L’activateur tissulaire du plasminogène (tPA) est une protéase initialement découverte dans le sang pour son rôle fibrinolytique. C’est pour cette fonction que le tPA recombinant est utilisé pour traiter la phase aigüe de l’accident vasculaire cérébral (AVC) ischémique, même s’il présente quelques limites. Le tPA exogène peut passer du compartiment vasculaire au parenchyme cérébral où il peut influencer des processus physiologiques, et participer au devenir neuronal, notamment aggraver la mort neuronale lors d’un AVC ischémique. Le laboratoire a montré que le tPA peut traverser la barrière-hémato-encéphalique (BHE), par transcytose au travers des cellules endothéliales de la BHE et cela sous le contrôle des récepteurs LRP1 (Low density lipoprotein receptor-related protein 1). D’autres barrières existent au sein du système nerveux central notamment la barrière sang-liquide cérébrospinal (BSLCS), formée par les plexus choroïdes (PCs). Les PCs sont une route de migration pour les cellules inflammatoires et le LCS peut véhiculer des solutés, via les espaces péri-artériels, vers le parenchyme cérébral. Ainsi, dans notre première étude, nous avons testé l’hypothèse d’un passage du tPA vasculaire par les PCs. Pour cela, nous avons produit un tPA traçable in vivo et in vitro. Nous avons commencé par étudier la distribution du tPA suite à une injection intraveineuse (IV) avec comme focus les PCs et le LCS. Nos résultats montrent que le tPA exogène, suite à une injection IV, est retrouvé de manière séquentielle dans les PCs puis dans le LCS. Le tPA est donc capable de traverser les PCs. Nous avons alors développé un modèle de culture primaire de cellules épithéliales de PCs (CPECs) de souris pour disséquer le(s) mécanisme(s) sous-jacents à l’internalisation du tPA par les CPECs. Ce modèle nous a permis de montrer que l’internalisation du tPA par les CPECs est un phénomène actif, médié par un membre de la famille des récepteurs LRP, mais qui n’est ni LRP1, ni LRP2. Nous avons également mis en évidence la nécessité du domaine Finger du tPA pour son internalisation par les CPECs. Une étude préliminaire dans un modèle d’AVC suggère que l’ischémie modifie la cinétique de passage du tPA, puisqu’il y a plus de tPA dans les PCs des souris ischémiées que les souris non ischémiées.Dans une deuxième étude nous nous sommes intéressés à l’effet du tPA endogène sur les PCs. Nous montrons que l’absence de tPA endogène n’influence ni la morphologie des PCs, ni la diffusion du LCS. De plus, nous montrons que cette absence de tPA n’influence pas le nombre de macrophages et de lymphocytes T dans les PCs en conditions basales. / Tissue-type plasminogen activator (tPA) is a protease initially discovered in the blood for its fibrinolytic role. Accordingly, recombinant tPA has become the gold standard to treat the acute phase of ischemic stroke, despite some limitations. Exogenous tPA can switch from the vascular compartment to the brain parenchyma, where it can influence physiological processes, and participate in neuronal fate, including a worsening of neuronal death during ischemic stroke. In the team, it has been shown that tPA can cross the blood-brain barrier (BBB), by a transcytosis through BBB endothelial cells, under the control of LRP1 receptors (Low density lipoprotein receptor-related protein 1). The central nervous system has other barriers, including the blood-cerebrospinal fluid barrier (BCSFB), that relies on choroid plexuses (CPs). CPs are a migration route for inflammatory cells and a major source of CSF, which carries solutes to the cerebral parenchyma, via peri-arterial spaces. Thus, in a first study, we tested the hypothesis of a passage of vascular tPA through CPs. We thus produced a fluorescent tPA that can be tracked in vivo and in vitro. We first studied the distribution of tPA following intravenous (IV) injection, focusing on CPs and CSF. We show that after an IV injection, exogenous tPA is sequentially found in the CPs and then in the CSF. tPA is therefore able to cross the CPs. We then developed a model of primary culture of mouse choroid plexus epithelial cells (CPECs) to dissect the mechanism (s) underlying the internalization of tPA. This model allowed us to demonstrate that the internalization of tPA by CPECs is an active phenomenon, mediated by a member of the family of LRP receptors, but which is neither LRP1 nor LRP2. We also highlight the requirement for the Finger domain of tPA for its internalization by CPECs. A preliminary study in a murine stroke model suggests that ischemia alters the tPA passage kinetics, since there is more tPA in ischemic CPs than non-ischemic CPs.In a second study, we investigated the effect of endogenous tPA on CPs. We show that the absence of endogenous tPA influences neither CPs morphology nor CSF diffusion . Moreover we show that the absence of tPA does not influence the number of macrophages and T cells in the stroma of PCs under basal conditions.

Optimal induction of coronary thrombolysis depends in part upon the nature of the specific plasminogen activator used. The two general classes of plasminogen activators available clinically differ in a fundamental respect delineated by the term, clot selectivity. Clot selective agents are less prone to induce plasminemia and consequent occult activation of the coagulation cascade than are non-selective agents. However, under clinical conditions, all plasminogen activators result in some activation of the cascade with consequent generation of thrombin. Accordingly, optimal therapy requires the use of conjunctive anticoagulation to preclude the deleterious effects of rebound generation of thrombin, which has been well documented biochemically. The potential value of antiplatelet agents that can attenuate the positive feedback loop between activation of platelets and markedly amplified generation of thrombin in the setting of coronary thrombolysis is under active exploration. With appropriate monitoring of the efficacy of such agents in vivo it should be possible to enhance even further the benefits that can be conferred by pharmacologically induced coronary thrombolysis.

No description available.

Urokinase-type plasminogen activator (uPA) protein is a serine protease of the trypsin family that is overexpressed by tumors cells seeking to metastasize. Molecular imaging methods using molecular imaging probe designed to target uPA could provide a method for the detection of aggressive cancers and monitoring response to treatment. Four classes of high affinity uPA inhibitors, three which were reversible and one irreversible, were used as platforms to develop radiolabeled probes for uPA. Based on structure-activity relationships, lead compounds were modified to allow for the introduction of a radiohalogen (radioiodine) at different sites in the corresponding molecules. Suitable synthetic strategies were developed to create libraries of iodinated phenyl guanidine, peptide, naphtamidine and phosphonate derivatives. For the phenylguanidines colorimetric assays showed the product had micromolar affinity while for the peptide derivatives low nanomolar affinity for the iodinated analogue was observed (1.4 nM to 2.53 nM). Unfortunately quantitative biodistribution studies showed low tumour uptake (<0.5% ID/g). More promising results were obtained for the irreversible iodinated phosphonated derivative which had an affinity of 2.1 nM. This reagent showed 1.95% ID/g tumour uptake and lower blood uptake in vivo which demonstrates advantageous properties over existing uPA probes in terms of tumour-to-blood ratios. A complementary development was also achieved in that the first example of a 125I-labelled tetrazine was prepared. This new reagent can be used in pre-targeted strategies that utilize bioorthogonal coupling between stained trans-cyclooctene (TCO) and tetrazines. The product was prepared using a concomitant oxidation iodo-destannylation reaction and the product isolated in 80% radiochemical yield. The reaction with transcycloctene proceeded rapidly to produce various isomers which were fully characterized through NMR analysis of the non-radioactive analogues. / Thesis / Doctor of Philosophy (PhD)

Excerpt: The recent study by Sahlas et al1 in the Journal of Stroke and Cerebrovascular Diseases concluded that “the estimation of a patient's weight in the acute setting can lead to overcalculation of the tissue plasminogen activator dose, which is associated with poorer functional outcomes.” However, this conclusion is not well supported by their study1 because the most severe ischemic stroke cases were the ones that were most likely not weighed, and this severity could have led to the increased mortality that was found2 and the majority of unweighed patients were actually given an underdose that was associated with better discharge outcomes, as explained below.

Studies demonstrated ADAMTS13 possesses unique properties with a mystifying regulatory mechanism. ADAMTS13’s role is in its proteolytic function to its VWF. The disparity in the hemostatic balance between ADAMTS13 activity and the distribution of VWF multimers could result in the bleeding disorder Von Willebrand Disease (VWD) or the thrombotic disorder thrombotic thrombocytopenic purpura (TTP). ADAMTS13 is constitutively secreted as an active protease, yet VWF retains its capacity to recruit platelets. This ability makes ADAMTS13 an enigmatic protease with an unknown regulatory mechanism. Currently, the postulated regulatory mechanism of ADAMTS13 is in its open/closed conformation, yet ADAMTS13 activity is retained in both forms. Literature showed that few proteases are capable of degrading ADAMTS13 in-vitro. We hypothesize that the partial degradation of ADAMTS13 regulates its activity, thereby stabilizing VWF and promoting thrombosis. The goals of this project were to develop and optimize in-vitro plasma BioID to identify novel interactions to ADAMTS13, validate novel interactions, identify proteases capable of degrading ADAMTS13 and their proteolytic sites, and develop protease-resistant ADAMTS13 mutants as novel therapeutics to thrombotic disorders. We optimized the BioID technique to be used in-vitro in plasma, to study novel interactions with ADAMTS13. Our results identified novel potential interactions with vitronectin or plasminogen. Validation studies disregarded vitronectin’s interaction and confirmed plasminogen’s interaction through the CUB and Kringle domains in a lysine-dependent manner. Further, the list of proteases capable of degrading ADAMTS13 was expanded to include FXIa and neutrophil-derived proteases including Cathepsin G, elastase, and hPR3. Activated neutrophils played a stronger role than coagulation proteases in degrading ADAMTS13 in vivo, while also demonstrating that elastase is a more potent regulator. Proteolytically degraded sites on ADAMTS13 were identified and proteolytic-resistant ADAMTS13 mutants were produced accordingly, which we aim to be utilized as a novel therapeutic to thrombotic disorders. / Thesis / Doctor of Philosophy (Medical Science)

No description available.

<p> Tissue plasminogen activator (tPA), is commonly used as thrombolytic agent for the treatment of various cardiovascular diseases. This thesis constitutes the first report on cloning and expression of tPA in the methylotrphic yeast Pichia pastoris. </p> <p> The tPA gene was first cloned into an E. coli/Pichia shuttle plasmid and then integrated into the Pichia genome. The recombinant Pichia was able to express tPA intracellularly, under methanol induction. The tPA produced by the Pichia had a similar molecular weight as native tPA and it had serine protease activity. At the shake flask scale, the recombinant Pichia strain was able to produce twice as much tPA as reported for E. coli. </p> <p> Elastin-like polypeptides (ELP) are proteins that have a peculiar characteristic: they are able to undergo a reversible inverse phase transition temperature within a very narrow temperature range. On a second aspect of heterologous protein, a construct composed of thioredoxin-intein-ELP was used to provide direct evidence, for the first time, that protein folding and activity, in this case the intein, was maintained when the tripartite fusion was present in the aggregated state. These results are important, since they provide the necessary degree of confidence to stimulate future work directed towards expression and maintenance of proper folding of aggregation-prone proteins when expressed in-vivo E. Coli as ELP directed inclusion bodies. It is also shown that the intein-ELP system may be a very interesting system to be used as a drug delivery vehicle. </p> / Thesis / Master of Applied Science (MASc)

Motivation: Cardiovascular heart disease (CVD) is the leading cause of death worldwide. Risk markers for CVD include, amongst others, the haemostatic factors tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor type 1 (PAI-1), factor VII (FVII) and fibrinogen and more recently, C-reactive protein (CRP), a sensitive marker of inflammation. Epidemiological studies have demonstrated an inverse association between dietary fibre (DF) consumption and risk factors for CVD and CVD prevalence. Some research indicates that this protection may be related to favourable changes in the haemostatic profile and inflammatory markers. This is applicable for the consumption of total DF, as well as soluble and insoluble fibre. However, clinical intervention trials report conflicting data on the effects of DF on t-PA, PAI-1, FVII, fibrinogen and CRP. In addition, available literature is not clear on the mechanisms through which DF may have favourable effects. Objective: The main objective of this study was to review the results of randomised controlled trials systematically on the effects of DF on the above-mentioned selected haemostatic variables and CRP in healthy adults and subjects with hypertriglyceridaemia and the metabolic syndrome. Methods: Human adult intervention trials, at least two weeks in duration, with an increased and measurable consumption of DF were included. Electronic databases were searched from the earliest record to May/July 2006 and supplemented by crosschecking reference lists of relevant publications. From the literature search, two reviewers identified studies that were rated for quality based on the published methodology. No formal statistical analysis was performed due to the large differences in the study designs of the dietary intervention trials. The primary outcome measures were percentage changes between intervention and control groups, or baseline to end comparisons for t-PA, PAI-1, FVII, fibrinogen and CRP. Results t-PA activity increased significantly (14-167%) over the short and long-term following increased fibre intakes. PAI-1 activity decreased significantly between 15-57% over periods ranging from two to six weeks. These favourable changes in t-PA and PAI-1 occurred in healthy, hypertriglyceridaemic and metabolic syndrome subjects following consumption of diets containing ≥3.3 g/MJ DF and ≥4.5 g/MJ DF respectively. Mechanisms through which DF may affect t-PA and PAI-1 include its lowering effect on insulinaemic and glycaemic responses, decreasing triglycerides which are a precursor of very-low-density lipoproteins, fermentation of DF to short-chain fatty acids, which may reduce free fatty acid concentrations, as well as the role of DF in promoting weight loss. High DF intakes did not have a significant effect on fibrinogen concentrations possibly because of relatively little weight loss, too low DF dosages and maintaining a good nutritional status. Inadequate study designs deterred from meaningful conclusions. Significant decreases in FVll coagulant activity (6-16%) were observed with DF intakes of ≥3.3 g/MJ and concomitant decreased saturated fat intakes and weight loss in healthy and hypertriglyceridaemic subjects. Confounding factors include weight loss and a simultaneous decreased intake of saturated fats. The type of fibre seems to play a role as well. Mechanisms through which DF may reduce FVll concentrations include its effects on triglyceride-rich lipoproteins, insulin and weight loss. Increased DF consumption with dosages ranging between 3.3-7.8 g/MJ were followed by significantly lower CRP concentrations (25-54%), however, simultaneous weight loss and altered fatty acid intakes were also present in all the studies. Mechanisms are inconclusive but may involve the effect of DF on weight loss, insulin, glucose, adiponectin, interleukin-6, free fatty acids and triglycerides. Conclusions: Epidemiological evidence indicates an association between DF and the CVD risk factors t-PA, PAI-1, FVII, fibrinogen and CRP. In general, the risk of CVD may improve with high-fibre intakes as indicated by the favourable changes in some of the parameters. However, simultaneous reduced fat intakes and weight loss presented difficulties in separating out the effects of specific components. Furthermore, DF is consumed in a variety of different forms and different dosages that may have different effects. Overall, the study designs used in the intervention trials prevented significant conclusions. DF did, however, play a role in modifying t-PA, PAI-1, FVII and CRP. Potential effects on fibrinogen were not quantifiable. Recommendations: The results from this investigation provide the motivation for additional controlled clinical research to establish the effect and mechanisms of DF on haemostatic variables and CRP. A critical aspect of future studies would be to set up suitable protocols. The amount of subjects, duration of the trials, confounding factors such as weight loss and altered fat intakes and differentiation between types and dosage of DF are important. DF supplemental studies are recommended as they may be the most suitable method to reach meaningful conclusions. / Thesis (Ph.D. (Nutrition))--North-West University, Potchefstroom Campus, 2007

Pang Petti. / "August 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.