Thesis

Exploration of novel Proteinase activated receptor-4 (PAR4) interacting proteins for identification of new pathways for regulating receptor activity

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Awarding institution
  • University of Strathclyde
Date of award
  • 2024
Thesis identifier
  • T17137
Person Identifier (Local)
  • 201993917
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Abstract
  • The role of Proteinase Activated Receptor-4 (PAR4) in eliciting platelet shape change upon stimulation with thrombin is well established, however functional characterisation of the pathways involved in coordinating this change is not. In particular proteins which sever actin allowing for the formation of new filamentous actin hasn’t been shown in the context of PARs. The present study aimed at experimentally identifying and validating novel interacting proteins downstream of PAR4. Proteomic analysis revealed actin remodelling and dynamic regulation proteins as standout pathways close to the PAR4 receptor, with the actin severing protein Cofilin emerging as a high-confidence hit. MEG-01 cells were used as a cell model expressing physiological levels of both PAR1 and PAR4 in order to validate the findings of the proteomic data. Results of western blotting revealed that stimulation of MEG-01 cells with thrombin or PAR4-AP (AYPGKF) lead to significant increases in the levels of Cofilin phosphorylation reaching a peak at 15 mins post stimulation. It was also shown that phosphorylation of Cofilin showed dose dependency with all concentrations used showing significant changes in Cofilins phosphorylation state. This was also confirmed using immunofluorescent analysis. Stimulation of just PAR1 with TFLRRN showed no significant increase in Cofilin phosphorylation through western blot analysis, however significance was shown through IF analysis but non-comparable to the changes seen in PAR4 stimulated MEG-01 cells. Stimulation of PAR4 showed significant activity regulation of Cofilin pathway proteins, including regulators of phosphorylation (ROCK1, LIMK2 and TESK1) and Cofilin phosphatases (SSHL1 and PDXP). In addition, MEG-01 cells were shown to contain the Cofilin-2 isoform and stimulation with thrombin showed dose dependant variation in both total Cofilin-2 and Cofilin-2 phosphorylation showing distinct regulatory signalling dynamics compared to Cofilin-1 The study identified a novel potential interacting protein with PAR4 and functionally validated this interaction within a physiologically relevant cell model.
Advisor / supervisor
  • Cunningham, Margaret Rose
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DOI

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