An investigation into the regulation of cellular homeostasis through modulation of cell-surface tissue factor

Madkhali, Yahya Awaji

Abstract

In recent years it has become evident that cell-derived microvesicles (MV) influence the recipient cells through inducing signalling mechanisms which lead to cell proliferation or apoptosis. In addition, the excessive release of procoagulant MV during chronic diseases including cancer, sepsis and cardiovascular disease, appears to advance the severity of these diseases. MV released in response to inflammatory stimuli, mostly carry the proteins tissue factor (TF), coagulation factor VIIa (fVIIa) and negatively charged phospholipids. The formation of the TF-fVIIa complex is known to activate protease activated receptor 2 (PAR2) which in turn promotes the release of additional TF-containing MV by cells. In this study, the ratio of fVIIa:TF carried on the surface of MV was measured and its influence on cell proliferation, apoptosis and PAR2 activation was assessed. Incubation of cells with MV containing the highest ratios of fVIIa:TF (38:1 and 54:1) were shown to be proliferative, while at lower ratios (10:1), the MV were shown to be pro-apoptotic. To investigate the requirements for the activation of PAR2 on cell lines and endothelial cells, the cDNA for PAR2 was cloned and expressed as a hybrid protein, in tandem with either mCherry or mEmerald. The digestion of PAR2 resulted in the release of the fluorescent group into the media which was then measured using a fluorescence plate-reader. Analysis of the cell lines and endothelial cells treated with procoagulant MV indicated that the activation of PAR2 on the surface of the transfected cells was proportionately dependent on the fVIIa:TF ratio. Furthermore, incubation of the transfected endothelial cells with either the MV, or combinations of fVIIa and recombinant TF confirmed the dependence of PAR2 activation on the fVIIa:TF ratio with which the cells come into contact. In the final section of the study, the association of TF-fVIIa complex with PAR2 and also with the caveolae on the cell surface was investigated, and alternative mechanisms for clearance of TF were explored. Knock-down of fVII using specific siRNA prevented the association of TF with PAR2 as examined by the proximity ligation assay (PLA). In addition, incubation of cells with excess amounts of Texas Red labelled-recombinant TF indicated the association of TF with lipid rafts as identified by NBD-cholesterol loading. The exogenous recombinant TF also co-localised with caveolin-1 as determined by PLA.Disruption of lipid rafts using methyl-β-cyclodextrin (MβCD) enhanced the pro-apoptotic influence of the TF in endothelial cells indicating that the TF associated with lipid rafts is maintained in a functionally inactive form. Additionally, a series of experiments indicated the potential of the currently used direct oral anticoagulant Apixaban, to reduce the damage arising from the procoagulant MV. In conclusion, this study has shown that cellular regulation of TF by cells determines the fate of the cells following injury. This is dependent on the ratio of fVIIa and TF carried on MV, which may lead to growth or clearance of cells. In addition, cells regulate the level of TF by two separate mechanisms, both of which are dependent on PAR2 signalling. While these mechanisms ensure the survival of cells following injury and trauma, the exhaustion of the cellular capacity, overwhelmed during chronic diseases, appears to be an underlying cause of vascular deterioration, but there is potential to modulate these interactions for therapeutic benefit. Finally, the study indicated the potential of Apixaban to reduce the damage arising from the procoagulant MV.

Citation

Madkhali, Y. A. An investigation into the regulation of cellular homeostasis through modulation of cell-surface tissue factor. (Thesis). University of Hull. https://hull-repository.worktribe.com/output/4836545

• Thesis Type: Thesis
• Deposit Date: Sep 28, 2024
• Publicly Available Date: Oct 25, 2024
• Keywords: Biomedical sciences
• Public URL: https://hull-repository.worktribe.com/output/4836545
• Additional Information: Department of Biomedical Sciences; University of Hull
• Award Date: Aug 20, 2020