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The treatment of acute myocardial infarction (AMI) has undergone a tremendous development during the last decades, and the most important factor is probably the introduction of reperfusion therapy aimed at preventing or limiting the myocardial injury. It is of vital importance that patients with AMI are adequately monitored regarding the development of ECG changes during and after treatment to identify successful or failed reperfusion and to detect episodes of recurrent ischemia. Vectorcardiography (VCG) is one method for this purpose. This series of studies was aimed at evaluating VCG as a method for detecting reperfusion and recurrent ischemia in patients with ST-elevation AMI who were treated with different reperfusion strategies. Specific changes in the VCG during the initial treatment phase, “reperfusion peaks,” were examined in detail. The influence of the fibrinolytic system and von Willebrand factor (vWF) on successful reperfusion and subsequent AMI and death after thrombolytic treatment with streptokinase (SK) was another main objective. From the data in these studies it can be concluded that: VCG is a relevant and easily used method for ischemia-monitoring in patients with AMI. A specific sign, the reperfusion peak, is associated with vectorcardiographic signs of reperfusion. This sign is observed both in patients treated with primary coronary angioplasty and in those who are treated with fibrinolytic agents. The reperfusion peak is associated with successful reperfusion and with larger infarcts, but by itself, the parameter has little prognostic significance. The recognition of the reperfusion peak is important since it can mimic severe ischemia. In an unfortunate situation the incorrect interpretation of the VCG could lead to premature treatment decisions that might even be harmful to the patient. Streptokinase treatment of patients with AMI induced profound changes in the fibrinolytic system and vWF. A high tissue plasminogen activator (tPA) activity level (>25 U/mL) early after the start of treatment, reflecting the fibrinolytic activity obtained by the given drug, was associated with successful reperfusion. Pre-existing neutralizing antibodies to SK were found to varying degrees in the previously SK-treated patients. No association between SK-neutralizing antibodies and the result of the treatment regarding successful reperfusion as judged by VCG was seen. Pre-treatment levels of tPA activity, PAI-1 activity, PAI-1 mass-concentration and vWF had no correlation with the success of reperfusion therapy with SK or on the incidence of recurrent ischemia during the first 24 hours. Recurrent ischemia, however, was shown to be an independent risk factor for death within the first 1 year. Elevated levels of PAI-1 mass-concentration, and to some extent PAI-1 activity, after the start of SK treatment, were associated with a higher risk for death at one year, though not at five years.

Multiple interacting mechanisms control the formation and dissolution of clots to maintain blood in a state of delicate balance. In addition to a myriad of biochemical reactions, rheological factors also play a crucial role in modulating the response of blood to external stimuli. The broad stimuli for clot formation were laid out, more than a century ago, in, what is now referred to as, Virchow’s triad. To date, a comprehensive model for clot formation and dissolution, that takes into account the biochemical, medical and rheological factors, has not been put into place, the existing models emphasizing either one or the other of the factors. In this dissertation, a model is developed for clot formation and dissolution that incorporates many of the relevant crucial factors that have a bearing on the problem. The model, though just a first step towards understanding a complex phenomenon goes further than previous models in integrating the biochemical, medical and rheological factors that come into play. The model is tested in some simple flow situations as part of an attempt to elucidate Virchow’s triad. Extensions to the model, along with detailed numerical studies, will hopefully aid in a clearer understanding of the phenomenon, and in making relevant clinical correlations.

Thesis (Ph. D.)--North Carolina State University. / Includes vita. Includes bibliographical references.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that acts as a molecular link between the coagulation and fibrinolytic cascades. TAFI can be activated by thrombin and plasmin but the reaction is enhanced significantly when thrombin is in a complex with the endothelial cofactor thrombomodulin (TM). The in vitro properties of TAFI have been extensively characterized. Activated TAFI (TAFIa) is a thermally unstable enzyme that attenuates fibrinolysis by catalyzing the removal of basic residues from partially degraded fibrin. The in vivo role of the TAFI pathway, however, is poorly defined and very little is known about the role of different activators in regulating the TAFI pathway. In the present study, we have constructed and characterized various TAFI mutants that are resistant to activation by specific activators. Based on peptide sequence studies, these mutants were constructed by altering key amino acid residues surrounding the scissile R92-A93 bond. We measured the thermal stabilities of all our mutants and found them to be similar to wild type TAFI. We have identified that the TAFI mutants P91S, R92K, and S90P are impaired in activation by thrombin or thrombin-TM, thrombin alone, and thrombin alone or plasmin, respectively. The TAFI mutants A93V and S94V were predicted to be resistant to activation by plasmin but this was not observed. The triple mutant, DVV was not activated by any of the aforementioned activators. Finally, we have used in vitro fibrin clot lysis assays to evaluate the antifibrinolytic potential of our variants and were able to correlate their effectiveness with their respective activation kinetics. In summary, we have developed activation resistant TAFI variants that can potentially be used to explore the role of the TAFI pathway in vivo. / Thesis (Master, Biochemistry) -- Queen's University, 2009-01-30 11:44:37.191

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like pro-enzyme that, once activated, attenuates fibrinolysis. TAFIa also possesses anti-inflammatory properties. Although liver is the main source of plasma TAFI, platelet-derived TAFI has also been reported. An alternatively spliced TAFI variant resulted from the skipping of exon 6 and a 52-base deletion in exon 10 of CPB2 mRNA (∆6+10) was described to be brain specific. This TAFI variant is reputed to possess a secretase-like activity that cleaves β-amyloid precursor protein to form β-amyloid, a process involved in the onset of Alzheimer's disease. In this thesis, we report the identification of CPB2 mRNA and TAFI protein in various vascular and inflammatory cells. Specifically, we describe the expression of CPB2 mRNA in the megakaryocytic cell lines MEG-01 and Dami, the monocytic cell line THP-1, and peripheral blood mononuclear cells. TAFI protein was detected in differentiated Dami and THP-1 cells. We next describe the effect of external stimuli such as phorbol myristate acetate (PMA) on CPB2 expression in Dami and THP-1 cells. We found that PMA treatment increases both CPB2 mRNA abundance and promoter activity in Dami cells, and decreases both CPB2 mRNA abundance and promoter activity in THP-1 cells. Deletion analysis of the CPB2 promoter indicated cell-type specific regulation of CPB2 gene expression. Finally, we evaluated the expression of alternatively spliced CPB2 mRNA variants in hepatic and non hepatic cells. We found that exon 6 skipping variants are expressed in all cell types of interest. The variant previously reported to be brain specific was also found to be expressed in platelets. We found that the alternatively spliced TAFI variants accumulated inside the cells in a non-secretable, hypoglycosylated form and showed no carboxypeptidase activity. Taken together, this thesis provides further evidence supporting the hypothesis that platelet-derived TAFI is originated from CPB2 gene expression in megakaryocytes. Moreover, our data imply a potential for site-specific anti-inflammatory control provided by macrophage-derived TAFI. Alternative splicing of the CPB2 mRNA may give rise to variants with an intracellular role, perhaps as a peptidase chaperone, and may modulate the synthesis of secretable TAFI. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-09-26 21:22:33.348

Autotransporters represent a large superfamily of known and putative virulence factors produced by Gram-negative bacteria. They consist of an N-terminal “passenger domain” responsible for the specific effector functions of the molecule and a C-terminal “β domain” responsible for translocation of the passenger across the bacterial outer membrane. The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent those autotransporters produced by Enterobacteriaceae where, as the name suggests, the passenger domain functions as a serine protease. Members of this family of autotransporters include among others the extracellular serine protease EspP produced by enterohemorrhagic Escherichia coli (EHEC) O157:H7. EHEC, especially those of serotype O157:H7, have been implicated as causative agents of hemorrhagic colitis and hemolytic-uremic syndrome, both of which include disruption of the normal processes in human blood responsible for maintaining good health. EspP has previously been shown to cleave human coagulation factors V and VIII and has been hypothesized to possibly contribute to the mucosal hemorrhage in patients infected with EHEC. This thesis aims to better understand the functional significance of EspP in EHEC pathogenesis by analyzing the crystallographic structure of the mature passenger domain of EspP and by investigating, in vitro, its effects on the coagulation and fibrinolytic processes in human blood. Like the previously determined autotransporter passenger domains, the EspP passenger domain is found to contain an extended right-handed parallel β-helix preceded by an N-terminal globular domain housing the catalytic function of the protease. Of note, however, is the absence of a second globular domain protruding from this β-helix. Furthermore, EspP is found to alter hemostasis in vitro by drastically decreasing the activities of human blood coagulation factors V, VII, VIII and XII, by enhancing platelet-fibrin clot formation, and by accelerating fibrinolysis. These results provide compelling evidence for a pathogenic role played by EspP during EHEC infection.

Autotransporters represent a large superfamily of known and putative virulence factors produced by Gram-negative bacteria. They consist of an N-terminal “passenger domain” responsible for the specific effector functions of the molecule and a C-terminal “β domain” responsible for translocation of the passenger across the bacterial outer membrane. The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent those autotransporters produced by Enterobacteriaceae where, as the name suggests, the passenger domain functions as a serine protease. Members of this family of autotransporters include among others the extracellular serine protease EspP produced by enterohemorrhagic Escherichia coli (EHEC) O157:H7. EHEC, especially those of serotype O157:H7, have been implicated as causative agents of hemorrhagic colitis and hemolytic-uremic syndrome, both of which include disruption of the normal processes in human blood responsible for maintaining good health. EspP has previously been shown to cleave human coagulation factors V and VIII and has been hypothesized to possibly contribute to the mucosal hemorrhage in patients infected with EHEC. This thesis aims to better understand the functional significance of EspP in EHEC pathogenesis by analyzing the crystallographic structure of the mature passenger domain of EspP and by investigating, in vitro, its effects on the coagulation and fibrinolytic processes in human blood. Like the previously determined autotransporter passenger domains, the EspP passenger domain is found to contain an extended right-handed parallel β-helix preceded by an N-terminal globular domain housing the catalytic function of the protease. Of note, however, is the absence of a second globular domain protruding from this β-helix. Furthermore, EspP is found to alter hemostasis in vitro by drastically decreasing the activities of human blood coagulation factors V, VII, VIII and XII, by enhancing platelet-fibrin clot formation, and by accelerating fibrinolysis. These results provide compelling evidence for a pathogenic role played by EspP during EHEC infection.

Thesis (doctoral)--Universiteit van Amsterdam.

Les microparticules sont des vésicules extracellulaires qui résultent du remodelage des phospholipides membranaires en réponse à une activation ou une apoptose. La vision initiale leur attribuant une activité uniquement procoagulante s’avère plus complexe, par la mise en évidence d'une activité plasminogénolytique portée par les MPs endothéliales, tumorales et leucocytaires (MPLs). Au cours de ce travail, nous avons démontré un nouveau mécanisme impliquant les MPLs comme des vecteurs d’une activité fibrinolytique capable de lyser un thrombus fibrino-plaquettaire en fonction de leur activité de génération de plasmine. Cette activité s’intègre dans un rôle protecteur des MPs capable de contrebalancer le risque de microthromboses associé au choc septique. Cette activité fibrinolytique identifie les MPs comme des biomarqueurs déterminants pour le pronostic vital mais aussi comme des cibles thérapeutiques potentielles, pouvant être à l’origine de biothérapies vésiculaires basées sur la génération de MPs fibrinolytiques de grade clinique. L’évaluation du bénéfice apporté par les MPs est actuellement limitée par un manque de standardisation. Dans une seconde partie de ce travail, nous avons proposé une nouvelle stratégie de standardisation de la cytométrie en flux. Cette stratégie a été évaluée dans le cadre d'une étude multicentrique internationale qui a montré pour la première fois l’absence de différence significative des numérations des MPs entre des instruments de configuration optique différente. Maitriser des protocoles standardisés est une étape indispensable pour accélérer le transfert de ces innovations diagnostiques et thérapeutiques au lit du patient. / The microparticles are extracellular vesicles resulting from the remodeling of membrane phospholipids in response to activation or apoptosis. The initial vision only assigning a procoagulant activity is more complex, by highlighting a range plasminogenolytic activity by endothelial, tumor and leukocyte (MPLS) MPs. In this work, we demonstrated a novel mechanism involving MPLS as vectors fibrinolytic activity able to lyse a fibrin-platelet thrombus on the basis of generation of plasmin activity. This activity is part of a protective role of MPs which may counterbalance the risk of microthromboses associated with septic shock. This fibrinolytic activity identifies MPs as key biomarkers for prognosis but also as potential therapeutic targets that can be the source of vesicular biotherapies based on generation of fibrinolytic MPs of clinical grade. The evaluation of the benefit provided by MPs is currently limited by a lack of standardization. In the second part of this work, we proposed a new strategy for standardization of flow cytometry. This strategy was evaluated in a multicenter international study that showed for the first time no significant difference in MPs counts between different optical configuration tools. To master standardized protocols is a necessary step to improve the transfer of these diagnostic and therapeutic innovations to the patient.