Thrombin-induced reactive oxygen species generation in platelets: A novel role for protease-activated receptor 4 and GPIbα

Abstract

BackgroundPlatelets 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.ObjectiveTo investigate the relative contribution of platelet GPIbα and PARs to thrombin-induced reactive oxygen species (ROS) generation.Methods and resultsHighly 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.ConclusionsBoth 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.

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“…Platelets were isolated using the centrifugation method as previously described [57]. Briefly, whole blood anticoagulated with acid citrate dextrose (ACD) was drawn from healthy volunteers then centrifuged at room temperature for 20 min at 150× g to obtain platelet-rich plasma (PRP).…”

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“…Minimal Inhibitory Concentrations (MICs) for the GAD and GA were determined following the microdilution method, detailed by the Clinical and Laboratory Standards Institute. 85 Briey, overnight cultures of bacteria were diluted to yield a nal concentration of about 5 Â 105 cells mL À1 . Samples were then added to equivalent volumes of various concentrations of GA and the GAD, previously appropriately diluted in dimethyl sulfoxide, distributed on a microplate and prepared from serial 2-fold dilutions, ranging from 256 mg mL À1 (1506 mM) to 16 mg mL À1 (94 mM) for GA and from 2048 mg mL À1 (120 mM) to 128 mg mL À1 (7,5 mM) for the GAD, respectively.…”