the relative contribution of platelet GPIbα and PARs to thrombin-induced reactive oxygen species (ROS) generation.
  
  
  RESULTS
  Highly specific antagonists targeting PAR1 and PAR4, and the GPIbα-cleaving enzyme, Naja kaouthia (Nk) protease, were used in quantitative flow cytometry assays of thrombin-induced ROS production. Antagonists of PAR4 but not PAR1, inhibited thrombin-derived ROS generation. Removal of the GPIbα ligand binding region attenuated PAR4-induced and completely inhibited thrombin-induced ROS formation. Similarly, PAR4 deficiency in mice abolished thrombin-induced ROS generation. Additionally, GPIbα and PAR4-dependent ROS formation were shown to be mediated through focal adhesion kinase (FAK) and NADPH oxidase 1 (NOX1) proteins.
  
  
  CONCLUSIONS
  Both GPIbα and PAR4 are required for thrombin-induced ROS formation, suggesting a novel functional cooperation between GPIbα and PAR4. Our study identifies a novel role for PAR4 in mediating thrombin-induced ROS production that was not shared by PAR1. This suggests an independent signalling pathway in platelet activation that may be targeted therapeutically.
authors.affiliation: Department of Experimental Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland.
authors.forename: Naadiya
authors.initials: N
authors.lastname: Carrim
authors.affiliation: Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.
authors.forename: Jane F
authors.initials: JF
authors.lastname: Arthur
authors.affiliation: Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.
authors.forename: Justin R
authors.initials: JR
authors.lastname: Hamilton
authors.affiliation: Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Gardiner
authors.affiliation: Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.
authors.forename: Robert K
authors.initials: RK
authors.lastname: Andrews
authors.affiliation: Department of Molecular Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin, Ireland.
authors.forename: Niamh
authors.initials: N
authors.lastname: Moran
authors.affiliation: Department of Experimental Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland; Faculty of Health Sciences, Curtin University, Perth, Australia.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Berndt
authors.affiliation: Department of Experimental Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland; Faculty of Health Sciences, Curtin University, Perth, Australia; Curtin Health Innovation Research Institute, Curtin University, Perth, Australia. Electronic address: pat.metharom@curtin.edu.au.
authors.forename: Pat
authors.initials: P
authors.lastname: Metharom
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D019556:COS Cells; D002522:Chlorocebus aethiops; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D006801:Humans; D051379:Mice; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D044463:Receptor, PAR-1 / Q000378:metabolism; D018179:Receptors, Thrombin / Q000502:physiology*; D013917:Thrombin / Q000502:physiology*
chemical_list: D019038:Platelet Glycoprotein GPIb-IX Complex; D017382:Reactive Oxygen Species; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; C094990:adhesion receptor; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C402799:NOX1 protein, human; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D013917:Thrombin; C112908:protease-activated receptor 4
keywords: PAR1 and PAR4; Platelets; Reactive oxygen species; Receptors GPIbα; Thrombin