Investigation of the effects of anticoagulants on tissue factor mediated cellular processes

Featherby, Sophie

Biomedical sciences
May 2021

Thesis or dissertation


Rights
© 2021 Sophie Featherby. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.
Abstract

Thrombotic complications are common in cancer patients and are amongst the leading causes of morbidity and mortality. Recently, it was reported that treatment of cancer patients with the low molecular weight heparin (LMWH) family of anticoagulants can result in a significant reduction in mortality compared with those receiving other conventional anticoagulants. Therefore, the mechanisms by which different anticoagulants interact with cancer cells were investigated. First, the influence of two preparations of LMWH (tinzaparin and dalteparin) and two direct fXa inhibitors (apixaban and rivaroxaban) on cancer invasion, angiogenesis and tumour growth was explored. Tinzaparin and to a lesser extent dalteparin, were shown to inhibit cancer cell invasion and angiogenesis, using matrix invasion and chorioallantoic membrane (CAM) assays, respectively. In addition, apixaban, but not rivaroxaban inhibited the growth of CAM-implanted xenografts which indicated a mechanism independent of the anti-fXa property of apixaban. The anti-tumour property of apixaban was further explored by comparing the influence of apixaban and rivaroxaban on cancer cell proliferation and microvesicle (MV) release. Apixaban was shown to inhibit the release of MV in response to exogenous fXa and fVIIa, as well as endogenous fVIIa expressed by the cancer cells. In contrast rivaroxaban was effective in suppressing MV release in response to fXa only. The mechanism of inhibition of endogenous fVIIa by apixaban was also shown to include the suppression of complex formation with TF and therefore, preventing the activation of protease activated receptor (PAR) 2 by the TF/fVIIa complex.
In addition to signalling through PAR2, the interaction of tissue factor (TF) with β1 integrin is known to be required for induction of cell proliferation and contact-independent survival of cancer cells. Therefore, in the next section of the study an attempt was made to identify the function of the potential interacting domains between TF and β1 integrin. The cDNA corresponding to the upper or lower fibronectin-like domains, and also that encoding the complete extracellular domain of TF were separately cloned into an expression plasmid. These were expressed in human dermal blood endothelial cells (HDBEC) and MDA-MB-231 cell line and the ability of the resulting peptides to bind to β1 integrin was assessed in situ, using the proximity ligation assay (PLA) and also, by co-immunoprecipitation. The upper fibronectin-like domain (UED) was shown to bind to the EGF4 domain of β1 integrin and the lower fibronectin-like domain (LED) with the βTD domain. To examine the proliferative properties of these peptides, the expression of cyclin D1 and ERK1/2 phosphorylation were measured and changes in cell numbers determined. The interaction of either the UED or the LED peptide with β1 integrin in HDBEC, which do not express endogenous TF, was shown to induce ERK1/2 phosphorylation. However, displacement of endogenous TF by the UED peptide in MDA-MB-231 cells resulted in a reduction in ERK1/2 phosphorylation, cyclin D1 expression and cell numbers. In contrast, expression of LED peptide in MDA-MB-231 cells did not influence these parameters for proliferative signalling, indicating that the TF-signalling was maintained by the LED peptide. Collectively, these findings suggested that although both the upper and lower extracellular domains of TF can induce cell signalling, the lower domain may be essential for the promotion of proliferation.
The last section of the study was based on previous work showing that prevention of the phosphorylation of TF can block the release of the protein in MV, resulting in a build-up of TF within cells. This study explored the role of TF depalmitoylation which is also known to precede and be essential for TF phosphorylation. It was hypothesised that such alterations may regulate the translocation of TF to lipid rafts and its release within MV. Mutant forms of TF with substitutions to prevent, or to mimic palmitoylation at cysteine 245, or with altered lengths of the transmembrane domain were prepared and expressed in cells. The influence of these proteins on cell proliferation and apoptosis were then examined. Expression of mutant forms of TF which could not be palmitoylated or containing a longer transmembrane domain resulted in decreased levels of apoptosis. However, neither the mutant mimicking palmitoylation, or containing a shortened transmembrane domain were not released within MV and resulted in increased cell apoptosis.
In conclusion, this study has demonstrated the anti-cancer properties and some of the mechanisms involved, in a set of anticoagulants. In particular, apixaban was shown to suppress cancer cell growth by preventing the activation of PAR2 by the TF/fVIIa complex. In addition, this study has elucidated the role of the various extracellular and intracellular domains within TF, in promoting the signals that regulate cell proliferation and apoptosis.

Publisher
Department of Biomedical Sciences, The University of Hull
Supervisor
Ettelaie, Camille
Sponsor (Organisation)
Bristol-Myers Squibb Company; LEO Pharmaceuticals; Castle Hill Hospital Cancer Trust Fund
Qualification level
Doctoral
Qualification name
PhD
Language
English
Extent
5 MB
Identifier
hull:18403
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