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Modulating Cytoprotective Signalling with Engineered Coagulation Proteases

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posted on 11.03.2022, 14:56 authored by Orla Willis Fox
Protein C (PC) is activated by the thrombin-thrombomodulin (thrombin-TM) complex on the surface of endothelial cells. Activated protein C (APC) generation is further enhanced by PC binding to the endothelial protein C receptor (EPCR). Once generated, APC interacts with phospholipids and protein S (PS) to mediate factor Va (FVa) proteolysis. In doing so, it supresses thrombin generation to reduce fibrin clot formation. APC can also initiate multiple signalling pathways via proteolysis of protease-activated receptor 1 (PAR1). PAR1 activation by thrombin results in proinflammatory signalling on endothelial cells, that is terminated by β-arrestin 1 recruitment. Conversely, APC-induced PAR1 interaction with b-arrestin 2 is critical for downstream cytoprotective signalling. Other coagulation proteases also activate PAR1, but the precise role of other membrane co-receptors, such as EPCR, in enabling their proteolysis of PAR1 and subsequent recruitment of specific β-arrestins, is not known.

In this thesis, I modified a novel reporter assay for PAR1 proteolysis dependent β- arrestin recruitment and demonstrated that this assay could be utilised to investigate β-arrestin recruitment to PAR1 activated by multiple coagulation enzymes. Furthermore, I generated novel APC and meizothrombin (mFIIa) variants to better understand the precise role of EPCR binding in enabling cytoprotective PAR1 signalling by coagulation proteases. Specifically, I generated a recombinant nonanticoagulant PC variant, (PCCIDRα1.4), in which the PC Gla domain was substituted with a Plasmodium falciparum adhesion protein (PfEMP1) domain (CIDRα1.4) that binds EPCR with 200-fold greater affinity than wild-type PC (PCWT). Enhanced and prolonged binding to EPCR by PCCIDRα1.4 resulted in significant inhibition of PC activation by the thrombin-TM complex. Moreover, APCCIDRα1.4 proteolysis of PAR1 resulted in enhanced EPCR-dependent β-arrestin 2 recruitment to activated PAR1. These findings suggest that APCCIDRα1.4 possesses unique functional properties that have potential for translation into novel therapeutics for both bleeding and inflammatory disorders. To investigate the effect of rapid PAR1 proteolysis combined with enhanced EPCR affinity, I generated a series of mFIIa variants with a spectrum of EPCR affinities by substituting the mFIIa Gla domain with either the PC Gla domain or PfEMP1 CIDRα1.4 domain (EPCR affinity; mFIIa < mFIIaPC-Gla < mFIIaCIDRα1.4).

Each mFIIa variant mediated rapid PAR1 proteolysis, but the efficacy with which they recruited β-arrestins was dependent upon their affinity for EPCR. Collectively, these results demonstrate that EPCR occupancy by any PAR1-cleaving protease enables enhanced β-arrestin recruitment to activated PAR1, independent of proteolysis site and cleavage rate. Notably, prolonged EPCR occupancy by mFIIaCIDRα1.4 or APC CIDRα1.4 increased β-arrestin 2 recruitment beyond that of wild-type versions of either protease. Further studies to delineate the molecular parameters underlying enhanced β-arrestin recruitment to activated PAR1 caused by prolonged EPCR occupation may contribute to the generation of targeted therapeutics aimed at modulating this important signalling pathway.

History

First Supervisor

Dr Roger Preston

Comments

Submitted for the Award of Doctor of Philosophy to the Royal College of Surgeons in Ireland, 2021.

Published Citation

Fox OW. Modulating Cytoprotective Signalling with Engineered Coagulation Proteases [PhD Thesis] Dublin: Royal College of Surgeons in Ireland; 2021

Degree Name

Doctor of Philosophy (PhD)

Date of award

31/05/2021

Programme

  • Doctor of Philosophy (PhD)