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

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

Regulation of tissue factor activity by interaction with the first PDZ domain of MAGI1

Background Tissue factor (TF) activity is stringently regulated through processes termed encryption. Post-transla-tional modification of TF and its interactions with various protein and lipid moieties allows for a multi-step de-encryp-tion of TF and procoagulant activation. Membrane-associated guanylate kinase-with inverted configuration (MAGI) proteins are known to regulate the localisation and activity of a number of proteins including cell-surface receptors. Methods The interaction of TF with MAGI1 protein was examined as a means of regulating TF activity. MDA-MB-231 cell line was used which express TF and MAGI1, and respond well to protease activated receptor (PAR)2 activation. Proximity ligation assay (PLA), co-immunoprecipitation and pull-down experiments were used to examine the interaction of TF with MAGI1-3 proteins and to investigate the influence of PAR2 activation. Furthermore, by cloning and expressing the PDZ domains from MAGI1, the TF-binding domain was identified. The ability of the recombinant PDZ domains to act as competitors for MAGI1, allowing the induction of TF procoagulant and signalling activity was then examined. Results PLA and fluorescence microscopic analysis indicated that TF predominantly associates with MAGI1 and less with MAGI2 and MAGI3 proteins. The interaction of TF with MAGI1 was also demonstrated by both co-immunoprecipitation of TF with MAGI1, and co-immunoprecipitation of MAGI1 with TF. Moreover, activation of PAR2 resulted in reduction in the association of these two proteins. Pull-down assays using TF-cytoplasmic domain pep-tides indicated that the phosphorylation of Ser253 within TF prevents its association with MAGI1. Additionally, the five HA-tagged PDZ domains of MAGI1 were overexpressed separately, and the putative TF-binding domain was identified as PDZ1 domain. Expression of this PDZ domain in cells significantly augmented the TF activity measured both as thrombin-generation and also TF-mediated proliferative signalling. Conclusions Our data indicate a stabilising interaction between TF and the PDZ-1 domain of MAGI1 and demonstrate that the activation of PAR2 disrupts this interaction. The release of TF from MAGI1 appears to be an initial step in TF de-encryption, associated with increased TF-mediated procoagulant and signalling activities. This mechanism is also likely to lead to further interactions and modifications leading to further enhancement of procoagulant activity, or the release of TF

Apixaban suppresses the release of TF-positive microvesicles and restrains cancer cell proliferation through directly inhibiting TF-fVIIa activity

The activation of protease-activated receptor (PAR)-2 by factor Xa (fXa) promotes the release of tissue factor-positive microvesicles (TF + MV), and contributes to proliferation in cancer cells. This study examined the ability of direct oral anticoagulants (DOACs), apixaban and rivaroxaban, to inhibit the release of TF + MV from two cell lines (MDA-MB-231 and AsPC-1) as well as cell proliferation. Activation of the cells with fXa (10 nM) enhanced the release of TF + MV but was suppressed in the presence of either DOAC. These MVs were found to contain fVIIa, but not fXa. Incubation of cell lines with apixaban (1.8 μM) but not rivaroxaban (1.8 μM), in the absence of fXa decreased the release of TF + MV below that of resting cells, in a PAR2-dependent manner. Furthermore, incubation with apixaban reduced the proliferation rate in both cells lines. Incubation of purified fVIIa with apixaban but not rivaroxaban resulted in complete inhibition of fVIIa proteolytic activity as measured using two fVIIa chromogenic substrates. Pre-incubation of the cells with an inhibitory anti-fVIIa antibody, with apixaban or the blocking of PAR2 suppressed the release of TF + MV to a comparable level, and reduced cell proliferation but the effect was not cumulative. This study has established that the activation of PAR2 by TF-fVIIa complex is the principal mediator in augmenting the release of TF + MV as well as cancer cell proliferation. Importantly, for the first time we have shown that apixaban selectively inhibits the proteolytic activity of fVIIa as well as the signalling arising from the TF-fVIIa complex

Peptidyl-prolyl isomerase 1 (Pin1) preserves the phosphorylation state of tissue factor and prolongs its release within microvesicles

© 2017 Elsevier B.V. The exposure and release of TF is regulated by post-translational modifications of its cytoplasmic domain. Here, the potential of Pin1 to interact with the cytoplasmic domain of TF, and the outcome on TF function was examined. MDA-MB-231 and transfected-primary endothelial cells were incubated with either Pin1 deactivator Juglone, or its control Plumbagin, as well as transfected with Pin1-specific or control siRNA. TF release into microvesicles following activation, and also phosphorylation and ubiquitination states of cellular-TF were then assessed. Furthermore, the ability of Pin1 to bind wild-type and mutant forms of overexpressed TF-tGFP was investigated by co-immunoprecipitation. Additionally, the ability of recombinant or cellular Pin1 to bind to peptides of the C-terminus of TF, synthesised in different phosphorylation states was examined by binding assays and spectroscopically. Finally, the influence of recombinant Pin1 on the ubiquitination and dephosphorylation of the TF-peptides was examined. Pre-incubation of Pin1 with Juglone but not Plumbagin, reduced TF release as microvesicles and was also achievable following transfection with Pin1-siRNA. This was concurrent with early ubiquitination and dephosphorylation of cellular TF at Ser253. Pin1 co-immunoprecipitated with overexpressed wild-type TF-tGFP but not Ser258 → Ala or Pro259 → Ala substituted mutants. Pin1 did interact with Ser258-phosphorylated and double-phosphorylated TF-peptides, with the former having higher affinity. Finally, recombinant Pin1 was capable of interfering with the ubiquitination and dephosphorylation of TF-derived peptides. In conclusion, Pin1 is a fast-acting enzyme which may be utili sed by cells to protect the phosphorylation state of TF in activated cells prolonging TF activity and release, and therefore ensuring adequate haemostasis

The Ratio of Factor VIIa:Tissue Factor Content within Microvesicles Determines the Differential Influence on Endothelial Cells

Tissue factor (TF)-positive microvesicles from various sources can promote cellular proliferation or alternatively induce apoptosis, but the determining factors are unknown. In this study the hypothesis that the ratio of fVIIa:TF within microvesicles determines this outcome was examined. Microvesicles were isolated from HepG2, BxPC-3, 786-O, MDA-MB-231, and MCF-7 cell lines and microvesicle-associated fVIIa and TF antigen and activity levels were measured. Human coronary artery endothelial cells (HCAECs) were incubated with these purified microvesicles, or with combinations of fVIIa-recombinant TF, and cell proliferation/apoptosis was measured. Additionally, by expressing mCherry-PAR2 on HCAEC surface, PAR2 activation was quantified. Finally, the activation of PAR2 on HCAEC or the activities of TF and fVIIa in microvesicles were blocked prior to addition of microvesicles to cells. The purified microvesicles exhibited a range of fVIIa:TF ratios with HepG2 and 786-O cells having the highest (54:1) and lowest (10:1) ratios, respectively. The reversal from proapoptotic to proliferative was estimated to occur at a fVIIa:TF molar ratio of 15:1, but HCAEC could not be rescued at higher TF concentrations. The purified microvesicles induced HCAEC proliferation or apoptosis according to this ruling. Blocking PAR2 activation on HCAEC, or inhibiting fVIIa or TF-procoagulant function on microvesicles prevented the influence on HCAEC. Finally, incubation of HCAEC with recombinant TF resulted in increased surface exposure of fVII. The induction of cell proliferation or apoptosis by TF-positive microvesicles is dependent on the ratio of fVIIa:TF and involves the activation of PAR2. At lower TF concentrations, fVIIa can counteract the proapoptotic stimulus and induce proliferation

Oligoubiquitination of tissue factor on Lys255 promotes Ser253-dephosphorylation and terminates TF release

Restriction of tissue factor (TF) activity at the cell surface and TF release are critical for prevention of excessive coagulation. This study examined the regulation of TF dephosphorylation and its release through ubiquitination. A plasmid containing the sequence to express the tandem protein TF-tGFP was mutated to include an arginine-substitution at Lys255 within TF. MDA-MB-231 cell line, and HCAEC endothelial cells were transfected and subsequently activated with PAR2-agonist peptide. The wild-type and mutant TF-tGFP were immunoprecipitated from the cell lysates and the ubiquitination and phosphorylation state of TF examined. Analysis of the proteins showed that arginine-substitution of Lys255 within TF prevented its ubiquitination while the wild-type TF-tGFP was oligoubiquitinated. The TF-associated oligoubiquitin chain was estimated to contain up to 4 ubiquitin units, with the linkage formed between Lys63 of one ubiquitin unit, and the C-terminus of the next unit. The Lys255 → Arg substitution of TF-tGFP prolonged the phosphorylation of Ser253 within TF, compared to the wild-type TF-tGFP, lengthened the presence of TF-tGFP at the cell surface and extended the duration of TF-tGFP release from cells following PAR2 activation. A biotinylated 19-mer peptide corresponding to the C-terminus of TF (TFc) was used as substrate to show that the ubiquitination of TF was mediated by the Ube2D family of E2-enzymes and involved Mdm2. Moreover, double-phosphorylation of TFc was prerequisite for ubiquitination, with subsequent dephosphorylation of Ser253 by phosphatase PP2A. In conclusion, oligoubiquitination of Lys255 within TF permits PP2A to bind and dephosphorylate Ser253 and occurs to terminate TF release and contain its activity

Low molecular weight heparin and direct oral anticoagulants influence tumour formation, growth, invasion and vascularisation by separate mechanisms

The bidirectional association between coagulation and cancer has been established. However, anticoagulant therapies have been reported to have beneficial outcomes by influencing the vascularisation of the tumours. In this study the influence of a set of anticoagulants on tumour formation, invasion and vascularisation was examined. WM-266-4 melanoma and AsPC-1 pancreatic cancer cell lines were treated with LMWH (Tinzaparin and Dalteparin), and DOAC (Apixaban and Rivaroxaban) and the rate of tumour formation, growth and invasion were measured in vitro. In addition, the influence of these anticoagulants on vascularisation was examined using the chorioallantoic membrane assay (CAM) model and compared to the outcome of treatment with Bevacizumab. Using this model the influence of pharmacological concentrations of the anticoagulant on the growth, invasion and vascularisation of tumours derived from WM-266-4 and AsPC-1 cells was also measured in vivo. Tinzaparin and Daltepain reduced tumour formation and invasion by the cell lines in vitro, but with dissimilar potencies. In addition, treatment of CAM with LMWH reduced the local vascular density beyond that achievable with Bevacizumab, particularly suppressing the formation of larger-diameter blood vessels. In contrast, treatment with DOAC was largely ineffective. Treatment of CAM-implanted tumours with LMWH also reduced tumour vascularisation, while treatment of tumours with Apixaban reduced tumour growth in vivo. In conclusion, LMWH and DOAC appear to have anti-cancer properties that are exerted through different mechanisms

Alteration in endothelial permeability occurs in response to the activation of PAR2 by factor Xa but not directly by the TF-factor VIIa complex

Alterations in the endothelial permeability occur in response to the activation of coagulation mechanisms in order to control clot formation. The activation of the protease activated receptors (PAR) can induce signals that regulate such cellular responses. PAR2 is a target for the coagulation factor Xa (fXa) and tissue factor-factor VIIa (TF-fVIIa) complex. By measuring the permeability of dextran blue across endothelial monolayer, we examined the mechanisms linking coagulation and endothelial permeability. Activation of PAR2 using the agonist peptide (PAR2-AP) resulted in increased permeability across the monolayer and was comparable to that obtained with VEGF at 60 min. Incubation of cells with activated factor Xa (fXa) resulted in an initial decrease in permeability by 30 min, but then significantly increased at 60 min. These responses required fXa activity, and were abrogated by incubation of the cells with a PAR2-blocking antibody (SAM11). Activation of PAR2 alone, or inhibition of PAR1, abrogated the initial reduction in permeability. Additionally, inclusion of Rivaroxaban (0.6 µg/ml) significantly inhibited the response to fXa. Finally, incubation of the endothelial monolayers up to 2 h with TF-containing microvesicles derived from MDA-MB-231 cells, in the presence or absence of fVIIa, did not influence the permeability across the monolayers. In conclusion, fXa but not TF-fVIIa is a noteworthy mediator of endothelial permeability. The rapid initial decrease in permeability requires PAR2 and PAR1 which may act to constrain bleeding. The longer-term response is mediated by PAR2 with increased permeability, presumably to enhance clot formation at the site of damage

Tumour-Associated Tissue Factor (TF)-mRNA Is A Precursor for Rapid TF-Microvesicle Release and A Potential Predictive Marker for the Risk of Pulmonary Embolism (PE) in Gastrointestinal Cancer Patients

Predicting which cancer patients are at risk of thrombosis remains a key challenge to effective thromboprophylaxis. This study was based on the hypothesis that the rapid release of TF-containing MV occurs in cancer cells that possess high levels of TF mRNA, permitting the transient but amplified TF-protein production in response to cellular activation. To gather preliminary clinical evidence for this hypothesis the correlation between the levels of tumour-associated TF mRNA and incidence of Pulmonary Embolism (PE) in Gastrointestinal cancer patients (GI) was assessed using stringently-selected patient cohorts. Furthermore, the rapid TF-MV release was assessed in three cell lines with high TF mRNA in which protein-translation or mRNA-transcription were inhibited separately. On applying the exclusion criteria, the study accrued 9 clinical samples with incidental PE (colonic n=5; gastroesophageal n=4) which were type, gender and stage of cancer matched one-to-one with patients without PE (9+9 samples). Total-RNA was extracted from the samples using a FFPE-RNA extraction kit and TF mRNA was quantified using a quantitative real-time PCR procedure along with a standard curve prepared using in vitro-transcribed TF mRNA. Relative amounts of PAR2 mRNA were also determined. Analysis of absolute amounts of tumour-associated TF mRNA showed significant increase in patients who developed PE (mean=26.931±15.371 pg/100ng-total RNA; median=5.340 pg/100ng-total RNA; range=0.4-131.43 pg/100ng-total RNA) compared to those who didn’t (mean=0.098±0.023 pg/100ng-total RNA; median=0.110 pg/100ng-total RNA; range=0-0.19 pg/100ng-total RNA). Receiver Operating Characteristic (ROC) analysis returned an area under the curve of 1. In contrast, no significant difference in PAR2 mRNA was recorded. To provide an explanation for these findings, inhibition of protein-translation using cycloheximide prevented the incorporation of TF but not the MV release. However, blocking of RNA-transcription did not prevent TF-MV release. In conclusion, this is a first demonstration of a strong correlation between the risk of PE in GI cancer and the levels of tumour-TF mRNA, and further supports the hypothesis that the process is driven by the rapid translation of mRNA into TF-protein, following stimulation

De-palmitoylation of tissue factor regulates its activity, phosphorylation and cellular functions

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 hydropho-bicity (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 PKCα-mediated Ser253 phosphorylation with Gö6976 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 activa-tion. 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