Thrombin-activatable Fibrinolysis Inhibitor (TAFI) is a 60 kDa plasma protein that can be activated to the enzyme, TAFIa, by thrombin, plasmin or trypsin. TAFIa is a carboxypeptidase B-like enzyme that attenuates fibrinolysis. Thrombomodulin (TM) is a cofactor which increases the overall efficiency of thrombin-mediated TAFI activation by 1250-fold. Thus, the thrombin-TM complex is believed to be the physiological TAFI activator. The minimal structure of TM required for efficient TAFI activation contains the EGF-like domains 3 through 6. New structure models have postulated that the C-loop of TM EGF-like domain 3 has a negatively charged molecular surface that could interact with several positively charged surface patches on TAFI. In this study, we constructed recombinant TAFI variants to assess whether the selected positively charged residues on TAFI complement the negative electrostatic potential of the TM EGF-like domain 3, thereby promoting the TAFI-TM interaction in the formation of the ternary thrombin/TM/TAFI complex. TAFI has exclusive triple lysine residues on its activation peptide. When they are substituted by alanine residues (K42/43/44A), compared to the wild-type, the catalytic efficiencies for TAFI activation by thrombin in the presence and absence of TM decreased by factors of 9 and 3.5, respectively. Other derivatives of TAFI with alanine point mutations at positions K133, K211, K212, and R220, which together represent one positively charged surface patch of TAFI, showed decreased catalytic efficiencies for TAFI activation by thrombin-TM complex from 2.4 to 2.9-fold. A second positive surface patch includes residues K240 and R275. Alanine mutations of these two residues caused decreased catalytic efficiencies by 1.7 and 2.1-fold, respectively. Together, our data show that no single mutation completely eliminates TM dependence in TAFI activation by thrombin, but each mutated residue contributes in the formation of the ternary thrombin/TM/TAFI complex. In addition, all TAFIa derivatives had half lives (8.1 ± 0.6 min) comparable to that of wild-type TAFIa (8.4 ± 0.3 min) at 37 ºC, suggesting that these residues are not involved in TAFIa inactivation by conformational instability. / Thesis (Master, Biochemistry) -- Queen's University, 2008-01-31 15:10:19.209

Atherosclerosis is a chronic inflammatory condition of larger arteries characterized by build-up of plaques in the vessel wall. Disruption of plaques results in superimposed thrombus formation, or atherothrombosis, leading to the occlusion of the blood vessels. Atherosclerosis is the primary cause of heart attacks and strokes. Recent studies show that various fibrinolytic factors influence atherosclerosis progression. A healthy fibrinolytic system is fundamental to mitigating the worsening of the lesion and atherosclerosis progression by rapidly removing unwanted microthrombi that is otherwise incorporated into the plaque. In this regard, the activated thrombin-activable fibrinolysis inhibitor (TAFIa), a potent antifibrinolytic metallocarboxypeptidase that attenuates clot dissolution, could influence atherosclerosis progression. Aside from its antifibrinolytic property, TAFIa also affects inflammation by inactivating anaphylatoxins and kinins. Therefore, TAFIa possesses the potential to influence atherosclerosis via antifibrinolytic and anti-inflammatory facets. We utilized mice deficient in apolipoprotein E (ApoE-/-) as a model of atherosclerosis. Characterization of murine TAFIa determined that the functional half-lives at 37 and 25 ˚C were about 4.0 and 12.7 minutes, respectively. TAFIa was stable indefinitely at 0 ˚C. In vitro clot lysis assays were performed on the plasmas of C57BL/6J (wild-type) and ApoE-/- mice, aged 5, 10, 15 and 30+ weeks. No differences in clot lysis times were observed between the two genotypic classifications. TAFIa was directly quantified using our in-house assay, which showed decreased TAFIa levels in the ApoE-/- mice. Closer examination revealed that the plasma from the ApoE-/- mice was negatively influencing the TAFIa assay. Total TAFI zymogen levels in these samples appeared to increase with age. Other biomarkers of inflammation were quantified using ELISAs; however, only IL-10 levels were measurable with slight elevation in the ApoE-/- mice across age, while IL-1beta, TNF-alpha, and IL-6 was unquantifiable. Overall, TAFIa does not appear to influence atherosclerosis progression in ApoE-/- mice. / Thesis / Master of Science (MSc) / Atherosclerosis is an inflammatory disease of large blood vessels that leads to unwanted clot formation on these weakened/compromised vessel walls. Eventually, the blood flow is hindered by these clots and the supply of oxygen and nutrients to vital organs are disrupted, leading to irreparable damage and death of these organs. As such, it, is the primary cause of heart attacks and strokes depending on the location of vessel occlusion. Thrombin activable fibrinolysis inhibitor (TAFI), when activated to TAFIa, contains both anti-inflammatory and antifibrinolytic properties. Therefore, it is likely that TAFI(a) would be involved in atherosclerosis development and/or progression, especially since other fibrinolytic factors have shown to be involved. Despite promising preliminary data, we found that TAFI(a) alone may minimally be involved in the overall atherosclerosis progression and development using a mouse model of atherosclerosis. We further investigate whether TAFI(a) can be used as a biomarker for early diagnosis of atherosclerosis.

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

The formation of the Salmonella extracellular matrix is a multicellular behavior important for environmental persistence. It is comprised of uniquely but ill-defined assembled thin aggregative fimbriae (Tafi), cellulose and uncharacterized polysaccharides. Consequently, investigations were launched into further clarifying Tafi assembly and the polysaccharide constituents of the extracellular matrix. In the Salmonella agfBAC Tafi operon, the transcription and role of agfC has been elusive. In this study using the clinical isolate, Salmonella Enteritidis 27655-3b, agfBAC transcripts were detected using a reverse transcriptase and transcription was not enhanced by replacement of a stem-loop structure immediately preceding agfC. AgfChis was purified, localized to the periplasm, and found to specifically bind noncrystalline cellulose suggesting an association with the extracellular matrix. An inframe ΔagfC mutant displayed an abundance of 20 nm fibers, which could be complemented with agfC in trans, in addition to Tafi and an increase in cell hydrophobicity. Depolymerization of purified 20 nm fibers required exceptionally stringent conditions to release what proved to be AgfA subunits revealing the 20 nm fibers as AgfA assemblages of unique morphology. The role of AgfC in Tafi assembly was investigated further via a novel, quantitative antibody-capture assay of in-frame agf mutants. A soluble antibody-accessible form of AgfA was captured in wt, ΔagfB and ΔagfF strains in support of the extracellular nucleation-precipitation pathway of Tafi assembly, but not in ΔagfC or ΔagfE mutants. These results suggest that AgfC and AgfE are required for AgfA’s extracellular assembly and thus may act as atypical AgfAspecific chaperones which facilitate Tafi assembly. The implications of these results are presented in an assembly model for Tafi. Additional investigations revealed that Salmonella produces an O-Antigen capsule co-regulated with the extracellular matrix. Structural analysis of purified extracellular polysaccharides (EPS) yielded a repeating oligosaccharide unit similar to iv that of lipopolysaccharide O-Antigen with modifications. Putative carbohydrate transport and regulatory operons important for capsule expression, designated emcA-H and emcIJ, were identified by screening a random transposon library with immune serum generated to the capsule. The absence of capsule was confirmed by generating various in-frame Δemc mutants where emcG and emcE were shown to be important in capsule assembly and translocation. Luciferase-based expression studies showed that, AgfD differentially regulated the emc operons in coordination with extracellular matrix genes. Survival assays demonstrated the capsule is important for desiccation tolerance. The emc genes were found to be conserved in Salmonellae and thus, the O-Antigen extracellular matrix capsule may be a conserved survival strategy important for environmental persistence. Finally, a compositionally unique acidic EPS was found associated with the extracellular matrix. In-frame ΔbcsA, ΔemcG and ΔagfA mutants but neither ΔagfAΔbcsA nor ΔagfD mutants bound calcofluor, a β-glucan binding fluorescent agent, suggesting that multicellular behavior itself and not necessarily AgfD alone was influencing EPS expression. A transposon library was screened by ELISA using serum generated against purified EPS. This identified mutations inactivating genes involved in quorum sensing AI-2 degradation, flagella repression and Tafi and TolA expression. All mutations resulted in the loss of multicellular behavior and immunologically decreased levels of Tafi. This is the first report that implicates quorum sensing AI-2 degradation and flagella repression as part of the regulatory circuit for Tafi expression. Together, the results reveal Tafi uses assembly factors to facilitate extracellular polymerization which likely assists the formation of a network of branched, amorphous fimbriae. Tafi together with EPS form the extracellular matrix: Tafi stabilizes the EPS on the microbial communities; EPS imparts it with physical properties such as hydration, charge and diffusion barriers that protect it from adverse environmental conditions such as desiccation and antimicrobials. This probably contributes to Salmonella survival in the environment and facilitates its cyclic lifestyle.

L'efficacité du rtPA dans le traitement de l’ischémie aigue est bien reconnue avec des effets secondaires graves nécessitant l’évaluation d’autres stratégies. Un modèle d’ischémie cérébrale focale a été décrit, réalisé par injection in situ de thrombine. Dans notre première étude, nous avons utilisé l’imagerie par résonance magnétique multimodale pour documenter les lésions et les zones de pénombre dans ce modèle. Malgré une occlusion de l’artère reproductible et une hypoperfusion marquée chez tous les sujets, une reperfusion spontanée est constatée dans 38% des cas, rendant l’IRM incontournable dans l’évaluation de ce modèle. La deuxième étude a comparé l'efficacité d’un TAFI inhibiteur seul ou en combinaison avec le rtPA à faible dose. Nous avons montré que la combinaison du TAFI inhibiteur avec le rtPA à faible dose n'est pas aussi efficace que la dose standard de rtPA, avec une tendance positive, tandis que le TAFI inhibiteur seul n'est pas efficace du tout. Le modèle thromboembolique présente un intérêt particulier dans l'évaluation des stratégies thérapeutiques associées au rtPA pour améliorer la thrombolyse, surtout lorsqu'il est évalué par un suivi longitudinal en IRM / The benefit of recombinant tissue plasminogen activator (rtPA) treatment in stroke is well known with serious side effects requiring the evaluation of alternative strategies. Injection of thrombin in the middle cerebral artery of mice has been proposed as a new model of thromboembolic stroke. In the first study, we used multiparametric Magnetic Resonance Imaging (MRI), performed immediately after thrombin injection, to document occlusion and area at risk in this model. Despite similar MCA occlusion and marked hypoperfusion, half of animals showed a cortical lesion on DWI, while the other half demonstrated no or very limited lesion. Therefore, MRI measurement of basal lesion size is required to use this animal model in therapeutic studies. The second study compared efficacy between TAFI inhibitor alone and TAFI inhibitor in combination with low-dose rtPA. In conclusion, we showed that the combination of TAFI-I with low-dose rtPA is not as effective as the standard dose of rtPA, with a positive trend, while TAFI inhibition alone is not effective at all. The present thromboembolic model is of particular interest in assessing strategies rtPA association to improve thrombolysis, especially when coupled with longitudinal MRI assessment