Simple Summary The relationship between cancer and blood clotting has been well established. The activation of blood coagulation proteins regulates the fate of cells and is known to be used by... Show moreSimple Summary The relationship between cancer and blood clotting has been well established. The activation of blood coagulation proteins regulates the fate of cells and is known to be used by cancer cells to enhance survival and proliferation. Cells strictly regulate the initiation of coagulation through controlling the action of the protein "tissue factor (TF)". In addition to initiating clotting, TF also acts as a deciding factor to determine the extent of damage and instructs cells to proliferate and repair or, when severely damaged, to die. Therefore, normal cells keep TF in a dormant state, achieved through mechanisms called "TF encryption". Understanding the mechanisms by which the cells control the activity of TF is crucial, especially since cancer cells bypass these regulatory mechanisms, ensuring survival and tumour growth. This study has elucidated essential molecular mechanisms by which cells regulate TF clotting activity, and also the cellular signals arising from these. In this study, the role of de-palmitoylation of tissue factor (TF) in the decryption of its activity was explored. TF-tGFP constructs were prepared by mutagenesis-substitution at Cys245 to prevent or mimic palmitolyation. Additionally, to reduce TF de-palmitoylation, the expression of palmitoyl-protein thioesterases (PPT) was suppressed. Other TF mutants were prepared with altered flexibility, hydrophobicity or length of the transmembrane domain. The outcome of these alterations on fXa-generation, fVIIa binding, Ser253 phosphorylation and TF-microvesicle release were assessed in endothelial cells, and the influence on endothelial and MCF-7 cell proliferation and apoptosis was analysed. Preventing TF palmitoylation (TFSer245-tGFP), increasing the hydrophobicity (TFPhe241-tGFP) or lengthening (TFLongTM-tGFP) of the transmembrane domain enhanced fXa-generation in resting cells compared to cells expressing TFWt-tGFP, but fXa-generation was not further increased following PAR2 activation. Extending the available length of the transmembrane domain enhanced the TF-tGFP release within microvesicles and Ser253 phosphorylation and increased cell proliferation. Moreover, prevention of PKC alpha-mediated Ser253 phosphorylation with Go6976 did not preclude fXa-generation. Conversely, reducing the hydrophobicity (TFSer242-tGFP), shortening (TFShortTM-tGFP) or reducing the flexibility (TFVal225-tGFP) of the transmembrane domain suppressed fXa-generation, fVIIa-HRP binding and Ser253 phosphorylation following PAR2 activation. PPT knock-down or mimicking palmitoylation (TFPhe245-tGFP) reduced fXa-generation without affecting fVIIa binding. This study has for the first time shown that TF procoagulant activity is regulated through de-palmitoylation, which alters the orientation of its transmembrane domain and is independent of TF phosphorylation. However, Ser253 phosphorylation is facilitated by changes in the orientation of the transmembrane domain and can induce TF-cellular signalling that influences cellular proliferation/apoptosis. Show less
Simple Summary Under normal conditions, blood coagulation is suppressed to prevent thrombosis. However, during inflammatory conditions such as injury or disease conditions, the protein "tissue... Show moreSimple Summary Under normal conditions, blood coagulation is suppressed to prevent thrombosis. However, during inflammatory conditions such as injury or disease conditions, the protein "tissue factor (TF)" is expressed on the surface of the cells and is also released into the bloodstream within cell-derived vesicles called "microvesicles". TF appears first at the site of trauma which makes TF suitable for determining the extent of damage and instructing cells to proliferate and repair, or if severely damaged, to die. The relationship between cancer and thrombosis was reported in the early part of the 19th century. Cancer cells and particularly those with aggressive tendencies have the ability to produce, and then optimise the amount of TF on the cell, in order to maximise the pro-survival and proliferative properties of this protein. This study has demonstrated some of the mechanisms by which cells control excessive amounts of TF, to levels ideal for tumour survival and growth. Procoagulant activity of tissue factor (TF) in response to injury or inflammation is accompanied with cellular signals which determine the fate of cells. However, to prevent excessive signalling, TF is rapidly dissipated through release into microvesicles, and/or endocytosis. To elucidate the mechanism by which TF signalling may become moderated on the surface of cells, the associations of TF, fVII/fVIIa, PAR2 and caveolin-1 on MDA-MB-231, BxPC-3 and 786-O cells were examined and compared to that in cells lacking either fVII/fVIIa or TF. Furthermore, the localisation of labelled-recombinant TF with cholesterol-rich lipid rafts was explored on the surface of primary human blood dermal endothelial cells (HDBEC). Finally, by disrupting the caveolae on the surface of HDBEC, the outcome on TF-mediated signalling was examined. The association between TF and PAR2 was found to be dependent on the presence of fVIIa. Interestingly, the presence of TF was not pre-requisite for the association between fVII/fVIIa and PAR2 but was significantly enhanced by TF, which was also essential for the proliferative signal. Supplementation of HDBEC with exogenous TF resulted in early release of fVII/fVIIa from caveolae, followed by re-sequestration of TF-fVIIa. Addition of labelled-TF resulted in the accumulation within caveolin-1-containing cholesterol-rich regions and was also accompanied with the increased assimilation of cell-surface fVIIa. Disruption of the caveolae/rafts in HDBEC using M beta CD enhanced the TF-mediated cellular signalling. Our data supports a hypothesis that cells respond to the exposure to TF by moderating the signalling activities as well as the procoagulant activity of TF, through incorporation into the caveolae/lipid rafts. Show less
Background Cell injury signal-induced activation and release of tissue factor (TF) on extracellular vesicles (EVs) from immune and vessel wall cells propagate local and systemic coagulation... Show moreBackground Cell injury signal-induced activation and release of tissue factor (TF) on extracellular vesicles (EVs) from immune and vessel wall cells propagate local and systemic coagulation initiation. TF trafficking and release on EVs occurs in concert with the release of cell adhesion receptors, including integrin beta(1) heterodimers, which control trafficking of the TF-activated factor VII (FVIIa) complex. Activation of the TF signaling partner, protease-activated receptor (PAR) 2, also triggers TF release on integrin beta(+)(1) EVs from endothelial cells, but the physiological signals for PAR2-dependent EV generation at the vascular interface remain unknown. Objective To define relevant protease ligands of TF contributing to PAR2-dependent release on EVs from endothelial cells. Methods In endothelial cells with balanced expression of TF and PAR2, we evaluated TF release on EVs by using a combination of activity and antigen assays, immunocapture, and confocal imaging. Results and Conclusions PAR2 stimulation generated time-dependent release of distinct TF+ EVs with high coagulant activity (early) and high antigen levels (late). Whereas PAR2 agonist peptide and a stabilized TF-FVIIa-activated FX complex triggered TF+ EV release, stimulation with FVIIa alone promoted cellular retention of TF, despite comparable PAR2 activation. On endothelial cells, FVIIa uniquely induced formation of a complex of TF with integrin alpha(5)beta(1). Internalization of TF by FVIIa or anti-TF and activating antibodies against integrin beta(1) prevented PAR2 agonist-induced release of TF on EVs. These data demonstrate that intracellular trafficking controlled by FVIIa forcing interaction with integrin beta(1) regulates TF availability for release on procoagulant EVs. Show less
