The clinical management of Factor XI (FXI) deficiency is problematic due to the marked phenotypic heterogeneity between individuals with this disorder and the lack of a reliable test to predict bleeding risk. FXI-deficient individuals are currently at risk of being over- or under treated, with associated risks of transfusion-related complications or haemorrhage respectively. The improvement of care of FXI-deficient patients requires the development of measures that can predict bleeding phenotype and enable the identification of individuals who need treatment at times of haemostatic challenge. In addition, for those requiring treatment, there is a need for development of tests which can determine the optimal type and dose of FXI replacement on an individually tailored basis, as well as assays which can accurately monitor the effect of treatment and guide clinicians in the requirement for further perioperative treatment. This thesis addresses these objectives by studying global haemostasis assays and bleeding scores as tools to predict bleeding tendency and by studying the utility of global haemostasis assays as potential tests by which FXI replacement treatment can be determined and monitored. For prediction of bleeding tendency, this research demonstrated that the thrombin generation assay (TGA) was able to differentiate bleeding tendency provided the sample conditions used in the assay were optimised to assess FXI involved coagulation pathways thought to be of relevance in vivo: using platelet rich plasma with inhibition of in vitro contact activation and a low tissue factor trigger. Thromboelastometry measured using the same sample type was similarly able to distinguish bleeding phenotype. However, when the potential clinical utility of the assays was compared using receiver operating characteristic curve analysis, thromboelastometry was inferior to TGA as an identifier of bleeding tendency. When the thromboelastometry sample type used was whole blood, or where assays were performed in the presence of tissue plasminogen activator the assays did not differentiate bleeding phenotype. For purposes of treatment planning, the potential of the TGA to determine the optimal dose of FXI replacement was assessed by in vitro spiking experiments using two commercially available FXI concentrates and samples from individuals with major FXI deficiency. Each concentrate improved thrombin generation, but dose response curves were found to differ, suggesting different properties for the two products. The clinical utility of the approach was then demonstrated with comparable TGA results obtained in ex vivo samples from patients treated with FXI concentrate and baseline samples spiked in vitro with equivalent amounts of the same FXI concentrate. The utility of global haemostasis assays to monitor the effect of FXI replacement in FXI-deficient individuals undergoing surgery was also tested. Improvement in assay parameters after treatment with solvent-detergent fresh frozen plasma or FXI concentrate was demonstrated suggesting assay value in FXI replacement monitoring. Finally the use of recently developed bleeding assessment tools and bleeding scores as descriptive, diagnostic or predictive measures was tested along with correlation with FXI:C levels and TGA parameters. This analysis confirmed that bleeding scores have a limited value in the clinical assessment of FXI deficiency.

INTRODUÇÃO: A HPN é uma patologia na qual a atividade do sistema complemento, sem oposição, leva a complicações sistêmicas. Ela é caracterizada por anemia hemolítica adquirida com hemoglobinúria intermitente, falência medular e fenômenos tromboembólicos (TE). A trombose venosa é a sua principal causa de mortalidade, entretanto o seu mecanismo fisiopatológico é apenas parcialmente elucidado. O grande número de pacientes com trombocitopenia dificulta o manejo da profilaxia antitrombótica secundária e primária. Optou-se por evidenciar o desequilíbrio hemostático associado ao clone HPN através de um teste de avaliação global da coagulação. MÉTODOS: Para a detecção do potencial hemostático de cada indivíduo foi utilizado um ensaio fluorogênico de geração de trombina, em amostra de plasma pobre em plaquetas na presença e na ausência de trombomodulina (TM). A eficiência na redução do potencial de trombina endógeno (ETP) e da concentração máxima de trombina (pico) pela TM foi utilizada para a identificação do estado de hipercoagulabilidade. O tempo para o início da geração de trombina (tempo de latência) e para a concentração máxima de trombina foram utilizados para a detecção do fenótipo hemorrágico. Os indivíduos foram categorizados em três grupos: HPN, se clone HPN >= 10%; anemia aplástica idiopática adquirida ou associada a clone < 10%, e normais. Os pacientes e controles foram submetidos a avaliação laboratorial que incluiu pesquisa de trombofilia (TB) e do clone HPN, hemograma, testes habituais de avaliação da hemostasia, e no grupo de pacientes análise bioquímica. Os participantes foram avaliados para a identificação da presença de fatores de risco para TE através de questionário. A análise dos resultados foi realizada em duas fases: a primeira incluiu apenas indivíduos com pesquisa de TB negativa e sem fatores de risco para TE; a segunda, realizada apenas no grupo de pacientes, também incluiu indivíduos em uso de contraceptivo hormonal, diagnóstico de infecção assintomática, evento de TE associado a fator de risco temporário, em período superior a 1 ano da inclusão no estudo, e com pesquisa de TB positiva. Esta última fase da análise teve como objetivo incluir um maior número de pacientes com o diagnóstico destas patologias, de baixa prevalência populacional. RESULTADOS: A presença do clone >= 10% foi associada à ineficiência da ação da TM em reduzir o ETP e o pico. O primeiro, de maior relevância científica e clínica, apresentou correlação positiva e negativa, respectivamente, com a atividade do fator von Willebrand (FvW:RCo) e com níveis plasmáticos de proteína C (PC). O grupo HPN apresentou menor tempo para atingir o pico. Na segunda fase, o tempo de latência apresentou correlação negativa com o número de plaquetas no grupo HPN, e houve correlação positiva do clone com a ineficiência da ação da TM na redução do ETP. CONCLUSÕES: o teste de geração de trombina é eficaz na detecção do fenótipo protrombótico associado ao clone HPN. As correlações encontradas com o FvW:RCo e a PC sugerem que a ativação endotelial e o sistema da PC, respectivamente, podem estar comprometidos. A inflamação secundária à ativação do sistema complemento pode levar à redução de expressão endotelial da TM e do receptor da PC. Entretanto os nossos achados, associados à descrição recente da redução de expressão e de atividade da TM, secundária à depleção de óxido nítrico (em estudos com estatinas), podem justificar a característica agressiva da trombofilia na HPN, e o desenvolvimento de TE mesmo nos pacientes em anticoagulação oral. Os parâmetros que avaliam o \'tempo\' no trombograma (tempo de latência e tempo para o pico) podem auxiliar na identificação do risco hemorrágico eventualmente associado à HPN / INTRODUCTION: PNH is a pathology in which the uncontrolled activity of the complement system leads to systemic complications. The pathology is characterized by an acquired hemolytic anemia with intermittent hemoglobinuria, bone marrow failure and thromboembolic event (TE). Venous thrombosis is the main cause of death, however, its physiopathological mechanism is only partially understood. The large number of patients with thrombocytopenia affects the management of primary and secondary antithrombotic prophylaxis. We chose to demonstrate the hemostatic unbalance associated with PNH clone through a global evaluation of the coagulation test. METHODS: To detect the hemostatic potential, we used a fluorogenic thrombin-generation assay, in platelet-poor plasma, with and without throbomodulin (TM). Analysis of the efficiency of TM in reducing the endogenous thrombin potential (ETP) and of the upper limit of thrombin concentration (peak) was done to identify the hypercoagulable state. Times to initiate thrombin generation (latency time-LT) and for reaching the peak were used to identify hemorrhagic phenotypes. Subjects were divided in three groups: PNH patients (if PNH clone >= 10%), patients with acquired idiopathic aplastic anemia or clone-associated (clone < 10%), and controls. Patients and controls were investigated for thrombophilia (TB) and PNH clone, underwent blood test, and regular exams to evaluate hemostasis. Patients were evaluated for the presence of risk factors for TE through questionnaires. Results were analyzed in two steps: the first included only patients negative for TB and with no risk factors for TE; the second step, done only in patients, included individuals using hormonal contraceptive, diagnosis of any asymptomatic infection, TE associated to temporary risk factors, and which have occurred in a period longer than one year since inclusion in the study, and positive TB. The aim of the second step was to gather the largest possible number of patients in these low prevalence pathologies. RESULTS: The presence of the clone >= 10% was associated with TM inefficiency in reducing the ETP (ETP+TM) and peak. In the first analysis, which had greater clinical relevance, we observed a positive correlation between ETP+TM and the activity of the von Willebrand factor (FvW:RCo), whereas a negative correlation was observed with the levels of protein C (PC). The PNH group presented the shortest time to reach the peak. In the second step of the analysis, the LT showed negative correlation with platelet counts in the PNH group, whereas a positive correlation between the clone and ETP+TM was observed. CONCLUSION: The thrombin-generation assay effectively detects the prothrombotic phenotype associated to PNH. The correlation found with both FvW:RCo and PC suggests that endothelial activation, and the PC system as well, may be deficient in these patients. Secondary inflammation to activation of the complement system may lead to lower endothelial expression of TM and of the PC receptor. However, our findings, together with recent descriptions of a reduced expression of the TM activity secondary to nitric oxide depletion (observed in studies on statin) may explain the aggressive nature of thrombophilia in PNH and the development of TE in these patients, even in those taking oral anticoagulants. The parameters that measure the time of thrombin generation may help identify the hemorrhagic risk that might be associated with PNH

INTRODUÇÃO: A HPN é uma patologia na qual a atividade do sistema complemento, sem oposição, leva a complicações sistêmicas. Ela é caracterizada por anemia hemolítica adquirida com hemoglobinúria intermitente, falência medular e fenômenos tromboembólicos (TE). A trombose venosa é a sua principal causa de mortalidade, entretanto o seu mecanismo fisiopatológico é apenas parcialmente elucidado. O grande número de pacientes com trombocitopenia dificulta o manejo da profilaxia antitrombótica secundária e primária. Optou-se por evidenciar o desequilíbrio hemostático associado ao clone HPN através de um teste de avaliação global da coagulação. MÉTODOS: Para a detecção do potencial hemostático de cada indivíduo foi utilizado um ensaio fluorogênico de geração de trombina, em amostra de plasma pobre em plaquetas na presença e na ausência de trombomodulina (TM). A eficiência na redução do potencial de trombina endógeno (ETP) e da concentração máxima de trombina (pico) pela TM foi utilizada para a identificação do estado de hipercoagulabilidade. O tempo para o início da geração de trombina (tempo de latência) e para a concentração máxima de trombina foram utilizados para a detecção do fenótipo hemorrágico. Os indivíduos foram categorizados em três grupos: HPN, se clone HPN >= 10%; anemia aplástica idiopática adquirida ou associada a clone < 10%, e normais. Os pacientes e controles foram submetidos a avaliação laboratorial que incluiu pesquisa de trombofilia (TB) e do clone HPN, hemograma, testes habituais de avaliação da hemostasia, e no grupo de pacientes análise bioquímica. Os participantes foram avaliados para a identificação da presença de fatores de risco para TE através de questionário. A análise dos resultados foi realizada em duas fases: a primeira incluiu apenas indivíduos com pesquisa de TB negativa e sem fatores de risco para TE; a segunda, realizada apenas no grupo de pacientes, também incluiu indivíduos em uso de contraceptivo hormonal, diagnóstico de infecção assintomática, evento de TE associado a fator de risco temporário, em período superior a 1 ano da inclusão no estudo, e com pesquisa de TB positiva. Esta última fase da análise teve como objetivo incluir um maior número de pacientes com o diagnóstico destas patologias, de baixa prevalência populacional. RESULTADOS: A presença do clone >= 10% foi associada à ineficiência da ação da TM em reduzir o ETP e o pico. O primeiro, de maior relevância científica e clínica, apresentou correlação positiva e negativa, respectivamente, com a atividade do fator von Willebrand (FvW:RCo) e com níveis plasmáticos de proteína C (PC). O grupo HPN apresentou menor tempo para atingir o pico. Na segunda fase, o tempo de latência apresentou correlação negativa com o número de plaquetas no grupo HPN, e houve correlação positiva do clone com a ineficiência da ação da TM na redução do ETP. CONCLUSÕES: o teste de geração de trombina é eficaz na detecção do fenótipo protrombótico associado ao clone HPN. As correlações encontradas com o FvW:RCo e a PC sugerem que a ativação endotelial e o sistema da PC, respectivamente, podem estar comprometidos. A inflamação secundária à ativação do sistema complemento pode levar à redução de expressão endotelial da TM e do receptor da PC. Entretanto os nossos achados, associados à descrição recente da redução de expressão e de atividade da TM, secundária à depleção de óxido nítrico (em estudos com estatinas), podem justificar a característica agressiva da trombofilia na HPN, e o desenvolvimento de TE mesmo nos pacientes em anticoagulação oral. Os parâmetros que avaliam o \'tempo\' no trombograma (tempo de latência e tempo para o pico) podem auxiliar na identificação do risco hemorrágico eventualmente associado à HPN / INTRODUCTION: PNH is a pathology in which the uncontrolled activity of the complement system leads to systemic complications. The pathology is characterized by an acquired hemolytic anemia with intermittent hemoglobinuria, bone marrow failure and thromboembolic event (TE). Venous thrombosis is the main cause of death, however, its physiopathological mechanism is only partially understood. The large number of patients with thrombocytopenia affects the management of primary and secondary antithrombotic prophylaxis. We chose to demonstrate the hemostatic unbalance associated with PNH clone through a global evaluation of the coagulation test. METHODS: To detect the hemostatic potential, we used a fluorogenic thrombin-generation assay, in platelet-poor plasma, with and without throbomodulin (TM). Analysis of the efficiency of TM in reducing the endogenous thrombin potential (ETP) and of the upper limit of thrombin concentration (peak) was done to identify the hypercoagulable state. Times to initiate thrombin generation (latency time-LT) and for reaching the peak were used to identify hemorrhagic phenotypes. Subjects were divided in three groups: PNH patients (if PNH clone >= 10%), patients with acquired idiopathic aplastic anemia or clone-associated (clone < 10%), and controls. Patients and controls were investigated for thrombophilia (TB) and PNH clone, underwent blood test, and regular exams to evaluate hemostasis. Patients were evaluated for the presence of risk factors for TE through questionnaires. Results were analyzed in two steps: the first included only patients negative for TB and with no risk factors for TE; the second step, done only in patients, included individuals using hormonal contraceptive, diagnosis of any asymptomatic infection, TE associated to temporary risk factors, and which have occurred in a period longer than one year since inclusion in the study, and positive TB. The aim of the second step was to gather the largest possible number of patients in these low prevalence pathologies. RESULTS: The presence of the clone >= 10% was associated with TM inefficiency in reducing the ETP (ETP+TM) and peak. In the first analysis, which had greater clinical relevance, we observed a positive correlation between ETP+TM and the activity of the von Willebrand factor (FvW:RCo), whereas a negative correlation was observed with the levels of protein C (PC). The PNH group presented the shortest time to reach the peak. In the second step of the analysis, the LT showed negative correlation with platelet counts in the PNH group, whereas a positive correlation between the clone and ETP+TM was observed. CONCLUSION: The thrombin-generation assay effectively detects the prothrombotic phenotype associated to PNH. The correlation found with both FvW:RCo and PC suggests that endothelial activation, and the PC system as well, may be deficient in these patients. Secondary inflammation to activation of the complement system may lead to lower endothelial expression of TM and of the PC receptor. However, our findings, together with recent descriptions of a reduced expression of the TM activity secondary to nitric oxide depletion (observed in studies on statin) may explain the aggressive nature of thrombophilia in PNH and the development of TE in these patients, even in those taking oral anticoagulants. The parameters that measure the time of thrombin generation may help identify the hemorrhagic risk that might be associated with PNH

Die vorliegende Arbeit untersucht in einer Population von 132 gesunden Probanden die Hämostase mittels Calibrated Automated Thrombogram (CAT) und Rotationsthromboelastometrie (ROTEM). CAT wurde im plätchenarmen Plasma mit einer tissue factor (TF) von 1 und 5 pM durchgeführt. Lag time, Thrombin peak, Time to thrombin peak und das endogene Thrombin Potential (ETP) wurden ermittelt. ROTEM wurde ohne Aktivator durchgeführt (NATEM) und die Daten für Gerinnungszeit (clotting time, CT), Gerinnselbildungszeit, Alpha Winkel und maximale Gerinnselfestigkeit (MCF) mit den Daten der Thrombingenerierung korreliert. Es zeigte sich eine positive aber nicht lineare Korrelation bezüglich Alter versus lag time und time to peak, sowie eine annähernd lineare Korrelation bezüglich Alter versus thrombin peak und ETP. Für ROTEM konnte eine positive Korrelation bezüglich Alter versus MCF und Alpha Winkel, aber eine negative Korrelation bezüglich Alter versus CT dargestellt werden. In der Gegenüberstellung beider Assays korrelierten Thrombin peak und ETP (aktiviert mit einer TF Konzentration von 5 pM) signifikant mit dem Alpha Winkel und der MCF. Alle signifikanten Korrelationen zeigten lediglich eine moderate Regressionssteigung. / Published data on thrombin generation variables and their correlation with thromboelastometry in the healthy population are scarce. This study aimed at assessing thrombin generation in adults and its correlation to classical rotational thromboelastometry (ROTEM). Methods: Thrombin generation was measured in platelet-poor plasma from healthy volunteers using the calibrated automated thrombogram (CAT) with 1 and 5 pmol/l tissue factor final concentration. Lag time, thrombin peak, time to thrombin peak and endogenous thrombin potential (ETP) were analyzed. ROTEM was performed without activator (NATEM) and data for clotting time, alpha angle, clot formation time and maximum clot firmness were correlated with those of thrombin generation. Results: Altogether 132 persons (72 men, 60 women; median age: 48.0 years) were included. There was a positive non-linear correlation for age versus lag time (p < 0.001) and time to peak (p = 0.001), and almost linear correlation for age versus thrombin peak (p = 0.024) and ETP (p = 0.001), although with a moderate regression slope. Regarding ROTEM, there was a positive correlation between age and maximum clot firmness and alpha angle (p = 0.001), but a negative correlation between age and clotting time (p = 0.039). Comparing both assays, thrombin peak and ETP measured with a final tissue factor concentration of 5 pmol/l correlated significantly with alpha angle and maximum clot firmness. Conclusion: The age-related changes in CAT and ROTEM variables among adults are not linear. There is a significant correlation, although with a moderate slope, between data from CAT measured with 5 pmol/l tissue factor and ROTEM.

La transplantation hépatique (TH) est une intervention à risque hémorragique, au cours de laquelle toutes les étapes de la coagulation (hémostase primaire, hémostase secondaire, fibrinolyse) sont perturbées. Nous avons d’abord montré que les facteurs de risque de saignement et transfusion étaient difficiles à identifier et n’étaient pas cliniquement très pertinents. Puis nous avons montré que la thromboélastométrie (ROTEM®) pouvait diagnostiquer la thrombopénie et l’hypofibrinogénémie au cours de la TH, même si l’utilisation d’un algorithme basé sur le ROTEM® ne diminuait pas le saignement ni la transfusion par rapport à un algorithme basé sur les résultats du laboratoire. De plus le ROTEM® manquait de sensibilité pour détecter l’hyperfibrinolyse. La thrombine est l’enzyme-clé de la cascade de la coagulation. Le Calibrated Automated Thrombogram (CAT®) est la méthode de référence de génération de thrombine. Nous avons cherché des moyens rapides d’évaluation de la génération de thrombine, utilisable en routine et sur échantillons individuels. Le Thrombodynamics-4D® (TD4D) permettait à la fois l’étude de la formation et de la propagation du caillot de fibrine dans le temps et l’espace et de la génération de thrombine. Une hyperfibrinolyse survient au cours de 20 à 66% des TH. Elle majore le saignement et la transfusion. Le diagnostic rapide de l’hyperfibrinolyse permettrait un traitement rapide et ciblé par antifibrinolytique. Le Lysis Timer était plus sensible que le ROTEM® pour détecter les hyperfibrinolyses. Le TD4D permettait également de visualiser la lyse du caillot. L’utilisation en routine de ces nouveaux appareils nécessite la validation des résultats sur plasma frais, après accélération des étapes pré-analytiques (centrifugation rapide) et des études cliniques pour les positionner au sein d’algorithmes transfusionnels. / Liver transplantation (LT) is a bleeding procedure, in which all the haemostatic steps (primary haemostasis, secondary haemostasis, fibrinolysis) are impaired. We have first shown the predictive factors of bleeding and transfusion were difficult to determine and were of poor clinical relevance. Then, we showed that thromboelastometry (ROTEM®) could detect thrombocytopenia and hypofibrinogenemia during LT. However, the utilisation of an algorithm based on ROTEM® results did not led to less bleeding and transfusion when compared to an algorithm based on laboratory results. Moreover, ROTEM® lacked sensitivity to detect hyperfibrinolysis. Thrombin is the key-enzyme of coagulation cascade. The Calibrated Automated Thrombogram (CAT®) is the reference test for thrombin generation. We searched for rapid tools to evaluate thrombin generation, usable in routine and on individual plasma samples. The Thrombodynamics-4D® (TD4D) enabled in the same time study of fibrin clot formation and propagation in time and space and thrombin generation. Hyperfibrinolysis is encountered in 20 to 66% of LT procedures. It is associated with more bleeding and transfusion. Rapid diagnosis of hyperfibrinolysis would allow a quick and target treatment with antifibrinolytic drugs. Lysis Timer was more sensitive than ROTEM® to detect hyperfibrinolysis. TD4D also visualized clot lysis. Routine utilization of these new devices now requires validation of its results in fresh plasma, after acceleration of pre-analytic steps (rapid centrifugation) and clinical studies to find their place in transfusion algorithms.

L’hémophilie est une maladie génétique se traduisant par la déficience des facteurs VIII et IX de la coagulation et conduisant à une tendance hémorragique. L’intensité des traitements substitutifs en facteur VIII et IX est définie essentiellement sur le taux basal du facteur déficitaire et non pas sur la capacité propre à chaque patient à générer de la thrombine qui est l’enzyme clé dans la formation du caillot de fibrine. Le test de génération de thrombine pourrait être utilisé pour permettre une individualisation du traitement anti-hémophilique. En effet, le taux de facteur VIII ou IX nécessaire à la normalisation de la génération de thrombine est potentiellement variable d’un patient à l’autre pour une même sévérité d’hémophilie. On peut donc se demander quelle approche expérimentale permettrait de mettre en exergue le lien entre taux de facteur anti-hémophilique et la génération de thrombine. Est-il possible de modéliser mathématiquement la coagulation pour obtenir une relation, soit explicite, soit implicite, entre taux de facteurs et génération de thrombine ? Les modèles existants permettent-ils d'obtenir une telle relation ? Une vaste campagne expérimentale a donc été menée pour mettre en place une base de données qui a permis d’identifier les facteurs déterminants de la génération de thrombine et la relation entre génération de thrombine et taux de facteur anti-hémophilique, de définir leurs valeurs de références, ainsi que d’évaluer et de paramétrer de manière sujet-spécifique des modèles mathématiques de la coagulation. / Haemophilia is a genetic disease corresponding to the deficiency of coagulation factor VIII or IX and leading to a bleeding tendency. The current substitutive treatment is defined essentially by the basal level of deficient factor and not the individual capacity to generate thrombin, a key enzyme of the clot formation. The thrombin generation assay could help in the individualisation of the anti-haemophilia treatment. Indeed, the factor VIII or IX level needed to normalise the thrombin generation vary potentially from one patient to another for a same degree of severity. We can wonder which experimental approach could emphasise the relation between level of anti-haemophilic factor and thrombin generation. Is it possible to mathematically model coagulation to obtain a relation, either explicit, or implicit, between factor level and thrombin generation? Could existing models provide this relation? An extensive experimental campaign was carried out to build a database that has been used to identify the determinant coagulation factors of thrombin generation and the individual relation between thrombin generation and anti-haemophilic factor level, to define their reference values, and also to evaluate and parametrise subject-specifically mathematical models of the coagulation cascade

Objectifs: Etude de l'influence des cellules du pancréas d'adénocarcinome (BXPC3) et des cellules de carcinome du sein humain (MCF7) sur l'efficacité antithrombotique de l'apixaban, du fondaparinux et de l'énoxaparine. Recherche des mécanismes procoagulants des BXPC3 et MCF7.Méthodes: Les cellules sont cultivées sur plaques 96 puits. Un plasma normal pauvre ou riche en plaquettes est surchargé par des anticoagulants. La génération de thrombine (GT) est réalisée dans différentes conditions par le test CAT. Le facteur tissulaire alternatif épissé (asTF), l'activité du FT (FTa) et le cancer procoagulant (CP) sont évalués. Les cellules HUVEC servent de contrôle normal.Résultats: La comparaison sur la base de l’IC50 a montré qu'en présence de BXPC3 ou de MCF7, l'efficacité de l'apixaban a été préservée. Le fondaparinux est plus vulnérable par la présence de cellules cancéreuses. Le FTa et l’asTF sont plus abondants pour les BXPC3 que les cellules MCF7. La GT est médiée plus fortement par le FVII pour les BXPC3 que les MCF7. Le facteur XII était plus important pour la GT médiée par les MCF7. La présence de MPs augmente considérablement la production de thrombine et cet effet est fonction du type de cellules et leur origine.Conclusion: Le type de cellules cancéreuses est déterminant pour l'efficacité anti-thrombotique des inhibiteurs spécifiques du facteur Xa. La GT par les BXPC3 est dominée par la voie du FT. Le rôle du FXII est plus impliqué pour les MCF7. L'hypercoagulabilité induite par les cellules cancéreuses est la résultante de la combinaison des propriétés procoagulantes des cellules cancéreuses elles-mêmes et des éléments procoagulants du microenvironnement. / Aims: study of influence of pancreas adenocarcinoma cells (BXPC3) and human breast carcinoma cells (MCF7) on the antithrombotic efficiency of apixaban, fondaparinux and enoxaparin. Dissection of procoagulant mechanisms of BXPC3 and MCF7.Methods: Cells were cultured and adhered in 96-well plates. Normal platelet poor or rich plasma were spiked whit apixaban, fondaparinux or enoxaparin. Thrombin generation (TG) was done with CAT¨ assay in different conditions. Alternatively spliced TF (asTF), TF activity (TFa) and cancer procoagulant (CP) were assessed. Primary human umbilical vein cells (HUVEC) were used as normal control.Results: Comparison on the basis of IC50 showed that in the presence of BXPC3 or MCF7 the efficiency of apixaban was preserved. Fondaparinux was more vulnerable to the presence of cancer cells. The TFa and asTF were found in abundant amounts in BXCP3 than MCF7 cells. TG enhancement by BXPC3 and MCF7 was mediated by FVII. Factor XII was more important for TG enhancement by MCF7.The presence of MPs drastically increases the generation of thrombin and this effect depending of the type of their original cells.Conclusion: The type of cancer cells is determinant for the antithrombotic efficiency of the specific factor Xa inhibitors. The mechanism of activation of blood coagulation by the BXPC3 is dominated by the TF pathway, MCF7 additionally imply also FXII activation. The hypercoagulability induced by cancer cells is the resultant of the combination of the procoagulant properties of cancer cells with procoagulant elements of the plasma microenvironment and highlight that circulating MVs are key players in the pathogenesis of cancer-associated thrombosis.

Haemostasis is maintained by a dynamic balance between pro- and anti-thrombotic mediators. Its dysregulation can lead to bleeding or thrombosis, and is a major cause of morbidity and mortality. Thus, elucidation of the mechanisms involved in maintaining or disrupting this balance have important implications in health and disease. Investigative tools enable characterization of the haemostatic system, but are often associated with limitations. For instance, haemostasis in animal models is often investigated by assessing bleeding responses in one particular vessel or tissue without a complete understanding of how the results translate to the regulation of haemostasis in other vascular beds. As a second example, microparticles (MPs) are a heterogeneous population of submicron-sized vesicles that may be important in thrombosis. With the exception of a few subtypes, MPs cannot be reliably characterized using widely accessible techniques. Finally, the thrombin generation assay (TGA), which measures ex vivo activation and inhibition of thrombin, is a promising tool for clinical assessment of thrombosis and haemostasis. However, characterization of thrombin generation in the general population, and the development of point of care testing are in their infancies. As a result, the TGA remains largely a research tool. The works described in this thesis specifically seek to address these three limitations in thrombosis and haemostasis research. The first isolated murine arterial bleeding model is presented and its characterization with respect to bleeding in other vascular tissues is described. In addition, a solid-phase capture assay for evaluating procoagulant, P-selectin-binding MPs, which are postulated to be mediators of thrombosis, was developed in order to determine whether these MPs associate with risk of recurrent venous thromboembolism. Lastly, a 25 x 20 mm chip that performs four individual thrombin generation assays using ~10 µl of capillary blood was developed as a proof of concept for point of care thrombin generation testing. / Thesis / Doctor of Philosophy (PhD)

Direct factor Xa inhibitors (FXaI) account for most oral anticoagulant use. FXaI-associated bleeding events are common and are associated with substantial morbidity and mortality. Nonspecific hemostatic therapies such as prothrombin complex concentrates (PCC) are often administered for FXaI-associated bleeding. The mechanism by which these agents improve hemostasis in the setting of direct oral anticoagulation is unclear. Thrombin generation assays may effectively measure the effect of anticoagulation reversal among FXaI-treated patients when bleeding cessation would otherwise be challenging to measure. To build a research program on the utility of thrombin generation assays to measure both the impact of direct oral anticoagulation and anticoagulation reversal, we completed a review of the literature with narrative synthesis and carried out a pilot study to determine the feasibility of a full scale prospective observational study of TGA responses among patients receiving PCC for FXaI-associated major bleeding or needing urgent surgery.

<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)