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Title: Endothelial cell signalling of APC and thrombin : the involvement of protein kinase C
Author: Yu, Yao
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Activated protein C (APC) is a natural anticoagulant. In addition to this well-described function, APC also mediates endothelial cell (EC) cytoprotection, including anti-apoptotic, anti-inflammatory and endothelial barrier integrity stabilising effects. APC confers these effects through proteolytic activation of protease activated receptor 1 (PAR1), but only when it is bound to its receptor, endothelial protein C receptor (EPCR). Interestingly, thrombin exerts opposing cellular responses (including induction of endothelial hyperpermeability) through activation of PAR1. How activation of the same receptor can mediate such different signals remains unclear. I hypothesised that, downstream of PAR1 cleavage, activation of different protein kinase C (PKC) isoforms may account for the divergent signal transduction. The aim of my thesis was to explore the contribution of specific PKC isozyme(s) that are involved in APC and thrombin mediated signalling. To do this, I expressed, purified and characterised a panel of APC variants lacking either anticoagulant activity, EPCR binding, PAR1 binding or proteolytic activity. I demonstrated that both APC and thrombin activate Erk1/2 pathway in the endothelial cells in a dose and time-dependent manner. APC also dose-dependently stabilised endothelial monolayer integrity, whereas thrombin disrupted this. APC’s cytoprotective properties, based on this established cell assay model, were found to be PAR1 and EPCR dependent. Using peptides that inhibit specific PKC isozymes, I found that the atypical PKCζ isozyme, was involved in Erk1/2 phosphorylation triggered by thrombin and both classical PKC isozymes (PKCβ1) and PKCζ were involved in the hyperpermeability induced by thrombin. In contrast, no PKC isozyme was found to play roles in the APC-mediated Erk1/2 activation as well as EC permeability reduction. These findings might indicate that the difference in PKC involvement may contribute to the diverse intracellular signalling pathways of APC and thrombin, and hence their opposite cellular responses.
Supervisor: Lane, David ; Crawley, Jim Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available