Introduction: Elevated levels of tissue factor positive (TF +) microparticles (MPs) are observed in plasma from a variety of patients with an increased risk of thrombosis. We and others have described the measurement of TF activity in MPs isolated from plasma. The aim of this study was to investigate the effects of pre-analytical and analytical variables on TF activity of MPs isolated from blood of healthy volunteers either untreated or treated ex vivo with bacterial lipopolysaccharide. Materials and methods: We evaluated the following parameters: use of different centrifugation speeds to isolate the MPs; comparison of TF activity of MPs isolated from platelet poor plasma versus platelet free plasma; effect of freeze/thaw on MP TF activity; and comparison of the MP TF activity assay with the measurement of TF protein by ELISA or flow cytometry. Results: MPs prepared from platelet poor plasma by centrifugation at 20,000 × g or 100,000 × g for 15 minutes had similar levels of TF activity. However, significantly less TF activity was found in MPs isolated from platelet free plasma compared with platelet poor plasma. Interestingly, freeze/thawing of the plasma showed donor to donor variation in MP TF activity, with a moderate increase in some individuals. Conclusion: TF + MPs can be quantitatively isolated from platelet poor or platelet free plasma by centrifugation at 20,000 × g for 15 minutes. Measurement of MP TF activity in plasma may be used to detect a prothrombotic state in patients with various diseases.

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Yes / This data article is related to the research article entitled Proteomics of Tissue Factor silencing in cardiomyocytic cells reveals a new role for this coagulation factor in splicing machinery control by Lento et al [1]. Tissue Factor (TF) is the key player in the coagulation cascade, but it has additional functions ranging from angiogenesis, tumor invasion and, in the heart, the maintenance of the integrity of cardiac cells. This article reports the nano-LC-MSE analysis of the cardiomyocytic HL-1 cell line proteome and describes the results obtained from a Gene Ontology analysis of those proteins affected by TF-gene silencing.

La transplantation d’hépatocytes est une nouvelle approche thérapeutique pour le traitement des maladies métaboliques. Elle peut être proposée en alternative à la transplantation de foie entier ou, à tout le moins, en attente de celle-ci chez les patients instables, à risque de décompensation métabolique. Les essais cliniques effectués chez 9 patients aux cliniques St Luc ainsi que ceux publiés dans la littérature démontrent l’intérêt de la transplantation de cellules hépatiques à court et moyen terme. La qualité de la suspension cellulaire transplantée reste le premier facteur limitant pour le développement clinique de la technique. La cryopréservation reste le moyen le plus approprié pour la conservation à long terme des cellules. Elle permet de constituer une banque de cellules pouvant être utilisées à tout moment. Nous avons d’abord analysé les protocoles de cryopréservation décrits dans la litérature, ainsi que leurs limites tant au niveau de la préservation de la qualité cellulaire après décongélation in vitro qu’après transplantation in vivo. Dans ce travail, nous avons démontré l’intérêt d’utiliser des cellules cryopréservées/décongelées, afin de stabiliser des patients atteints de maladies du cycle de l’urée, avant la greffe de foie entier. Les tests de contrôle de qualité effectués sur ces cellules ont cependant montré une altération aux niveaux biochimique et cellulaire, après décongélation. Nous avons ainsi démontré une chute des concentrations intracellulaires d’ATP, signe d’une atteinte mitochondriale. Nos travaux ont également permis de mettre en évidence une diminution de la consommation d’oxygène des hépatocytes en suspension, due plus particulièrement à une atteinte du complexe 1 de la chaîne respiratoire. Cette atteinte mitochondriale peut déjà être observée après l’incubation de la suspension cellulaire à –20°C. Aux alentours de cette température critique se fait le passage de l’état aqueux à l’état cristallin suggérant que les dégâts mitochondriaux observés sont dès lors vraisembablement dus à la formation de glace intracellulaire durant le processus de cryopréservation ou de décongélation. Diverses tentatives visant à améliorer les paramètres mitochondriaux affectés par le processus de congélation/décongélation par l’addition d’agents protecteurs du complexe 1 (Bilobalide), d’ inhibiteurs du pore de transition de perméabilité (Ciclosporine A), d’ anti-oxydants ou encore de solutions hyperosmotiques à la solution de cryopréservation, n’ont pas permis d’améliorer la qualité cellulaire. Le tri de sous-types de populations hépatocytaires ou l’isolement de foies hépatectomisés n’ont pas permis de révéler de différences de capacité de résistance à la cryopréservation. Toujours dans le but d’améliorer le rendement de la transplantation d’hépatocytes et d’augmenter l’efficacité d’implantation dans le parenchyme receveur, nous avons démontré dans la deuxième partie de la thèse la capacité des hépatocytes isolés (fraîchement isolés ou cryopréservés/décongelés) à induire un phénomène de coagulation dépendant du facteur tissulaire. Cette activité pro-coagulante, inhibée in vitro par lea N-acetyl-L-cystéine, pourrait être le point de départ d’une réaction inflammatoire aspécifique influençant ainsi la réussite de la transplantation cellulaire. En conclusion, nous proposons dans ce travail différentes stratégies en vue de l’amélioration du rendement de la thérapie cellulaire. La vitrification, autre technique de cryopréservation, permettrait d’éviter la formation d’eau intracellulaire. Enfin la modulation de l’activité pro-coagulante par la N-acetyl-L-cystéine, due à la transplantation cellulaire, constitue une piste intéressante pour essayer d’améliorer l’implantation des cellules transplantées et ainsi le rendement de la greffe. / Liver cell transplantation provides clinical benefit to patients with congenital metabolic abnormalities and currently represents an alternative to orthotopic liver transplantation or at least an interim measure for unstable patients awaiting transplantation. Our team and others have already demonstrated that transplanted hepatocytes can achieve metabolic control in the short or medium term. The quality of transplanted cells remains the first limiting factor for the success of liver cell transplantation. Because the use of freshly isolated cells is restricted by contemporary organ donation, cryopreservation remains necessary for long-term storage and permanent availability of the cells. In this thesis, we have first reviewed and discussed established hepatocyte cryopreservation protocols, especially the cooling procedure, and have focussed on the in vitro and in vivo assays used for the evaluation of post-thawing hepatocyte quality. Amongst 9 cell transplanted patients in our center, several received exclusively or predominantly cryopreserved/thawed hepatocytes. We demonstrated post-transplantation benefits of using these cells in control patients with congentital abnormalities in the urea cycle, particularly with respect to clear evidence of cell engraftment and de novo appearance of enzyme activity. However, despite these clinical benefits, we found an in vitro relationship between the low post-thawing quality of cryopreserved /thawed hepatocytes and an alteration in their mitochondrial function. This post-thawing mitochondrial damage was already evident after the first −20°C cryopreservation step of our protocol, suggesting it occurrs early in the process, around the nucleation point, by intracellular ice formation. Cellular impairment could therefore be possibly explained by mechanical alteration of mitochondria due to water crystallisation during the cryopreservation process or thawing procedure. We also observed a poor efficacy of cryopreserved/thawed hepatocytes (as compared to freshly isolated cells) when used liver engraftment in two mice transplantation models. The marked reductions in intracellular ATP concentrations and the decreases in oxygen consumption by hepatocytes were therefore used as markers for the evaluation of the effects of several compounds such as bilobalide, hyperosmotic or anti-oxidant molecules, pore transition permeability inhibitors, and for the evaluation of the resistance of selected hepatocyte subtypes to cryopreservation protocols. We also demonstrated that isolated hepatocytes exert tissue factor-dependent pro-coagulant activity, which may contribute to the early loss of infused cells. We observed that the addition of N-acetyl-L-cysteine to hepatocyte suspensions inhibits coagulation activation. In conclusion, this work has identified several ways to improve the clinical benefit of liver cell transplantation, including new cryopreservation strategies, such as vitrification. In addition, modulation of the pro-coagulant activity induced by cell infusion with N-acetyl-L-cysteine might beneficially enhance cell engraftment.

Malignancy is associated with a hypercoagulable state manifested clinically by an increased incidence of venous thromboembolism (VTE). Colorectal cancer (CRC) patients who develop VTE have reduced survival. This increased mortality extends beyond the acute VTE event, suggesting that VTE is associated with aggressive tumour biology. Tissue factor (TF) and other clotting factors have been implicated in this process. However, the significance of clotting factors in the tumour microenvironment (TME) remains unknown. The aim of this thesis is to i) determine if a procoagulant TME is a biomarker for poor prognosis and VTE in patients undergoing resectional surgery for CRC and ii) determine the effect of TF, thrombin and FXa on proliferation and migration in vitro in CRC and if their inhibitors have potential as anticancer therapies. In the in vitro studies, epithelial expression of TF had a modest effect on proliferation and migration when quantified using the PrestoBlue proliferation and transwell migration assays. Exogenous TF, FXa and thrombin all increased migration in DLD-1 wild type cells. In addition, exogenous thrombin increased proliferation amongst SW620 wild type cells. This suggests that coagulation factors from the TME, rather than epithelial expression, may influence tumour biology. Moreover, dabigatran, a direct thrombin inhibitor, abrogated the pro-proliferative effects of thrombin, which highlights its potential role as an anticancer therapy. In a multicentre, prospective cohort study of 159 CRC patients undergoing resectional surgery, rates of duplex screen detected deep vein thrombosis (DVT) were correlated to plasma and tumour markers of hypercoagulability. TF is upregulated in the stroma of cancer compared to normal tissue. However, stromal TF expression decreased in more advanced (T4) tumours. This suggests that a procoagulant TME has a role in early tumourigenesis. In total, 5.4%, 7.0% and 9.1% of patients had an asymptomatic DVT pre- operatively, at six weeks post-surgery and after the commencement of adjuvant chemotherapy respectively. The development of a post-operative complication was a risk factor for DVT, whilst locally advanced tumours resulted in a prolonged hypercoagulable state i.e. raised D-dimer at six weeks. This highlights a possible role for pre- and post- operative screening duplex ultrasonography and super-extended VTE prophylaxis in selected patients. In conclusion, this thesis establishes a role for exogenous coagulation factors in promoting tumour biology in CRC. VTE is more common amongst patients undergoing resectional surgery for CRC than previously estimated. The utility of tumour and plasma hypercoagulabilty as biomarkers for survival in CRC will be further analysed when long term follow-up data is available.

Des résultats d’expériences cliniques de reconstruction de l’os maxillaire faites à partir de la greffe d’une "pâte osseuse" gélifiée par l’ajout de facteur tissulaire ont été le primum movens de ce travail. Cette "pâte osseuse", faite d’os en poudre et de plasma enrichi en plaquette (PRP) à laquelle on ajoute du facteur tissulaire, est un modèle à la fois de la coagulation et de la régénération osseuse. Pour analyser des effets de la coagulation, nous avons utilisé un modèle connu : la culture primaire de cellules endothéliales (HUVEC). Les effets in vitro des facteurs de coagulation, dénommés protéases de la coagulation, pris séparément, ont été bien étudiés dans ces cellules, néanmoins aucune information sur l’effet combiné de ces protéases ou du plasma en coagulation n’était connue. Nous avons mesuré la "signalisation calcium" comme réponse cellulaire aux différents agents et ces mesures de la signalisation calcium ont été complétées par la mesure d’une autre réponse biologique, à savoir la sécrétion de cytokines pro-inflammatoires (IL-6 et IL-8). Pour l’étude de la régénération osseuse, la signalisation calcium a été mesurée sur une lignée d’ostéosarcomes humains (SaOS-2), stimulée par des protéases de la voie extrinsèque de la coagulation (facteur VIIa, facteur Xa et thrombine). Comme réponse biologique complémentaire, nous avons évalué l’effet des protéases d’intérêt sur l’apoptose induite par l’absence de sérum dans le milieu de culture. Les premiers travaux, réalisés sur les HUVEC, nous ont permis de montrer que le facteur Xa et la thrombine induisaient des signaux calcium différents sur ces cellules en mono couches, alors que le complexe facteur tissulaire – facteur VIIa ne provoquait aucune signalisation calcium. Nous avons également pu montrer une addition des signaux calcium induits par le facteur Xa et la thrombine. L’activation in situ du facteur Xa et de la thrombine à partir de leur zymogène a permis à la fois de confirmer les résultats précédents et de se rapprocher de l’in vivo. Finalement, au plus proche de l’in vivo, les expériences faites avec du plasma en coagulation ont également permis de détecter un signal calcium. La réponse biologique (sécrétion d’IL-6 et d’IL-8) en aval du signal calcium a confirmé les résultats calcium. En ce qui concerne la régénération osseuse étudiée à partir de SaOS-2, nous avons démontré l’expression du facteur tissulaire sur la lignée SaOS-2 et nous avons montré que le facteur VIIa, le facteur Xa et la thrombine induisaient tous des signaux calcium. Ces signaux présentaient des caractéristiques propres suivant la ou les protéase(s) utilisée(s) pour la stimulation. Les mesures ont également permis de caractériser, sur ces cellules, les récepteurs activés par les protéases d’intérêt, à savoir les "protease-activated receptors" 1 et 2 (PAR-1 et PAR-2). Comme réponse biologique, nous avons mesuré la diminution de l’apoptose induite par les protéases en absence de sérum dans le milieu de culture. Il a ainsi été montré que seule l’activation du récepteur PAR-1 permettait de diminuer l’apoptose. Finalement, nous avons caractérisé la voie suivie, qui passait par la phosphoinositide 3-kinase et la voie des MAPK Raf/MEK/ERK 1/2. En conclusion, cette thèse a permis de montrer, d’une part, que le facteur Xa et la thrombine provoquent des réponses calciques et proinflammatoires additifs dans les cellules endothéliales et, d’autre part, que le complexe facteur tissulaire – facteur VIIa, le facteur Xa et la thrombine induisent des signaux calcium caractéristiques dans les ostéosarcomes par l’activation des récepteurs PAR, l’activation de PAR-1 diminuant l’apoptose induite par l’absence de sérum dans le milieu de culture.

Cancer and thrombosis are common co-occurrences in healthcare today. Cancer is the second leading cause of death in the United States with thrombosis being the second leading killer of cancer patients behind tumor progression. Cancer patients as a whole are 4 to 7 times more likely to develop thrombosis and 20%-30% of all first-time thrombosis diagnoses are cancer-related. Risk assessment and treatment options have much room for improvement. The lack of success for conventional antithrombotic prophylaxis suggests that hyperthrombosis in cancer works through a discrete pathway. A discovery by our group in recent years correlated the increased activity of Aryl hydrocarbon receptor (AHR) in the body to increase thrombotic phenotypes in patients with chronic kidney disease. Another group had published a manuscript about Kynurenine (Kyn), an activating ligand of the receptor that was produced by tumor cells to promote tumor growth through an AHR pathway (Opitz 2011). The link between the findings of these two groups could show that Kyn—AHR pathway is causing the increase in thrombosis in cancer patients. We used an animal model of thrombosis in cancer and created a new variation of it to test the Kyn—AHR pathway. We hypothesized that cancerous animals would show an increase in thrombosis and increased levels of AHR and Kyn along with downstream elements such as Tissue Factor (TF). Cancer was induced on nude mice via xenograft injection of cancer cells and 4-5 weeks of incubation to allow the tumors to proliferate. After the incubation period, mice underwent inferior vena cava (IVC) ligation, and were then euthanized 48 hours later. Two types of cancer were tested: HT-29 colon adenocarcinoma and A549 non small cell lung adenocarcinoma. There were 4 animal groups: mice that were injected with cancer cells and operated on, mice that were injected with cancer cells but not operated on, mice that were not injected but were operated on, and mice that did not receive neither the injections or the operation. After euthanasia, blood, tumors, and major organs were harvested to assess markers and pathways of thrombosis associated with cancer. We were able to successfully grow xenograft tumors in the nude mice. The HT-29 tumors grew very aggressively while A549 tumors experienced a small latent period before starting to proliferate. Animals with HT-29 and A549 xenograft tumors displayed greater thrombosis, measured by the weight of the blood clot formed in the IVC due to ligation (p=0.04 and p=0.05, respectively). HT-29 also displayed significant increases in Kyn, AHR activity, indoxyl sulfate (IS), and showed increased staining of tissue factor with immunohistochemistry. A549 did not have significant p-values in these experiments, but did show upward trends in all categories besides IS sera levels. In summary, we developed a new animal model of thrombosis in colon adenocarcinoma and showed significant increases in thrombosis as well as multiple markers of thrombosis. This is an exciting and complex way to study thrombosis in cancer in an in vivo approach with opportunities for future therapeutic testing. / 2020-07-03T00:00:00Z

Introduction: Cancer associated thrombosis is a well-recognized phenomenon that results in considerable patient morbidity and mortality. Malignancy conveys an increased risk for thrombosis and chemotherapy further elevates this risk. The pathophysiological mechanisms underlying this process remain poorly defined. Materials and Methods: A human acute monocytic leukemia cell line (THP-1) was treated with commonly used anthracycline chemotherapeutics at concentrations similar to those found in the plasma of cancer patients. Cells were analyzed for tissue factor (TF) mRNA, protein, and activity. Microparticle (MP) TF activity was also measured. Phosphatidylserine (PS) exposure on cells and MPs was analyzed by flow cytometry. PS levels on MPs was also evaluated in an annexin V capture assay. Results: Anthracycline treatment of THP-1 cells resulted in a concentration-dependent increase in cellular TF activity without a change in TF protein, which was associated with increased PS exposure on the cell surface and apoptosis. The increase in TF activity was abolished by annexin V or lactadherin indicating that PS exposure was required. Anthracycline treatment of THP-1 cells also increased the number of TF-positive MPs. Conclusion: Treatment of THP-1 cells with anthracyclines induces apoptosis and increases cellular TF activity. The increased activity required an increase in exposure of PS. Additionally, anthracyclines increase the release of TF-positive MPs from THP-1 cells. We propose that the increase in cellular TF activity in circulating leukemic cells, combined with increased numbers of TF-positive MPs, may contribute to thrombosis in cancer patients receiving chemotherapy.

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