Myocardial infarction (MI) is often caused by a disruption of an atherosclerotic plaque with activation of coagulation, platelets and inflammation. The aims were; to investigate whether the oral direct thrombin inhibitor, ximelagatran affected markers for coagulation, platelet and inflammation in a patient cohort with recent MI and if the coagulation markers could identify patients with increased risk of new ischemic events; to evaluate some of the mechanisms involved in formation of platelet-monocyte aggregates (PMAs). In a biomarker substudy patients with recent MI were randomized to 24-60 mg of ximelagatran or placebo for six months. There was a persistent dose-independent reduction of coagulation markers (F1+2, D-dimer) by ximelagatran treatment. 60 % reduced their D-dimer levels after one week and that group had less ischemic events during treatment. There was an early increase of the platelet activation marker and ximelagatran in higher doses attenuated these increased levels. Both in vivo and in vitro the direct thrombin inhibitor diminished procoagulant activity and tissue factor (TF) presenting microparticles. In contrast, the inflammatory markers increased after six months of ximelagatran treatment. The PMA-levels were elevated for long-term after MI. In vitro thrombin inhibition diminished formation of PMAs. Formation of PMAs in stimulated whole blood was P-selectin dependent and induced TF expression through phosphorylation of the Src-family member Lyn in monocytes. Addition of an oral direct thrombin inhibitor reduces coagulation and platelet activation markers for long-term after a MI together with reduced procoagulant activity which may contribute to the clinical benefit of the drug. Early reduction of D-dimer levels seems to be suitable to identify patients with reduced risk of new ischemic events independent of antithrombotic treatment. Circulating PMAs persist after a MI connecting coagulation to inflammation. Within these aggregates P-selectin induces TF, the main initiator of coagulation, partly through phosphorylation of Lyn.

The aim of this study was to examine the expression of Thrombomodulin, Tissue Factor, Tissue Factor Pathway Inhibitor and Endothelial Protein C Receptor in placenta throughout the three phases of the third trimester in the normal placenta and in IUGR placenta from full term. Twenty-five normal placenta samples and twenty-five IUGR placenta samples were obtained and each sample was stained by immunohistochemistry using monoclonal antibodies. Each antibody was optimised for antigen retrieval method and for optimal dilution, before been applied to the test tissue. The results showed that each of the antibodies mentioned was expressed in normal placenta and in IUGR placenta. No significant difference could be established concerning the expression of each antibody mentioned between normal and IUGR placenta.

This thesis have explored the functions of the protein Tissue Factor (TF), which together with its ligand coagulation factor VII/VIIa (FVII/FVIIa) forms a proteolytic complex that functions in initiation of blood coagulation and activation of cell signaling. In paper I, the mechanisms behind the observation that TF/FVIIa signaling protects cells from apoptosis were further investigated. Using cell culture models, we found that antiapoptotic signaling by TF/FVIIa requires signaling by the Insulin-like growth factor I receptor (IGF-1R), as synthetic IGF-1R inhibitors and IGF1-R siRNA knock-down abolished the antiapoptotic effect of FVIIa. Furthermore, the IGF-1R translocated to the cell nucleus after FVIIa stimulation, implying a role in regulation of gene expression. Papers II and III describe the discovery that the Eph tyrosine kinase receptors EphB2 and EphA2 are proteolytically cleaved directly by TF/FVIIa. By using mass spectrometry and N-terminal Edman sequencing, the exact cleavage site was identified after a conserved arginine residue in the EphA2/EphB2 ligand binding domains, in agreement with the cleavage preferences of FVIIa. TF and EphA2/EphB2 co-localized in cancer cell lines and FVIIa potentiated ligand-dependent Eph signaling by increasing cytoskeletal remodeling and cell repulsion, demonstrating a novel proteolytical event that modulates Eph receptor signaling. In paper IV, expression of TF was investigated in colorectal cancer in both the stromal and tumor cell compartments by immunohistochemistry using an anti-TF-antibody developed and validated by the Human Protein Atlas project. In normal large intestine, TF was strongly expressed in the innermost pericryptal sheath cell layer lining the epithelium, in a cell population distinct from intestinal pericryptal myofibroblasts. We evaluated TF expression in two colorectal cancer materials, and found that TF was variably present in both the stromal and tumor cell compartments. TF expressed by pericryptal sheath cells was progressively lost after the adenoma-to-carcinoma transition and was a strong predictor of survival in rectal but not colon cancer patients independently of disease stage, histological tumor grade and age. In summary, this thesis demonstrates novel signaling mechanisms for the TF/FVIIa complex, and provides evidence of a hitherto unknown role of TF expressed by a specific population of stromal cells in colorectal cancer.

Définie comme étant le rétrécissement de la valve, la sténose aortique (SA) est la 3ème pathologie cardiovasculaire dans les pays industrialisés. Touchant essentiellement les personnes âgées de plus de 65 ans, cette pathologie représente un véritable problème de santé publique compte tenu du vieillissement de la population. Considérée initialement comme issue d’un processus passif de dégénérescence, il est désormais établi que la sténose aortique est une pathologie dite « atherosclerosis-like » caractérisée par les processus d’inflammation, de fibrose, de néo-angiogenèse et de calcification. Certaines protéines de la voie de coagulation tel que le facteur tissulaire (FT) sont connues pour avoir un rôle pro-fibrotique et participent activement au développement des lésions athéroscléreuses. Leurs rôles dans la SA semblent donc probables et restent à être identifiés.Composante cellulaire majeure de la valve aortique, les VICs présentent 5 sous-populations distinctes : les cellules progénitrices embryonnaires (EPCs), les cellules progénitrices (pVICs), quiescentes (qVICs), activées (aVICs) et ostéoblastiques (obVICs). Au cours de la valvulogenèse, les EPCs permettent la cellularisation de la valve en se différenciant en qVIC. Celles-ci maintiennent l’homéostasie valvulaire et, en cas de lésion, s’activent (aVICs) pour réparer efficacement le tissu valvulaire. L’inflammation valvulaire et l’activation des VICs initient la sécrétion de protéines pro-calcifiantes induisant la différenciation des aVICs en obVICs. Enfin, les pVICs, naturellement présentes au sein de la valve (appelées résidantes) ou issues de la circulation sanguine (appelées hématopoïétiques), semblent favoriser le renouvellement cellulaire et peuvent être impliquées dans les processus angiogénique et ostéoblastique.Bien que décrites, la validation de la culture primaire des VICs par le suivi de ces sous-populations n’avait pas été réalisé et à constituer notre premier objectif. Nous avons ensuite étudié l’implication des voies de signalisation du FT dans le développement de la SA.Dans le cadre du suivi longitudinal des VICs depuis les valves aortiques humaines contrôles et pathologiques jusqu’à la culture in vitro réalisée sur plastique et sur collagène, nous avons tout d’abord montré que les différentes sous-populations étaient présentes au sein de ces valves avec des localisations et des proportions différentes selon l’état physiopathologique du tissu. Après digestion enzymatique de la valve, elles sont toutes retrouvées mais lors de la mise en culture, les pVICs hématopoïétiques ont disparu, quel que soit le support. Nous avons ainsi validé le modèle de culture primaire des VICs tout en mettant en lumière ses limites : absence des pVICs hématopoïétique, activation et différenciation ostéoblastique spontanée des VICs au cours de la culture.Dans le cadre de l’’étude de l’implication du FT dans le développement de la SA, nous avons montré sa colocalisation avec la thrombine et les calcifications de valves pathologiques. A partir de la culture primaire de VICs issues de valves humaines contrôles et pathologiques, nous avons montré que l’expression et l’activité du FT étaient constitutivement plus importantes pour les VICs pathologiques et que son expression pouvait être induite par l’IL1β. De plus, l’activation du FT, en présence de son ligand le facteur VII, induit, directement et via le récepteur PAR2, différentes voies de signalisation impliquées dans la prolifération cellulaire et les processus de fibrose et de calcification. Cette étude suggère ainsi que le FT produit par les VICs est un médiateur clef dans le développement de la sténose aortique. / Defined as the narrowing of the aortic valve, aortic stenosis (AS) is the third cardiovascular pathology in industrialized countries. Affecting mainly people aged over 65 years, AS represents a major public health problem because of the aging of the population. After initially been considered as a passive degenerative process, it is now established that AS is an "atherosclerosis-like " disease characterized by the processes of inflammation, fibrosis, neo-angiogenesis and calcification. Some proteins of the coagulation pathway such as tissue factor (TF) are known to have a pro-fibrotic role and actively participate in the development of atherosclerotic lesions. Their implication in AS seems, therefore, probable and remain to be identified.Prevalent cellular component of the aortic valve, VICs have five distinct subpopulations: embryonic progenitor cells (EPCs), progenitor cells (pVICs) quiescent (qVICs), activated (aVICs) and osteoblastic (obVICs). During the valvulogenesis, EPCs allow the cellularization of the valve, differentiating into qVICs. These cells maintain the valvular homeostasis and, in case of damage, are activated (aVICs) to effectively repair the valve tissue. The valvular inflammation and VICs activation initiate the secretion of pro-calcifying proteins inducing the differentiation of aVICs into obVICs. Finally, pVICs, naturally present within the valve (called resident) or from the blood circulation (called hematopoietic), seem to promote cell renewal and may be involved in the angiogenic and osteoblastic processes.Although described, these subpopulations have never been studied longitudinally, in respect to their behavior in vitro. Our first objective was to perform this investigation. Our second objective was to study the potential role of TF pathway in the deleterious mechanisms of AS.As part of the longitudinal follow-up of VICs from control and pathological human aortic valves to the in vitro culture performed on plastic and collagen, we first showed that different subpopulations were present in these valves with different locations and proportions according to the pathophysiological state of the tissue. After enzymatic digestion, all subpopulations are found but, in culture, hematopoietic pVICs disappeared, whichever the support. Thus, we validated the primary culture model of VICs while highlighting its limitations: lack of hematopoietic pVICs, spontaneous osteoblastic differentiation and activation of VICs in culture.As part of the study the involvement of FT in the AS development, we showed its colocalization with thrombin and calcifications of pathological valves. We showed that the expression and activity of TF were constitutively more important in VICs from fibrocalcified valves than control ones and that IL-1β for pathological VICs and that its expression could be induced by IL1 beta. In addition, TF activation in the by its ligand FVII, induced, directly and via the PAR-2 receptor, different signaling pathways involved in cell proliferation and the processes of fibrosis and calcification. Thus, our findings suggest that the FT expressed by VICs mediates fibrocalcific processes of aortic stenosis.

No description available.

Ce travail est consacré au rôle pléotropique des microparticules endothéliales dans la réponse et l'homéostasie vasculaire. Un modèle de sénescence réplicative a été caractérisé en utilisant des cellules endothéliales de coronaires de porc en culture primaire. Ce modèle a mis en évidence des changements drastiques du phénotype endothélial avec la production de ROS, la dépolarisation de la membrane mitochondriale et la surexpression de régulateurs clés du cycle cellulaire incluant p53, p21 et p16. La sénescence a transformé le phénotype endothélial vers un statut procoagulant indiqué par la libération de microparticules (MP). L'induction d'activité Facteur Tissulaire (FT) et une réduction drastique de l'inhibition de l'agrégation plaquettaire due à la sécrétion réduite de NO endothélial. Simultanément, une augmentation importante des protéines du système de l'angiotensine à la surface des cellules sénescentes et des MPs qu'elles émettent a été mesurée. D'autres résultats obtenus avec les MPs circulantes de patients transplantés ou atteints de syndrome coronarien suggèrent l'existence dune boucle d'amplification des effets délétères des MPs au travers de la signalisation Redox et l'altération des fonctions vasculaires résultant d'une sénescence exacerbée. En plus de ses qualités reconnues d'immunosuppresseur, la cyclosporine A (CsA) est un inhibiteur puissant de l'ouverture des pores mitochondriaux (mPTP). Certaines études ont présenté le traitement bref et contrôlé par CsA comme un moyen de limiter les dommages vasculaires de l'ischémie reperfusion.Nos données suggèrent une possible modulation de la sénescence endothéliale induite par les MPs grâce au préconditionnement avec des concentrations faibles de CsA. nos résultats suggèrent aussi que de faibles doses de Cs A peuvent avoir un effet bénéfique dans les pathologies cardiovasculaires lorsque la sénescence est exacerbée et contribue les fonctions vasculaires de l’endothélium. / This scientific work has tackled the issue of the pleitropic role mediated by endothelial microparticles function and homeostasis. A replicative model of senescence using coronary endothelial cells was set showing drastic phenotype changes characterized by ROS production, mitochondrial membrane depolarization and the up-regulation of key regulators of cell cycle arrest including p53, p21 and p16.Replicative senescence shifted the coronary endothelial phenotype toward a procoagulant status as evidenced by (i) procoagulant MP shedding (ii) enhanced tissue factor (TF) expression and (iii) a marked decrease in the endothelial NO-mediated inhibition of platelet aggregation. In parallel, a drastic up regulation of the angiotensin system could be evidenced at the surface of senescent cells or derived MP. Results obtained with MPs from patients with acute coronary artery syndrome and from grafted patients,suggested a feedback loop disseminating the deleterious effect of circulating MPs redox signaling and alteration of vascular function owing to exaggerated senescence. In addition to its well-known immunosuppressive properties, cyclosporine A (Cs A) is a potent inhibitor of the opening of the mitochondrial permeability transition pore (mPTP), and several reports have indicated that a brief and timely administration of Cs A can limit ischemia-reperfusion injuries. Our data evidenced the possible pharmacological modulation of endothelial MP-mediated senescence by cell preconditioning with low concentrations of Cs A. our data are thus suggestive of a beneficial effect of CsA in cardiovascular disorders where senescence is altering the endothelial vascular functions.

Thrombosis and colorectal cancer have a bi-directional relationship. The presence of a colorectal malignancy results in an increased risk of developing a thrombosis and the presence of a thrombosis results in a worse cancer prognosis. The physiology causing this is at present unclear but it is proposed that proteins from the tissue factor (TF) pathway may be the instigator of this bi-directional relationship. The in-vitro studies have shown that in colorectal cancer TF impairs that action of colorectal cancer stem cells as demonstrated by reduced cancer sphere formation and also lower expression of the stem cell marker ALDH. The ability for a colorectal cell to avoid anoikis is impaired by a reduced TF level. Proliferation is affected by the level of expression of TF with a significant increase in proliferation with additional expression of TF. The increase in proliferation is further increased by the presence of TF’s ligand factor VIIa. Paradoxically reduced expression of TF also increases colorectal cancer expression. The ERK1/2 pathway offers a possible method by which TF and factor VIIa may exert their proliferative effects. In the prospective clinical cohort study (CHAMPion) abnormal expression of TF pathway proteins (TF, PAR1, PAR2 and thrombin) by both malignant epithelial and cancer associated stromal cells has been demonstrated. The stromal expression was independent of the epithelial expression and was only in stroma in close contact (0.1mm) with epithelial cells suggesting that the TF pathway proteins may have a role in stromal/epithelial communication. There was no link between the expression of TF pathway proteins and clinicopathological markers of a poor prognosis. The plasma expression of markers of TF pathway activation did not demonstrate any role as a biomarker for colorectal cancer or prognosis. The CHAMPion study has demonstrated that 7% of patients undergoing surgery for colorectal cancer have asymptomatic pre-operative DVTs present. A further 6% who were DVT free pre-operatively developed a DVT in the peri-operative period despite receiving venous thromboprophylaxis in line with current national guidelines. Pre-operative d-dimer may have the potential to identify those patients at risk of a post-operative VTE.This thesis establishes the role that TF has in promoting proliferation and anoikis resistance. It also confirms the abnormal expression of TF pathway proteins by colorectal cancer epithelial cells and for the first time demonstrates abnormal expression by the cancer associated stroma. The interaction between the stroma and epithelial cells, combined with the cellular effects of TF suggests that targeting this interaction may have a therapeutic role. The incidence of DVTs pre-operatively suggests that screening patients for the asymptomatic presence of a DVT may have an impact on their clinical outcome. The development of DVTs despite prophylaxis suggests that the level of anticoagulation is insufficient and current guidelines need to be revisited.

No description available.

This thesis explores the pathogenesis of calciphylaxis, a rare and potentially fatal complication of chronic kidney disease (CKD) characterized by calcification and thrombosis of small- to medium-sized arteries. A range of bench techniques, including cell culture, genetic analysis, and immunofluorescence, were utilized in combination with human samples from patients with calciphylaxis and healthy controls. The results revealed a pathway that may modulate the thrombotic phenotype in these patients and, in turn, may serve as a targetable therapeutic axis. This work provides a foundation for further research and clinical advances in the field of calciphylaxis. Moreover, this study has the potential to inform the development of therapeutic interventions that could greatly improve the outcomes of CKD patients suffering from calciphylaxis. / 2026-03-05T00:00:00Z

<p>Thrombosis is a common complication of chemotherapy for breast cancer patients. However, the specific mechanisms by which chemotherapy agents modulate these hemostatic pathways are not well understood. In this thesis, we investigated the mechanism(s) by which chemotherapy agents can upregulate procoagulant pathways (tissue factor (TF), phosphatidylserine exposure, and cell-free DNA (CFDNA) release) and impair the protein C (PC) anticoagulant pathway. We examined the effects of chemotherapy agents doxorubicin, epirubicin and the cyclophosphamide metabolite acrolein on cell surface procoagulant activity. We found that treatment of endothelial cells with the chemotherapy drugs increased phosphatidylserine exposure and TF activity on treated endothelial cells, blood monocytes and/or smooth muscle cells. This corresponded to an increase in thrombin generation on chemotherapy-treated cells exposed to recalcified, defibrinated plasma. We also found that found that doxorubicin and epirubicin can increase CFDNA release from breast cancer chemotherapy patients and healthy mice, which corresponds to an increase in thrombin-antithrombin levels. Treatment of venous whole blood and isolated neutrophils with doxorubicin and epirubicin increased CFDNA release. We found that exposure of recalcified plasma to CFDNA isolated from epirubicin-treated whole blood increased thrombin generation by activating the contact pathway. We investigated the effects of chemotherapy on the PC anticoagulant pathway. We found that acrolein decreased EPCR while increasing thrombomodulin expression on treated endothelial cells. A corresponding decrease in activated PC generation was measured on acrolein-treated endothelial cells exposed to recalcified, defibrinated plasma. Healthy mice treated with acrolein and cyclophosphamide increased PC antigen levels, but no measurable increase in plasma APC levels. Breast cancer chemotherapy drugs elevate thrombin generation by activating coagulation through the TF and contact pathways, and by promoting phosphatidylserine exposure, as well as by impairing PC activation EPCR expression. These studies provide insight into the mechanisms of breast cancer chemotherapy-induced hypercoagulation.</p> / Doctor of Philosophy (Medical Science)