Les plaquettes sanguines sont les principaux acteurs de l’hémostase primaire et de la thrombose, deux éléments majeurs de la physiopathologie vasculaire. Plusieurs médicaments régulent les fonctions plaquettaires mais peu de tests sont validés pour suivre leur efficacité en fonction de l’évolution clinique des patients. Mon doctorat a eu pour but de développer de nouvelles approches d’évaluation de la fonction plaquettaire. Deux essais cliniques réalisés sur des patients atteints de syndrome coronarien stable ont constitué la première partie de mon doctorat. La première étude met en évidence la nécessité d'une standardisation des tests biologiques pour la détection de patients répondant moins au clopidogrel, un inhibiteur du récepteur plaquettaire de l'ADP P2Y12. L’étude suivante montre le potentiel thérapeutique, chez ces patients, de l’inhibition conjointe de P2Y12 et du second récepteur plaquettaire de l'ADP P2Y1, sur la fonction plaquettaire. De plus, le suivi en temps réel par vidéomiscroscopie a permis de distinguer des effets précoces et tardifs des antiplaquettaires sur la formation du thrombus en chambre de perfusion. La seconde partie de mon doctorat concerne les microdomaines membranaires de type « lipid rafts » qui tiennent une place fondamentale dans les fonctions cellulaires et plaquettaires. Ainsi plusieurs récepteurs dépendent de ces microdomaines, régulant la fonction plaquettaire et les effets des médicaments antiplaquettaires. Cependant, les techniques d’étude de ces microdomaines sont complexes et peu adaptées aux études cliniques. Profitant de nouvelles sondes fluorescentes sensibles au niveau d’ordre liquide membranaire (OLM), nous avons développé une méthode de mesure de l’OLM par cytométrie de flux spectrale. Grâce à cette approche, nous avons montré que l’activation plaquettaire diminue l’OLM alors qu’il est augmenté chez des patients traités par un inhibiteur de la synthèse du cholestérol ou par le clopidogrel. Nous avons également mis en évidence, en condition de forces de cisaillement élevées correspondant à celles retrouvées au niveau de sténoses artérielles, une sous-population plaquettaire présentant un OLM plus bas. Le passage dans le domaine clinique de ces approches fondamentales qui privilégient l’étude dynamique des plaquettes pourrait permettre d’améliorer le diagnostique et le suivi de traitement de pathologies cardiovasculaires. / Blood platelets play a central role in primary hemostasis and thrombosis, two major elements of vascular physiopathology. Although a number of drugs regulate platelet functions, there are no validated tests that monitor their efficacy on the basis of the patients' clinical course. This doctorate, therefore, aims to develop novel approaches to evaluate platelet function. The first part of my work consisted of two clinical trials involving patients with stable coronary syndrome. The first study demonstrated the need for standardized biological tests to screen for patients who respond less well to clopidogrel, an ADP P2Y12 receptor antagonist. The second study showed the therapeutic potential of the joint inhibition of P2Y12 and P2Y1 receptors on platelet adhesion, activation, and aggregation for these patients. Furthermore, a video microscopy model using perfusion chambers made it possible to monitor the course of thrombosis in real time, and enabled us to dissociate the early and late effects of the antiplatelet drugs. Lipid raft membrane microdomains play a pivotal role in many cell functions. At the platelet level, many receptors depend on these microdomains and thus modulate the function as well as the drug sensitivity of platelets. However, current techniques for the study of these microdomains are complex and limit their clinical applications. By taking advantage of new fluorescent probes that are sensitive to the level of the membrane order, we developed a method of measuring the membrane order using spectral flow cytometry. Through this approach we showed that platelet activation reduced the lipid order of the membranes, whereas it was increased in patients treated with a cholesterol synthesis inhibitor or clopidogrel. It was also possible for us to demonstrate the appearance, under shear stress similar to that of stenotic arteries, of a platelet sub-population with a very low membrane order. These approaches which privilege the dynamic study of thrombi and platelets could be applied to the clinical practice and thus widen the fields of clinical studies.

Les anticorps anti-phospholipides (aPL), tels que les anticoagulants lupiques (LAC), sont associés au développement récurrent de thromboses chez les patients atteints du lupus érythémateux disséminé (LED). Il a été observé que des titres élevés d’auto-anticorps antilamine B1 (anti-LB1), chez des patients porteurs de LAC, diminuent le risque de ces manifestations thrombotiques. Toutefois, la relation existant entre la lamine B1 (LB1), les anti-LB1 et la thromboprotection n’est toujours pas expliquée. Dans cette étude, nous avons donc cherché à comprendre comment la LB1 et les anti-LB1 induisent cette thromboprotection. Nous avons testé les effets d'anti-LB1 purifiés et de LB1 recombinante sur l'activation des cellules endothéliales et des plaquettes. Nous avons été en mesure de déterminer que la LB1, contrairement aux anti-LB1, possède une activité anti-plaquettaire. En effet, la LB1 réduit l’activation et l’agrégation plaquettaires in vitro et in vivo. Cette activité est due à une liaison directe de la LB1 aux plaquettes, suivie par une internalisation rapide dans des vésicules de clathrine. Par co-immunoprécipitation, nous avons découvert que la LB1 interagit avec le récepteur de l’insuline situé sur la membrane plaquettaire. La liaison de la LB1 à ce récepteur entraîne vraisemblablement son internalisation et l'inhibition d'une des cascades de signalisation normalement induite par le récepteur de l’insuline, menant éventuellement à l’inhibition des fonctions plaquettaires. L’ajout d’anti-LB1 purifiés dans nos expériences a permis d'augmenter de façon significative la persistance de la LB1 dans les plaquettes, une observation confirmée par la détection de LB1 uniquement dans les lysats de plaquettes prélevées chez des patients anti-LB1 positifs. iv Nos résultats suggèrent que la LB1 prend part aux mécanismes régulateurs des processus d’hémostase chez des sujets sains et que la présence d’anti-LB1, chez les patients lupiques, prolonge la persistance de cet auto-antigène dans les plaquettes, les empêchant ainsi de s’activer. Ce mécanisme expliquerait la diminution du risque de thrombose chez les patients LAC positifs porteurs d’anti-LB1 circulants. / Anti-phospholipid antibodies such as lupus anticoagulant antibodies (LAC) are associated with recurrent thrombotic events in systemic lupus erythematosus (SLE) patients. However, the risk of thrombosis in LAC positive patients is markedly reduced in the presence of high titers of autoantibodies to lamin B1 (anti-LB1). To date, the implication of lamin B1 (LB1) and anti-LB1 in thromboprotection remains unclear. Our objective was to examine the mechanism whereby LB1 and anti-LB1 induced thromboprotection. Functional platelet and endothelial cell activation assays were used to determine the effects of recombinant LB1 and affinity purified anti-LB1 on these two cell types. LB1, contrarily to anti-LB1, was found to possess an intrinsic anti-platelet activity. This protein reduced the activation and aggregation of platelets in vitro and in vivo. This activity was likely due to the direct binding of LB1 to platelets, followed by its rapid internalization within clathrin coated-pits. Coimmunoprecipitation revealed that LB1 interacted with the insulin receptor located within the platelet membrane. The binding of LB1 to this receptor induced its internalization and inhibited at least one of the phosphorylation cascade stimulated by the receptor, which in turn inhibited platelet functions. The addition of affinity-purified anti-LB1 in our model markedly increased the persistence of LB1 within platelets, a finding supported by the detection of LB1 only in platelets from anti-LB1 positive SLE patients. Our results suggest that LB1 regulates haemostasis in normal subjects. The presence of anti-LB1 in SLE patients prolongs the persistence of LB1 within platelets, thus possibly vi preventing further platelet activation. This mechanism likely explains the reduced risk of thrombotic events in LAC positive SLE patients with circulating anti-LB1 autoantibodies.

Lors du phénomène d’hémostase primaire ou de thrombose vasculaire, les plaquettes sanguines doivent adhérer aux parois afin de remplir leur fonction réparatrice ou pathologique. Pour ce faire, certains facteurs rhéologiques et hémodynamiques tels que l’hématocrite, le taux de cisaillement local et les contraintes de cisaillement pariétal, entrent en jeu afin d’exclure les plaquettes sanguines de l’écoulement principal et de les transporter vers le site endommagé ou enflammé. Cette exclusion pourrait aussi être influencée par l’agrégation de globules rouges qui est un phénomène naturel présent dans tout le système cardiovasculaire selon les conditions d’écoulement. La dérive de ces agrégats de globules rouges vers le centre des vaisseaux provoque la formation de réseaux d’agrégats dont la taille et la complexité varient en fonction de l’hématocrite et des conditions de cisaillement présentes. Il en résulte un écoulement bi-phasique avec un écoulement central composé d’agrégats de globules rouges avoisinés par une région moins dense en particules où l’on peut trouver des globules rouges singuliers, des petits rouleaux de globules rouges et une importante concentration en plaquettes et globules blancs. De ce fait, il est raisonnable de penser que plus la taille des agrégats qui occupent le centre du vaisseau augmente, plus il y aura de plaquettes expulsées vers les parois vasculaires. L'objectif du projet est de quantifier, in vitro, la migration des plaquettes sanguines en fonction du niveau d’agrégation érythrocytaire présent, en faisant varier l’hématocrite, le taux de cisaillement et en promouvant l’agrégation par l’ajout d’agents tels que le dextran à poids moléculaire élevé. Cependant, le comportement non Newtonien du sang dans un écoulement tubulaire peut être vu comme un facteur confondant à cause de son impact sur l’organisation spatiale des agrégats de globules rouges. De ce fait, les études ont été réalisées dans un appareil permettant de moduler, de façon homogène, la taille et la structure de ces agrégats et de quantifier ainsi leur effet sur la migration axiale des plaquettes. Du sang de porc anti coagulé a été ajusté à différents taux d’hématocrite et insérer dans un appareil à écoulement de Couette, à température ambiante. Les plaquettes sanguines, difficilement isolables in vitro sans en activer certains ligands membranaires, ont été remplacées par des fantômes en polystyrène ayant un revêtement de biotine. La quantification de la migration de ces fantômes de plaquettes a été réalisée grâce à l’utilisation de membranes biologiques fixées sur les parois internes de l’entrefer du rhéomètre de Couette. Ces membranes ont un revêtement de streptavidine assurant une très forte affinité d’adhésion avec les microparticules biotynilées. À 40% d’hématocrite, à un cisaillement de 2 s-1, 566 ± 53 microparticules ont été comptées pour un protocole préétabli avec du sang non agrégeant, comparativement à 1077 ± 229 pour du sang normal et 1568 ± 131 pour du sang hyper agrégeant. Les résultats obtenus suggèrent une nette participation de l’agrégation érythrocytaire sur le transport des fantômes de plaquettes puisque l’adhésion de ces derniers à la paroi du rhéomètre de Couette augmente de façon quasi exponentielle selon le niveau d’agrégation présent. / During the primary hemostatis or thrombosis phenomenon, the human blood platelets must adhere to the vascular wall in order for them to perform their repairing or pathological function. To do so, certain rheological and hemodynamic factors such as the hematocrit, local shear rate and the wall shear stress, must come into play to exclude blood platelets from the main blood stream and transport them to the vicinity of the damaged or inflamed site. This exclusion could also be influenced by red blood cell aggregation which is a natural process present throughout the entire cardiovascular system under certain flow conditions. The displacement of these rouleaux of red blood cells towards the centre of the vessel induces the formation of 3D networks of aggregates whose size and complexity vary as a function of the hematocrit and the shearing conditions present. It results in a two phase flow with an inner core composed of red blood cell aggregates surrounded by single red blood cells or small aggregates and large numbers of white blood cells and platelets. It is therefore reasonable to believe that the larger the inner core becomes, the more platelets will be expulsed towards the vascular wall. The objective of the study was to quantify, in vitro, the lateral migration of blood platelets as a function of the level of red blood cell aggregation present, by changing the hematocrit, the shear rate and by promoting red blood cell aggregation with the use of agents such as high molecular weight dextran. However, the non Newtonian behavior of blood in tube flow can be seen as a confounding factor to the understanding of the spatial organization of the red blood cell aggregates. In this study, whole blood was circulated in a simple shear flow apparatus, which allowed to homogeneously modulate the red blood cell aggregate sizes and structure, and quantify their effect on the axial migration of blood platelets. Anticoagulated porcine bloods were adjusted to different hematocrits and inserted into a Couette flow apparatus, at room temperature. Blood platelets, difficult to isolate in vitro without activating in a non reproducible manner specific membrane ligands, were replaced with biotin coated fluorescent polystyrene beads. The quantification of the migration of these platelet ghosts was conducted with the use of biological membranes fixed on the interior walls of the Couette apparatus. These streptavidin coated membranes ensure a strong adhesive affinity with the biotynilated beads. At 40% hematocrit and at a shear of 2 s-1, 566 ± 53 micro particles were counted for non aggregated erythrocytes, 1077 ± 229 for aggregating red blood cells and 1568 ± 131 for hyper aggregating blood. The results obtained suggest a strong participation of the red blood cell aggregation on the transport of platelet ghosts since the number of ghost cells fixed on the wall of the Couette rheometer increases almost exponentially with the level of aggregation present.

L’agrégation érythrocytaire est le principal facteur responsable des propriétés non newtoniennes sanguines pour des conditions d’écoulement à faible cisaillement. Lorsque les globules rouges s’agrègent, ils forment des rouleaux et des structures tridimensionnelles enchevêtrées qui font passer la viscosité sanguine de quelques mPa.s à une centaine de mPa.s. Cette organisation microstructurale érythrocytaire est maintenue par des liens inter-globulaires de faible énergie, lesquels sont brisés par une augmentation du cisaillement. Ces propriétés macroscopiques sont bien connues. Toutefois, les liens étiologiques entre ces propriétés rhéologiques générales et leurs effets pathophysiologiques demeurent difficiles à évaluer in vivo puisque les propriétés sanguines sont dynamiques et fortement tributaires des conditions d’écoulement. Ainsi, à partir de propriétés rhéologiques mesurées in vitro dans des conditions contrôlées, il devient difficile d’extrapoler leurs valeurs dans un environnement physiologique. Or, les thrombophlébites se développent systématiquement en des loci particuliers du système cardiovasculaire. D’autre part, plusieurs études cliniques ont établi que des conditions hémorhéologiques perturbées constituent des facteurs de risque de thrombose veineuse mais leurs contributions étiologiques demeurent hypothétiques ou corrélatives. En conséquence, un outil de caractérisation hémorhéologique applicable in vivo et in situ devrait permettre de mieux cerner et comprendre ces implications. Les ultrasons, qui se propagent dans les tissus biologiques, sont sensibles à l’agrégation érythrocytaire. De nature non invasive, l’imagerie ultrasonore permet de caractériser in vivo et in situ la microstructure sanguine dans des conditions d’écoulements physiologiques. Les signaux ultrasonores rétrodiffusés portent une information sur la microstructure sanguine reflétant directement les perturbations hémorhéologiques locales. Une cartographie in vivo de l’agrégation érythrocytaire, unique aux ultrasons, devrait permettre d’investiguer les implications étiologiques de l’hémorhéologie dans la maladie thrombotique vasculaire. Cette thèse complète une série de travaux effectués au Laboratoire de Biorhéologie et d’Ultrasonographie Médicale (LBUM) du centre de recherche du Centre hospitalier de l’Université de Montréal portant sur la rétrodiffusion ultrasonore érythrocytaire et menant à une application in vivo de la méthode. Elle se situe à la suite de travaux de modélisation qui ont mis en évidence la pertinence d’un modèle particulaire tenant compte de la densité des globules rouges, de la section de rétrodiffusion unitaire d’un globule et du facteur de structure. Ce modèle permet d’établir le lien entre la microstructure sanguine et le spectre fréquentiel du coefficient de rétrodiffusion ultrasonore. Une approximation au second ordre en fréquence du facteur de structure est proposée dans ces travaux pour décrire la microstructure sanguine. Cette approche est tout d’abord présentée et validée dans un champ d’écoulement cisaillé homogène. Une extension de la méthode en 2D permet ensuite la cartographie des propriétés structurelles sanguines en écoulement tubulaire par des images paramétriques qui mettent en évidence le caractère temporel de l’agrégation et la sensibilité ultrasonore à ces phénomènes. Une extrapolation menant à une relation entre la taille des agrégats érythrocytaires et la viscosité sanguine permet l’établissement de cartes de viscosité locales. Enfin, il est démontré, à l’aide d’un modèle animal, qu’une augmentation subite de l’agrégation érythrocytaire provoque la formation d’un thrombus veineux. Le niveau d’agrégation, la présence du thrombus et les variations du débit ont été caractérisés, dans cette étude, par imagerie ultrasonore. Nos résultats suggèrent que des paramètres hémorhéologiques, préférablement mesurés in vivo et in situ, devraient faire partie du profil de risque thrombotique. / The aggregation of erythrocytes is the main determinant of blood non Newtonian behaviour under low shearing flow conditions. When red blood cells (RBCs) aggregate, they form « rouleaux » and complex tridimensional structures that increase blood viscosity from a few mPa.s to a hundred mPa.s. The reversible RBC aggregation phenomenon is attributed to weak adhesive links between erythrocytes that are readily broken by increasing flow shearing. Blood bulk rheological properties have been comprehensively studied. However, the in vivo physiological impacts of abnormal clustering of RBCs are more difficult to assess. Clinical studies have identified altered hemorheology as a risk factor for thrombosis, but a clear etiological relationship between abnormal aggregation and thrombosis has not yet been established, in part because clinical conclusions were derived from correlative findings. It is to note that cardiovascular diseases such as deep venous thrombosis generally occur at specific locations within the vascular bed, suggesting a hemodynamic contribution to the development of this disease. Consequently, it is postulated that in vivo hemorheological characterization may help shed some light on the role of RBC hyper-aggregation on cardiovascular disorders. Ultrasound imaging, a non-invasive method relying on the propagation of mechanical waves within biological tissues, is sensitive to RBC aggregation. Indeed, the study of backscattered waves allows characterizing blood microstructure in vivo and in situ under physiological flow conditions. The work described in this thesis is based on prior simulation studies, performed at the Laboratory of Biorheology and Medical Ultrasonics of the University of Montreal Hospital Research Center, in which the backscattering of ultrasound from aggregating RBCs was modeled by considering a particle scattering strategy. In this approach, each RBC is a weak ultrasound scatterer (Born assumption) and the backscattering coefficient is modeled as the product of the RBC number density, the RBC backscattering cross section and a structure factor. This model relates variations in the backscattering coefficient to the RBC spatial organisation through the structure factor, which is the only parameter that changes during the aggregation process. A second order expansion in frequency of the structure factor was used to describe blood microstructure in terms of a packing factor W and an ensemble averaged aggregate diameter D. The model was first presented and validated by considering a homogenous shear flow condition using three broadband mono-element transducers. It was then extended in 2D to allow computation of parametric images in tube flow. An extrapolation based on the assumption that viscosity is related to the level of aggregation was used to compute local viscosity maps. Finally, a last contribution was the demonstration that a sudden increase in aggregation tendency directly promoted the formation of venous thrombosis in an experimental animal model. In that study, RBC aggregation, thrombus formation and flow variations were monitored longitudinally for two weeks using ultrasound. The results reported in this thesis suggest that rheological parameters on RBC clustering, ideally assessed in vivo and in situ, should be included in thrombosis risk profiling.

L’accident thromboembolique veineux, tel que la thrombose veineuse profonde (TVP) ou thrombophlébite des membres inférieurs, est une pathologie vasculaire caractérisée par la formation d’un caillot sanguin causant une obstruction partielle ou totale de la lumière sanguine. Les embolies pulmonaires sont une complication mortelle des TVP qui surviennent lorsque le caillot se détache, circule dans le sang et produit une obstruction de la ramification artérielle irriguant les poumons. La combinaison d’outils et de techniques d’imagerie cliniques tels que les règles de prédiction cliniques (signes et symptômes) et les tests sanguins (D-dimères) complémentés par un examen ultrasonographique veineux (test de compression, écho-Doppler), permet de diagnostiquer les premiers épisodes de TVP. Cependant, la performance de ces outils diagnostiques reste très faible pour la détection de TVP récurrentes. Afin de diriger le patient vers une thérapie optimale, la problématique n’est plus basée sur la détection de la thrombose mais plutôt sur l’évaluation de la maturité et de l’âge du thrombus, paramètres qui sont directement corrélées à ses propriétés mécaniques (e.g. élasticité, viscosité). L’élastographie dynamique (ED) a récemment été proposée comme une nouvelle modalité d’imagerie non-invasive capable de caractériser quantitativement les propriétés mécaniques de tissus. L’ED est basée sur l’analyse des paramètres acoustiques (i.e. vitesse, atténuation, pattern de distribution) d’ondes de cisaillement basses fréquences (10-7000 Hz) se propageant dans le milieu sondé. Ces ondes de cisaillement générées par vibration externe, ou par source interne à l’aide de la focalisation de faisceaux ultrasonores (force de radiation), sont mesurées par imagerie ultrasonore ultra-rapide ou par résonance magnétique. Une méthode basée sur l’ED adaptée à la caractérisation mécanique de thromboses veineuses permettrait de quantifier la sévérité de cette pathologie à des fins d’amélioration diagnostique. Cette thèse présente un ensemble de travaux reliés au développement et à la validation complète et rigoureuse d’une nouvelle technique d’imagerie non-invasive élastographique pour la mesure quantitative des propriétés mécaniques de thromboses veineuses. L’atteinte de cet objectif principal nécessite une première étape visant à améliorer les connaissances sur le comportement mécanique du caillot sanguin (sang coagulé) soumis à une sollicitation dynamique telle qu’en ED. Les modules de conservation (comportement élastique, G’) et de perte (comportement visqueux, G’’) en cisaillement de caillots sanguins porcins sont mesurés par ED lors de la cascade de coagulation (à 70 Hz), et après coagulation complète (entre 50 Hz et 160 Hz). Ces résultats constituent les toutes premières mesures du comportement dynamique de caillots sanguins dans une gamme fréquentielle aussi étendue. L’étape subséquente consiste à mettre en place un instrument innovant de référence (« gold standard »), appelé RheoSpectris, dédié à la mesure de la viscoélasticité hyper-fréquence (entre 10 Hz et 1000 Hz) des matériaux et biomatériaux. Cet outil est indispensable pour valider et calibrer toute nouvelle technique d’élastographie dynamique. Une étude comparative entre RheoSpectris et la rhéométrie classique est réalisée afin de valider des mesures faites sur différents matériaux (silicone, thermoplastique, biomatériaux, gel). L’excellente concordance entre les deux technologies permet de conclure que RheoSpectris est un instrument fiable pour la mesure mécanique à des fréquences difficilement accessibles par les outils actuels. Les bases théoriques d’une nouvelle modalité d’imagerie élastographique, nommée SWIRE (« shear wave induced resonance dynamic elastography »), sont présentées et validées sur des fantômes vasculaires. Cette approche permet de caractériser les propriétés mécaniques d’une inclusion confinée (e.g. caillot sanguin) à partir de sa résonance (amplification du déplacement) produite par la propagation d’ondes de cisaillement judicieusement orientées. SWIRE a également l’avantage d’amplifier l’amplitude de vibration à l’intérieur de l’hétérogénéité afin de faciliter sa détection et sa segmentation. Finalement, la méthode DVT-SWIRE (« Deep venous thrombosis – SWIRE ») est adaptée à la caractérisation de l’élasticité quantitative de thromboses veineuses pour une utilisation en clinique. Cette méthode exploite la première fréquence de résonance mesurée dans la thrombose lors de la propagation d’ondes de cisaillement planes (vibration d’une plaque externe) ou cylindriques (simulation de la force de radiation par génération supersonique). DVT-SWIRE est appliquée sur des fantômes simulant une TVP et les résultats sont comparés à ceux donnés par l’instrument de référence RheoSpectris. Cette méthode est également utilisée avec succès dans une étude ex vivo pour l’évaluation de l’élasticité de thromboses porcines explantées après avoir été induites in vivo par chirurgie. / The venous thromboembolism such as the lower limb deep venous thrombosis (DVT) is a vascular pathology characterized by a blood clot formation that induces partial or total vessel lumen occlusion. Pulmonary embolism is a fatal complication of DVT where the clot detaches from the wall, circulates in the blood flow, and produces an obstruction of pulmonary arterial branches. The combination of clinical prediction rules (signs or symptoms) and blood tests (D-dimer testing) coupled to venous ultrasonography (i.e. compression ultrasonography, color Doppler) allows an accurate diagnosis of first DVT. Nevertheless, such clinical tools present poor results to detect recurrent thrombotic events. Then, in order to guide patients towards optimal therapy, the problem is no more to detect the presence of thrombus, but to evaluate its maturity and its age, which are correlated to their mechanical properties (e.g. elasticity, viscosity). The dynamic elastography (DE) has been recently proposed as a novel non-invasive imaging modality capable to characterize the quantitative mechanical properties of tissues. The DE is based on the analysis of acoustical parameters (i.e. velocity, attenuation, wave pattern) of low frequency (10-7000 Hz) shear waves propagating within the probed medium. Such shear waves generated by external vibration, or remotely using ultrasound beam focalisation (radiation force), were tracked using ultra-fast ultrasound or magnetic resonance imaging. A method based on DE and adapted to mechanical characterization of venous thrombosis may allow the quantification of diseases severity in order to improve the final diagnosis. This thesis presents the works related to the development and complete validation of a novel non-invasive elastography imaging method for the quantitative and reliable estimation of mechanical properties of venous thrombosis. In order to fulfil the main objective, it is first necessary to improve knowledge about mechanical behaviours of blood clot (coagulated blood) subjected to a dynamic solicitation similar to DE. The shear storage (elastic behaviour, G’) and loss (viscous behavior, G’’) moduli of porcine blood clots are measured by DE during the blood coagulation kinetics (at 70 Hz) and after completely coagulation (between 50 Hz and 160 Hz). These results are the first dynamic behaviour measurements of blood clots in such wide frequency range. The subsequent step consists in introducing an innovative reference instrument (« gold standard »), called RheoSpectris, dedicated to measure the hyper-frequency viscoelasticity (between 10 Hz and 1000 Hz) of materials and biomaterials. This tool is indispensable to validate new dynamic elastography techniques. A comparative study between RheoSpectris and classical rheometry is performed to validate the measurements on different materials (silicon, thermoplastic, biomaterials, gel). The excellent agreement between both technologies allows to conclude that RheoSpectris is a reliable instrument for mechanical measurements at high frequencies, which is not always possible with current tools. The theoretical basis of a novel elastographic imaging modality, labelled SWIRE (« shear wave induced resonance dynamic elastography ») is presented and validated on vascular phantoms. Such approach allows the characterization of mechanical properties of a confined inclusion (e.g. blood clot) from its resonance (displacement amplification) due to the propagation of judiciously oriented shear waves. SWIRE has also the advantage to amplify the vibration amplitude within the heterogeneity to help for its detection and segmentation. Finally, the method DVT-SWIRE ((« Deep venous thrombosis – SWIRE ») is adapted to the quantitative elasticity estimation of venous thrombosis in the context of clinical use. DVT-SWIRE exploits the first resonance frequency measured within the thrombosis during the plane (vibration of rigid plate) or cylindrical (simulating supersonic radiation force generation) shear waves propagation. The technique is applied on DVT phantoms and the results are compared to those given by the RheoSpectris reference instrument. This method is also used successfully in an ex vivo study for the elasticity assessment of explanted porcine thrombosis surgically induced in vivo.

Thrombosis is the most important pathological process underlying many cardiovascular diseases, which are responsible for high mortality worldwide. In this theses the computer-aided design of new anti-thrombotic agents able to inhibit two types of receptors located on the surface of the platelets has been applied. The first one - αIIbβ3 - is responsible for the interaction of activated platelets with fibrinogen to form clots, whereas the second one - thromboxane A2 - is responsible for platelet activation by one of agonists excreted by activated platelets. To achieve this, different types of models have been developed using experimentally available information and structure of protein-ligand complexes. This concerns: QSAR models, structure-based and ligand-based 3D pharmacophore models, 2D pharmacophore models, shape-based and molecular field-based models. The ensemble of the developed models were used in virtual screening. This study resulted in suggestion of new potential antagonists of αIIbβ3 and thromboxane A2 receptors. Suggested antagonists of αIIbβ3 able to bind either open or closed form of the receptor have been synthesized and tested experimentally. Experiments show that they display high activity; moreover some of theoretically designed compounds are more efficient than Tirofiban - the commercialized drug molecule. The recommended antagonists of thromboxane A2 receptor have been already synthesized but biological tests have not been completed yet.

Orientador: Regina Moura / Resumo: A Síndrome de Cockett (SC) se deve a uma compressão extrínseca da veia ilíaca comum esquerda (VICE) pela artéria ilíaca comum direita (AICD). Em consequência há o desenvolvimento de lesões intraluminais. Este cruzamento entre a artéria e a veia é considerada uma condição anatômica usual, porém em cerca de 29% da população ela pode torna-se sintomática. Os sintomas causados pela SC são: edema, dor, claudicação venosa, hiperemia, varizes e úlceras, podendo evoluir também com complicações como trombose venosa, embolia pulmonar, e mais raramente com Flegmasia Cerúlea Dolens. Até hoje não existe consenso sobre a melhor forma de tratar pacientes com SC na presença ou ausência de trombose da VICE, dividindo as opiniões se deve ser feito o tratamento clínico e/ou endovascular. Preferencialmente alguns autores optam pelas técnicas endovasculares em todos os casos e sugerem que a mesma seja realizada, na tentativa de melhorar a fluxo venoso evitando as complicações tardias. Á curto prazo, os resultados do tratamento endovascular com stents, tem mostrado excelente perviedade, embora estejam alguns deles associados ao uso de antiagregantes, anticoagulantes e ao tratamento clinico convencional com a meia elástica. Objetivo: O presente estudo teve a finalidade de comparar os resultados de perviedade e a taxa de complicações ocorridas em pacientes portadores da SC, tratados com angioplastia e stent, na presença e ausência de trombose da VICE, utilizando a Revisão Sistemática e Meta-análise ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

This thesis was about thrombomebolic disease in pregnancy. The incidence of venous thromboembolism (VTE) probably increases 2- to 4-fold when a woman becomes pregnant. I looked on this problem from many aspects. I looked on health care from her gynaecologist and eventually hematologist, recognition her VTE risk, laboratory tests and her medication. I looked on effectivity of medication too.

Objetivo: A trombose da veia porta é uma causa importante de hiper-tensão porta em crianças e adolescentes, porém, em uma proporção importante dos casos, não apresenta fator etiológico definido. O objetivo desse estudo é determinar a freqüência de deficiência das proteínas inibidoras da coagulação – proteínas C, S e antitrombina − e das mutações fator V Leiden, G20210A no gene da protrombina e C677T da metileno-tetraidrofolato redutase em crianças e adolescentes com trom-bose da veia porta, definir o padrão hereditário de uma eventual deficiência das pro-teínas inibidoras da coagulação nesses pacientes e avaliar a freqüência da deficiên-cia dessas proteínas em crianças e adolescentes com cirrose. Casuística e Métodos: Foi realizado um estudo prospectivo com 14 crianças e adolescentes com trombose da veia porta, seus pais (n = 25) e dois gru-pos controles pareados por idade, constituídos por um grupo controle sem hepato-patia (n = 28) e um com cirrose (n = 24). A trombose da veia porta foi diagnosticada por ultra-sonografia abdominal com Doppler e/ou fase venosa do angiograma celíaco seletivo. A dosagem da atividade das proteínas C, S e antitrombina foi determinada em todos os indivíduos e a pesquisa das mutações fator V Leiden, G20210A da pro-trombina e C677T da metileno-tetraidrofolato redutase, nas crianças e adolescentes com trombose da veia porta, nos pais, quando identificada a mutação na criança, e nos controles sem hepatopatia. Resultados: Foram avaliados 14 pacientes caucasóides, com uma média e desvio padrão de idade de 8 anos e 8 meses ± 4 anos e 5 meses e do diagnóstico de 3 anos e 8 meses ± 3 anos e seis meses. Metade dos pacientes pertenciam ao gênero masculino. O motivo da investigação da trombose da veia porta foi hemorra-gia digestiva alta em 9/14 (64,3%) e achado de esplenomegalia ao exame físico em 5/14 (35,7%). Anomalias congênitas extra-hepáticas foram identificadas em 3/14 (21,4%) e fatores de risco adquiridos em 5/14 (35,7%) dos pacientes. Nenhum pa-ciente tinha história familiar de consangüinidade ou trombose venosa. A deficiência das proteínas C, S e antitrombina foi constatada em 6/14 (42,9%) (p < 0,05 vs con-troles sem hepatopatia), 3/14 (21,4%) (p > 0,05) e 1/14 (7,1%) (p > 0,05) pacientes com trombose da veia porta, respectivamente. A deficiência dessas proteínas não foi identificada em nenhum dos pais ou controles sem hepatopatia. A mutação G20210A no gene da protrombina foi identificada em um paciente com trombose da veia porta e em um controle sem hepatopatia (p = 0,999), mas em nenhum desses foi identificado a mutação fator V Leiden. A mutação C677T da metileno-tetraidrofo-lato redutase foi observada na forma homozigota, em 3/14 (21,4%) dos pacientes com trombose da veia porta e em 5/28 (17,9%) controles sem hepatopatia (p = 0,356). A freqüência da deficiência das proteínas C, S e antitrombina nos pacientes com cir-rose foi de 14/24 (58,3%), 7/24 (29,2%) e 11/24 (45,8%), respectivamente (p < 0,05 vs controles sem hepatopatia), sendo mais freqüente nos pacientes do subgrupo Child-Pugh B ou C, que foi de 11/12 (91,7%), 5/12 (41,7%) e 9/12 (75%), respectivamente (p < 0,05 vs controles sem hepatopatia). Conclusões: A deficiência de proteína C foi freqüente nas crianças e adolescentes com trombose da veia porta e não parece ser de origem genética. A deficiência de proteína S, antitrombina e as presenças das mutações G20210A da protrombina e C677T da metileno-tetraidrofolato redutase foram observadas mas não apresentaram diferença estatística significativa em relação ao grupo controle sem hepatopatia. O fator V Leiden não foi identificado. Os resultados deste estudo sugerem que a deficiência da proteína C pode ocorre como conseqüência da hiper-tensão porta. Os distúrbios pró-trombóticos hereditários não parecem apresentar um papel importante em relação à trombose nas crianças e adolescentes estudadas. / Objective: Portal vein thrombosis is a major cause of portal hypertension in children and adolescents; yet, its etiology is not clearly defined in a considerable number of cases. The present study aims at determining the prevalence of blood coagulation disorders – protein C, protein S and antithrombin – and factor V Leiden, G20210A prothrombin, and C677T methylenetetrahydrofolate reductase mutations in children and adolescents with portal vein thrombosis, as well as assessing the hereditary character of these disorders in these patients, and also evaluating the prevalence of blood coagulation disorders in children and adolescents with cirrhosis. Study design: A prospective study was carried out, including children and adolescents with portal vein thrombosis (n = 14), their parents (n = 25), two age-matched control groups, one without liver disease (n = 28), and another with cirrhosis (n = 24). Portal vein thrombosis was diagnosed through abdominal Doppler ultrasonography and/or venous phase of selective coeliac angiograms. The activity of protein C, protein S and antithrombin was evaluated for all individuals; the presence of factor V Leiden, G20210A prothrombin, and C677T methylenetetrahydrofolate reductase gene mutations was investigated in children and adolescents with portal vein thrombosis, in parents when their respective children presented any of these mutations, and in the control group without liver disease. Results: 14 Caucasian patients were assessed. The mean and standard deviation for age were 8 years and 8 months ± 4 years and 5 months while the mean and standard deviation for diagnosis were 3 years and 8 months ± 3 years and six months. Half of the patients were males. Initial clinical manifestations upon diagnosis were digestive hemorrhage in 9/14 (64.3%) and splenomegaly on physical examination in 5/14 individuals (35.7%). Patients presented extrahepatic anomalies in 3/14 (21.4%) and acquired risk factors in 5/14 (35.7%) of the cases. None of the patients had a family history of consanguinity or venous thrombosis. The frequency of protein C, protein S and antithrombin deficiency was observed in 6/14 (42.9%) (p < 0.05 vs. controls without liver disease), 3/14 (21.4%) (p > 0.05) and 1/14 (7.1%) (p > 0.05) of patients, respectively. None of the portal vein thombosis patients or controls presented protein C, S or antithrombin deficiency. One portal vein patient and one control (p = 0.999) presented G20210A prothrombin mutation. None of these patients presented the factor V Leiden. The homozygous form of C677T methylenetetrahydrofolate reductase mutation was observed in 3/14 patients with portal vein thrombosis (21.4%) and in 5/28 controls (17.9%) (p = 0.356). The frequency of coagulation inhibitor deficiency was high in cirrhotic patients (14/24 (58.3%) PC, 7/24 (29.2%) PS and 11/24 (45.8%) AT; p < 0.05 vs. controls), especially in Child-Pugh B and C patients (11/12 (91.7%) PC, 5/12 (41.7%) PS and 9/12 (75%) AT; p < 0.05 vs. controls). Conclusions: Protein C deficiency was frequent in children and adolescents with portal vein thrombosis and does not seem to be an inherited condition. Protein S and antithrombin deficiency, and G20210A prothrombin and C677T methylenetetrahydrofolate reductase mutations were observed but did not present statistically significant differences when compared to the controls without liver disease. Factor V Leiden was not observed. The results suggest the protein C deficiency may originates from portal hypertension. The hereditary prothrombotic disorders do not seem to play a vital role in thrombosis in children and adolescents with portal vein thrombosis.

Objetivo: A trombose da veia porta é uma causa importante de hiper-tensão porta em crianças e adolescentes, porém, em uma proporção importante dos casos, não apresenta fator etiológico definido. O objetivo desse estudo é determinar a freqüência de deficiência das proteínas inibidoras da coagulação – proteínas C, S e antitrombina − e das mutações fator V Leiden, G20210A no gene da protrombina e C677T da metileno-tetraidrofolato redutase em crianças e adolescentes com trom-bose da veia porta, definir o padrão hereditário de uma eventual deficiência das pro-teínas inibidoras da coagulação nesses pacientes e avaliar a freqüência da deficiên-cia dessas proteínas em crianças e adolescentes com cirrose. Casuística e Métodos: Foi realizado um estudo prospectivo com 14 crianças e adolescentes com trombose da veia porta, seus pais (n = 25) e dois gru-pos controles pareados por idade, constituídos por um grupo controle sem hepato-patia (n = 28) e um com cirrose (n = 24). A trombose da veia porta foi diagnosticada por ultra-sonografia abdominal com Doppler e/ou fase venosa do angiograma celíaco seletivo. A dosagem da atividade das proteínas C, S e antitrombina foi determinada em todos os indivíduos e a pesquisa das mutações fator V Leiden, G20210A da pro-trombina e C677T da metileno-tetraidrofolato redutase, nas crianças e adolescentes com trombose da veia porta, nos pais, quando identificada a mutação na criança, e nos controles sem hepatopatia. Resultados: Foram avaliados 14 pacientes caucasóides, com uma média e desvio padrão de idade de 8 anos e 8 meses ± 4 anos e 5 meses e do diagnóstico de 3 anos e 8 meses ± 3 anos e seis meses. Metade dos pacientes pertenciam ao gênero masculino. O motivo da investigação da trombose da veia porta foi hemorra-gia digestiva alta em 9/14 (64,3%) e achado de esplenomegalia ao exame físico em 5/14 (35,7%). Anomalias congênitas extra-hepáticas foram identificadas em 3/14 (21,4%) e fatores de risco adquiridos em 5/14 (35,7%) dos pacientes. Nenhum pa-ciente tinha história familiar de consangüinidade ou trombose venosa. A deficiência das proteínas C, S e antitrombina foi constatada em 6/14 (42,9%) (p < 0,05 vs con-troles sem hepatopatia), 3/14 (21,4%) (p > 0,05) e 1/14 (7,1%) (p > 0,05) pacientes com trombose da veia porta, respectivamente. A deficiência dessas proteínas não foi identificada em nenhum dos pais ou controles sem hepatopatia. A mutação G20210A no gene da protrombina foi identificada em um paciente com trombose da veia porta e em um controle sem hepatopatia (p = 0,999), mas em nenhum desses foi identificado a mutação fator V Leiden. A mutação C677T da metileno-tetraidrofo-lato redutase foi observada na forma homozigota, em 3/14 (21,4%) dos pacientes com trombose da veia porta e em 5/28 (17,9%) controles sem hepatopatia (p = 0,356). A freqüência da deficiência das proteínas C, S e antitrombina nos pacientes com cir-rose foi de 14/24 (58,3%), 7/24 (29,2%) e 11/24 (45,8%), respectivamente (p < 0,05 vs controles sem hepatopatia), sendo mais freqüente nos pacientes do subgrupo Child-Pugh B ou C, que foi de 11/12 (91,7%), 5/12 (41,7%) e 9/12 (75%), respectivamente (p < 0,05 vs controles sem hepatopatia). Conclusões: A deficiência de proteína C foi freqüente nas crianças e adolescentes com trombose da veia porta e não parece ser de origem genética. A deficiência de proteína S, antitrombina e as presenças das mutações G20210A da protrombina e C677T da metileno-tetraidrofolato redutase foram observadas mas não apresentaram diferença estatística significativa em relação ao grupo controle sem hepatopatia. O fator V Leiden não foi identificado. Os resultados deste estudo sugerem que a deficiência da proteína C pode ocorre como conseqüência da hiper-tensão porta. Os distúrbios pró-trombóticos hereditários não parecem apresentar um papel importante em relação à trombose nas crianças e adolescentes estudadas. / Objective: Portal vein thrombosis is a major cause of portal hypertension in children and adolescents; yet, its etiology is not clearly defined in a considerable number of cases. The present study aims at determining the prevalence of blood coagulation disorders – protein C, protein S and antithrombin – and factor V Leiden, G20210A prothrombin, and C677T methylenetetrahydrofolate reductase mutations in children and adolescents with portal vein thrombosis, as well as assessing the hereditary character of these disorders in these patients, and also evaluating the prevalence of blood coagulation disorders in children and adolescents with cirrhosis. Study design: A prospective study was carried out, including children and adolescents with portal vein thrombosis (n = 14), their parents (n = 25), two age-matched control groups, one without liver disease (n = 28), and another with cirrhosis (n = 24). Portal vein thrombosis was diagnosed through abdominal Doppler ultrasonography and/or venous phase of selective coeliac angiograms. The activity of protein C, protein S and antithrombin was evaluated for all individuals; the presence of factor V Leiden, G20210A prothrombin, and C677T methylenetetrahydrofolate reductase gene mutations was investigated in children and adolescents with portal vein thrombosis, in parents when their respective children presented any of these mutations, and in the control group without liver disease. Results: 14 Caucasian patients were assessed. The mean and standard deviation for age were 8 years and 8 months ± 4 years and 5 months while the mean and standard deviation for diagnosis were 3 years and 8 months ± 3 years and six months. Half of the patients were males. Initial clinical manifestations upon diagnosis were digestive hemorrhage in 9/14 (64.3%) and splenomegaly on physical examination in 5/14 individuals (35.7%). Patients presented extrahepatic anomalies in 3/14 (21.4%) and acquired risk factors in 5/14 (35.7%) of the cases. None of the patients had a family history of consanguinity or venous thrombosis. The frequency of protein C, protein S and antithrombin deficiency was observed in 6/14 (42.9%) (p < 0.05 vs. controls without liver disease), 3/14 (21.4%) (p > 0.05) and 1/14 (7.1%) (p > 0.05) of patients, respectively. None of the portal vein thombosis patients or controls presented protein C, S or antithrombin deficiency. One portal vein patient and one control (p = 0.999) presented G20210A prothrombin mutation. None of these patients presented the factor V Leiden. The homozygous form of C677T methylenetetrahydrofolate reductase mutation was observed in 3/14 patients with portal vein thrombosis (21.4%) and in 5/28 controls (17.9%) (p = 0.356). The frequency of coagulation inhibitor deficiency was high in cirrhotic patients (14/24 (58.3%) PC, 7/24 (29.2%) PS and 11/24 (45.8%) AT; p < 0.05 vs. controls), especially in Child-Pugh B and C patients (11/12 (91.7%) PC, 5/12 (41.7%) PS and 9/12 (75%) AT; p < 0.05 vs. controls). Conclusions: Protein C deficiency was frequent in children and adolescents with portal vein thrombosis and does not seem to be an inherited condition. Protein S and antithrombin deficiency, and G20210A prothrombin and C677T methylenetetrahydrofolate reductase mutations were observed but did not present statistically significant differences when compared to the controls without liver disease. Factor V Leiden was not observed. The results suggest the protein C deficiency may originates from portal hypertension. The hereditary prothrombotic disorders do not seem to play a vital role in thrombosis in children and adolescents with portal vein thrombosis.