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 système d’activation du plasminogène (SAP), initialement décrit dans la circulation sanguine, intervient dans la dégradation des caillots de fibrine (fibrinolyse). Ce système permet de convertir un zymogène inactif (le plasminogène) en enzyme active (la plasmine) via l’action de l’activateur tissulaire du plasminogène (tPA), lui-même inhibé par la serpine inhibitrice de l’activateur du plasminogène de type 1 (PAI-1). Il a été suggéré qu’une dérégulation du SAP dans le système nerveux central (SNC) pouvait être un processus physiopathologique dans la sclérose en plaques (SEP). Cependant, l’origine et les conséquences de cette dérégulation restent peu caractérisées dans cette pathologie. Durant cette thèse, nous avons donc cherché à mieux caractériser la dérégulation du SAP dans la SEP et à déterminer son rôle dans la physiopathologie de cette maladie. Les travaux réalisés au cours de ce doctorat ont permis de montrer qu’une forte surexpression de PAI-1 survient dans le SNC lors des phases symptomatiques de modèles murins de SEP, entraînant une inhibition de la fibrinolyse intraparenchymateuse. Cette altération du SAP est une cause de la formation des zones de lésion, et coïncide temporellement avec les phases symptomatiques des modèles murins de SEP. Les astrocytes réactifs pro-inflammatoires sont les responsables de cette surexpression parenchymateuse délétère de PAI-1. Il apparaît qu’une intervention à visée thérapeutique permettant la restauration de l’activité fibrinolytique intraparenchymateuse, via l’inhibition de PAI-1, s’avère bénéfique dans des modèles animaux de SEP. En conclusion, nos travaux révèlent que la présence d’astrocytes réactifs est à l’origine de la surexpression de PAI-1 engendrant l’inhibition de la fibrinolyse intraparenchymateuse constatée dans la SEP. Ce processus physiopathologique délétère est une des causes menant à la formation de zones lésionnelles dans la SEP. / Degradation of fibrin blood clots (fibrinolysis) is mediated by the plasminogen activation system (PAS), initially discovered in the blood. This system allows the inactive plasminogen conversion into active plasmin by tissue-type plasminogen activator (tPA). The plasminogen activator inhibitor type 1 (PAI-1) is able to inhibit tPA. It has been suggested that in the central nervous system (CNS), a PAS dysregulation could be a physiopathological mechanism of multiple sclerosis (MS). However, this dysregulation cause and consequences are poorly described in this disease. This work aimed to characterise the PAS dysregulation occurring in MS, and to better define its role in MS physiopathology. Our results describe a strong PAI-1 overexpression in the CNS during symptomatic periods in animal models of MS, leading to an intraparenchymal fibrinolysis inhibition. The PAS dysregulation is a cause of lesion formation, and temporally coincides with symptomatic periods in these models. Pro-inflammatory reactive astrocytes overexpress PAI-1. It appears that an increase of parenchymal fibrinolysis by inhibiting PAI-1 reduces EAE severity. To conclude, our results highlight a role of reactive astrocytes in MS, leading to an over-expression of PAI-1 and an impairment of parenchymal fibrinolysis. This physiopathological mechanism is implied in lesion formation in MS.

No description available.

Carboxypeptidase N (CPN) is a plasma carboxypeptidase that was discovered in the 1960s as a regulator of inflammation and vascular tone. Through the removal of carboxy-terminal basic residues, CPN alters the activity or binding specificity of inflammatory mediators and vasoactive peptides. CPN shares significant homology with carboxypeptidases known to mediate antifibrinolysis through the removal of basic residues from fibrin clots, which would otherwise stimulate fibrinolysis. Despite the similarity of these enzymes, CPN is generally regarded as lacking a role in fibrinolysis. This thesis demonstrates that CPN is indeed a capable antifibrinolytic enzyme, and that the antifibrinolytic activity of CPN was previously undisclosed due to the presence of a circulating CPN inhibitor, which is likely the free CPN2 subunit. This inhibitor is described for the first time here. Furthermore, potential mechanisms of inhibition and mechanisms of enhancing activity of CPN are proposed based upon the additional structural characterization of CPN presented here.

Le système d’activation du plasminogène (AP) comprenant les protéases uPA et tPA, et leur inhibiteur PAI-1, génère une activité protéolytique dans la matrice extracellulaire et contribue au remodelage tissulaire dans une grande variété de processus physiopathologiques, y compris la myogenèse squelettique, et la différenciation adipocytaire.Nous avons évalué son rôle spécifique dans la différenciation des cellules souches embryonnaires (ES) de souris. On a trouvé que les activités d’uPA et de tPA ainsi que les niveaux protéiques de PAI-1 sont maximaux dans les cellules différenciées, contrairement aux cellules ES indifférenciées où ils sont indétectables et augmentent progressivement dès le jour 3 de la différenciation. La différenciation adipocytaire dans le modèle des cellules ES est inhibée par le traitement par l’amiloride, un inhibiteur spécifique de l’uPA. Egalement, les cellules ES surexprimant une forme active du PAI-1 sous le contrôle d’un système d’expression inductible, montrent des capacités adipogéniques réduites après l’induction du gène. Nos résultats démontrent que le contrôle de l’adipogenèse des cellules ES par le système AP correspond à des étapes successives, différentes, depuis les cellules indifférenciées jusqu’aux cellules bien différenciées. De plus, les capacités de la différenciation adipogénique des cellules pluripotentes induites déficientes en PAI-1 sont augmentées par rapport aux cellules contrôles.Similairement, la myogenèse squelettique est réduite par l’inhibition de l’uPA par l’amiloride ou par la surexpression du PAI-1 durant l’étape terminale de la différenciation du jour 7 au jour 24. Cependant, l’interférence avec l’uPA durant les jours 0 à 3 de la différenciation, stimule la formation des myotubes. Les différenciations cardiomyocyotaire, neuronale, endothéliale et du du muscle lisse ne sont pas affectées par le traitement à l’amiloride ou la surexpression du PAI-1.Nos résultats montrent que le système AP est capable de moduler spécifiquement l’adipogenèse et la myogenèse squelettique des cellules ES par des mécanismes moléculaires successifs différents. / Regulation of the extracellular matrix (ECM) plays an important functional biological role either in physiological or pathological conditions. The plasminogen activation (PA) system, comprising the uPA and tPA proteases and their inhibitor PAI-1, is one of the main suppliers of extracellular proteolytic activity contributing to tissue remodeling. Although its function in development is well documented, its precise role in mouse embryonic stem cell (ESC) differentiationin vitro is unknown. We found that uPA and tPA activities and PAI-1 protein are very low in undifferentiated ESCs and increase strongly during the differentiation, reaching a maximum in well differentiated cells. Adipocyte formation by ESCs is inhibited by amiloride treatment, a specific uPA inhibitor. Likewise, ESCs expressing ectopic PAI-1 under the control of an inducible expression system, display reduced adipogenic capacities after induction of the gene. Our results demonstrate that the control of ESC adipogenesis by the PA system correspond to different successive steps from undifferentiated to well differentiated ESCs. Furthermore, the adipogenic differentiation capacities of PAI-1-/- induced pluripotent stem cells (iPSCs) are augmented as compared to wt iPSCs. Similarly, skeletal myogenesis is decreased by uPA inhibition or PAI-1 overexpression during the terminal step of differentiation. However, interfering with uPA during days 0 to 3 of the differentiation process augments ESC myotube formation. Neither neurogenesis, cardiomyogenesis, endothelial cell nor smooth muscle formation are affected by amiloride or PAI-1 induction. Our results show that the PA system is capable to specifically modulate adipogenesis and skeletal myogenesis of ESCs by successive different molecular mechanisms.