Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are associated with increased risk of atherothrombotic disease. Lp(a) is a unique lipoprotein consisting of a low density lipoprotein-like moiety covalently linked to apolipoprotein(a) (apo(a)), a homologue of the fibrinolytic proenzyme plasminogen. Apo(a) is extremely heterogeneous in size with small isoforms being independently associated with increased cardiovascular risk. Several in vitro and in vivo studies have shown that Lp(a)/apo(a) can inhibit tissue-type plasminogen activator (tPA)-mediated plasminogen activation on fibrin surfaces, although the mechanism of inhibition by apo(a) remains controversial. Essential to fibrin clot lysis are a number of plasmin-dependent positive feedback reactions that enhance the efficiency of plasminogen activation, including the plasmin-mediated conversion of Glu1-plasminogen to Lys78-plasminogen. Additionally, abnormal fibrin clot structures have been associated with both an increased risk of cardiovascular disease and elevated Lp(a) levels. Similarly, oxidized phospholipids have been implicated in the development of cardiovascular disease, and are not only preferentially carried by Lp(a) in the plasma but have also been shown to covalently-modify both apo(a) and plasminogen. In this thesis, we built upon the understanding of the role of apo(a) in plasminogen activation on the fibrin/degraded fibrin surface by determining that: (i) apo(a) inhibits plasmin-mediated Glu1-plasminogen to Lys78-plasminogen conversion and identifying the critical domains in apo(a) responsible for this effect, (ii) apo(a) isoform size does not affect either the inhibition of tPA-mediated plasminogen activation or the inhibition of plasmin-mediated Glu1-plasminogen to Lys78-plasminogen conversion, (iii) apo(a) modifies fibrin clot structure to form more dense clots with thinner fibers and reduced permeability, modifications that enhance the ability of apo(a) to inhibit tPA-mediated plasminogen activation and (iv) the phosphorus content of apo(a) affects its ability to inhibit tPA-mediated plasminogen activation and the phosphorus content of plasminogen affects its ability to be activated by tPA. By understanding these individual reactions, each of which has the potential to affect the broader fibrin clot lysis process, we have expanded our understanding of the overall effect of Lp(a)/apo(a) in the inhibition of plasminogen activation on the fibrin/degraded fibrin surface and thus broadened our understanding of how Lp(a)/apo(a) may mediate the inhibition of thrombolysis in vivo. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-12-14 08:26:54.99

Le rôle physiologique du caillot est d’éviter un épanchement excessif de sang en présence d’une brèche vasculaire. Une fois cette fonction remplie, il doit pouvoir être facilement détruit, afin qu’il ne passe pas dans le système veineux et ne gêne la circulation sanguine. La formation d’un caillot de fibrine et sa lyse, processus clés de l’hémostase, impliquent à la fois la polymérisation des monomères de fibrinogène en un réseau de fibres de fibrine, et la résorption du réseau de fibres de fibrine constitué. Bien que ce réseau contrôle l’ensemble des propriétés physiques et mécaniques du caillot, sa structure aux échelles inférieures au micron est très mal caractérisée. Le principal verrou à la caractérisation physique du caillot en environnement clinique est l’absence de méthode de mesure quantitative, fiable, sensible et reproductible. Il est donc nécessaire de produire une méthode de mesure adéquate, couplée à un système de mesure sensible. Nous avons démontré dans ce travail, grâce à notre méthode utilisant plusieurs longueurs d’onde, que l’analyse du spectre de lumière visible transmis à travers un caillot permet de déterminer simultanément, quantitativement et en conditions quasi-physiologiques, plusieurs paramètres essentiels de structure du caillot de fibrine, à savoir le nombre de protofibrilles par fibre de fibrine, le rayon et la densité de ces fibres, ainsi que les temps de formation et de lyse du caillot. Cette technique a été validée via les résultats avec des CV inférieurs dans l’ensemble à 6% sous plusieurs conditions de tests et différents profils plasmatiques : normaux, hypo/hyper coagulants et hypo/hyper fibrinolytiques, attestant de la robustesse et de la fiabilité de la technique de mesure aussi bien pour le suivi de la coagulation que de la lyse. Cette méthode de spectrophotométrie a pu être implantée sur un automate modifié à des fins de diagnostic et à vocation hospitalière pour des plasmas de patients présentant des troubles de l’hémostase. Les informations cliniques et intérêts attendus de ce nouveau test, concernent à la fois la qualité du réseau de fibrine, sa lyse accélérée ou sa résistance à la fibrinolyse ainsi que la résultante de la balance coagulo-lytique. / The physiological role of the clot is to avoid excessive bleeding in the presence of a vascular breach. Once this function is filled, the clot must be able to be easily destroyed, so that it is not transported in the venous system and does not hamper blood circulation. The formation of a fibrin clot and its lysis are key processes of hemostasis, implying simultaneously the polymerization of the fibrinogen monomers in a fibrin fibers network, and the destruction of this constituted network.Although this network controls the physical and mechanical properties of the clot, its structure at scales smaller than the micron is poorly characterized. The main problem in the physical characterization of clot in clinical settings is the current absence of a quantitative, sensitive and reproducible measurement method.We demonstrated in this work, thanks to our method using several wavelengths, that the analysis of the visible spectra of light transmitted through a clot allows to determine simultaneously, quantitatively and in quasi-physiological conditions, several essential parameters of structure of the fibrin clot, namely the number of protofibrils per fibrin fibers, the radius and the density of fibers, and various times of clotting and lysis of the clot. This method was validated by the results with CV inferior to 6 % under all test conditions and various plasmatic profiles: normal, hypo / hyper coagulant and hypo / hyper fibrinolytic. This demonstrates the robustness and reliability of the measurement method when measuring both clotting and clot lysis.This spectrophotometric method was implemented on a modified automaton dedicated to diagnosis of patients presenting hemostatic disorders. The clinical information and the interests expected from this new test concern at the same time the quality of the fibrin network, its accelerated lysis or its resistance to fibrinolysis, and the resultant of the coagulo-lytic balance.