Platelets play an essential role in haemostasis by adhering to the damaged vessel wall and forming a platelet plug to arrest bleeding. Although platelets are traditionally thought of as pro-coagulant, they possess the ability to harbour functional proteins that are key to fibrinolysis, the breakdown of the blood clot, on their surface. They are therefore substantially well equipped to regulate local fibrinolysis. This thesis aims to further define the role of platelets in fibrinolysis, in particular platelet-derived plasminogen activator inhibitor 1 (PAI-1) and plasminogen. PAI-1 is the principal physiological inhibitor of tissue-type plasminogen activator (tPA), and plasminogen is the zymogen for plasmin. In Chapter 3, we show that platelet-derived PAI-1 is released from platelet α-granules by an αIIbβ3 and fibrin dependent mechanism. We found that a significant portion of α-granular PAI1 is retained on the surface of highly activated PS-positive platelets, and activity analysis revealed the majority of PAI-1 on the platelet surface was in its active form. The functional role of platelet PAI-1 was investigated by analysis of tPA-mediated lysis of Chandler model thrombi. Our data revealed a striking dependence for platelet PAI-1 in stabilising platelet-rich thrombi against degradation. Chapter 4 characterises the expression of a novel transmembrane receptor, Plg-RKT, on the surface of human and mouse platelets. This revealed that plasminogen and Plg-RKT augment one another's binding to the platelet surface. Furthermore, analysis of plasminogen binding to the platelet surface revealed two distinct binding sites: 1) via Plg-RKT and 2) via a fibrin and αIIbβ3 dependent mechanism. Finally, Chapter 5 of this thesis discusses the optimisation of a system that monitors thrombus formation and fibrinolysis under flow. Use of this model will help to further elucidate the complex role that platelets play in controlling the balance between coagulation and fibrinolysis.

Investigations of specific and nonspecific interactions of biomolecules at liquid/solid interfaces are presented. To investigate specific multivalent ligand-receptor interactions, bivalent antibodies and haptens bound to solid supported membrane were used as models for ligand-receptor coupling. Novel microfabrication strategies, which included spatially addressed bilayer arrays and heterogeneous microfluidic assays, in conjunction with total internal reflection microscopy, was employed to achieve this goal. These high throughput techniques allow thermodynamic data of binding interactions to be acquired with only a few microliters of analyte and superior signal to noise. The results yield both the first and second dissociation constant for bivalent IgG antibodies with membrane bound hapten molecules. Studies were conducted both as a function of hapten density and cholesterol content in the membrane. Another research area of this dissertation is the molecular level description of nonspecific adsorption and displacement of the model protein, fibrinogen, onto hydrophilic surfaces. Techniques such as atomic force microscopy, immunochemical assays, fluorescence microscopy, and vibrational sum frequency spectroscopy were employed to probe this system. The results demonstrate that the protein's αC domains play the critical role. When fibrinogen is adsorbed to a hydrophilic surface via these moieties, its displacement rate in the presence of human plasma is approximately 170 times faster than when these domains are not in direct surface contact. Even more significantly, spectroscopic studies show evidence for highly aligned Arg and Lys residues interacting with the negatively charged substrate only when the αC domains make direct surface contact. The interfacial ordering of these residues appears to be the hallmark of a weak and labile electrostatic attraction between the substrate and the adsorbed macromolecule.

It is widely accepted that the initial event that significantly influences biocompatibility is the nearly instantaneous adsorption of proteins from biological fluids onto the biomaterial surface. For blood-contacting devices, the complex layer of adsorbed plasma proteins is generally unfavourable and leads to major complications, including thrombus formation, inflammatory tissue responses, and microbial infections. Furthermore, protein interaction with passive films on metallic biomaterial surfaces may contribute to enhanced in vivo corrosion. To gain a better understanding of this phenomenon, the present thesis investigated the fundamental aspects of the interaction of the serum protein fibrinogen with a medical-grade stainless steel 316LVM surface using electrochemical and IR spectroscopy techniques. Aspects of this interaction included the thermodynamics and kinetics of fibrinogen adsorption, the effect of fibrinogen adsorption on the corrosion behavior of 316LVM stainless steel, and the conformational changes of fibrinogen upon its adsorption onto the stainless steel surface. / It was shown that fibrinogen readily adsorbs onto the 316LVM stainless steel surface. Increases in the bulk protein concentration resulted in a corresponding increase of the surface coverage, a dependence that was described by the Langmuir isotherm. Large, negative values of the calculated Gibbs energy of adsorption indicated a highly spontaneous and strong adsorption of fibrinogen onto the 316LVM stainless steel at all investigated temperatures. Although the adsorption process was shown to be endothermic under the applied experimental conditions, the primary driving force for the adsorption process was found to be the positive entropy gain that arises from structural loss and/or rearrangement of the protein upon adsorption, as well as dehydration of the protein and stainless steel surface during the adsorption process. Kinetic measurements indicated that fibrinogen adsorption occurs rapidly. / It was determined that for short contact times (1 hour), the addition of fibrinogen to the electrolyte enhanced the corrosion rate of the 316LVM stainless steel at the open circuit potential. For longer contact times (24 hours), an increase in the polarization resistance values was obtained, indicating an enhanced corrosion resistance of the material. It was postulated that the protein was not capable of complexing the well-stabilized passive film, and instead remained adsorbed to form a protective barrier to diffusion of oxygen-containing species from the electrolyte to the stainless steel surface. / The secondary structure of the surface-adsorbed fibrinogen molecules was investigated by modeling the experimental PM-IRRAS spectra. It was shown that the protein lost a certain extent of its secondary structure upon adsorption to the steel surface. Fibrinogen molecules adsorbed from more dilute solutions were also shown to possess a lower alpha-helical content than those adsorbed from more concentrated solutions, suggesting they laid on the stainless steel surface in a more linear configuration.

The multi-functional FGL2 can be expressed as either a type II membrane-associated glycoprotein or a secreted tetrameric molecule. As an important effector of regulatory T cells, secreted FGL2 inhibits dendritic cell maturation and T cell proliferation. The mechanism of its immunomodulatory function remains unclear. The goals of this thesis are to identify receptor(s) of secreted FGL2, key biological functions and signaling pathways, and mechanism of FGL2 oligomerization. Soluble FGL2 was critical for all studies, and the production of recombinant FGL2 was compared in E. coli, insect cells and mammalian cells. Soluble and stable FGL2 was secreted only by mammalian cells, indicating the importance of post-translational modification. In flow cytometry and surface plasmon resonance assays, recombinant FcFGL2 and albumin tagged FGL2 fusion proteins bound to Fc gamma RIIB and Fc gamma RIII receptors expressed by antigen presenting cells (APCs), including lipopolysaccharide (LPS)-stimulated B lymphocytes, endothelial cells, thioglycollate-stimulated peritoneal macrophages, and bone marrow-derived dendritic cells (BM-DCs). The binding of recombinant FGL2 to Fc gamma RIIB and Fc gamma RIII was specific, dependent on receptor expression and blocked by anti-Fc gamma RIIB/III antibody. FcFGL2 inhibited the maturation of BM-DC derived from fc gamma riib wild type mice but not from fc gamma riib knock out mice. It also induced apoptosis of the A20 mouse B cell line (Fc gammaRIIB+), but not the A20IIA1.6 cell line (Fc gamma RIIB-). The activation of caspases induced by FcFGL2 binding to A20 cells was confirmed by flow cytometry, Western blotting and analysis of DNA fragmentation. The role of Fc gammaRIIB in FGL2-mediated immunosuppression was confirmed in vivo. Infusion of FcFGL2 into fc gamma riib+/+, but not fc gamma riib-/- C57BL/6J mice (H-2b) inhibited the rejection of fully mismatched BALB/cJ (H-2d) skin and heart allografts. Studies on the mechanism of FGL2 oligomerization employed site-directed mutagenesis and revealed that cysteines at positions 94, 97, 184, and 187 were critical. Mutation of these cysteines resulted in secretion of monomeric FGL2. Computer modeling of FGL2 tetramers predicted an asymmetric arrangement that was similar to the structure of multimeric ficolin. The data presented in this thesis provide mechanistic insights into the immunosuppressive activity of soluble FGL2, and a foundation for the development of a novel and potentially highly effective immunosuppressive therapy.

The multi-functional FGL2 can be expressed as either a type II membrane-associated glycoprotein or a secreted tetrameric molecule. As an important effector of regulatory T cells, secreted FGL2 inhibits dendritic cell maturation and T cell proliferation. The mechanism of its immunomodulatory function remains unclear. The goals of this thesis are to identify receptor(s) of secreted FGL2, key biological functions and signaling pathways, and mechanism of FGL2 oligomerization. Soluble FGL2 was critical for all studies, and the production of recombinant FGL2 was compared in E. coli, insect cells and mammalian cells. Soluble and stable FGL2 was secreted only by mammalian cells, indicating the importance of post-translational modification. In flow cytometry and surface plasmon resonance assays, recombinant FcFGL2 and albumin tagged FGL2 fusion proteins bound to Fc gamma RIIB and Fc gamma RIII receptors expressed by antigen presenting cells (APCs), including lipopolysaccharide (LPS)-stimulated B lymphocytes, endothelial cells, thioglycollate-stimulated peritoneal macrophages, and bone marrow-derived dendritic cells (BM-DCs). The binding of recombinant FGL2 to Fc gamma RIIB and Fc gamma RIII was specific, dependent on receptor expression and blocked by anti-Fc gamma RIIB/III antibody. FcFGL2 inhibited the maturation of BM-DC derived from fc gamma riib wild type mice but not from fc gamma riib knock out mice. It also induced apoptosis of the A20 mouse B cell line (Fc gammaRIIB+), but not the A20IIA1.6 cell line (Fc gamma RIIB-). The activation of caspases induced by FcFGL2 binding to A20 cells was confirmed by flow cytometry, Western blotting and analysis of DNA fragmentation. The role of Fc gammaRIIB in FGL2-mediated immunosuppression was confirmed in vivo. Infusion of FcFGL2 into fc gamma riib+/+, but not fc gamma riib-/- C57BL/6J mice (H-2b) inhibited the rejection of fully mismatched BALB/cJ (H-2d) skin and heart allografts. Studies on the mechanism of FGL2 oligomerization employed site-directed mutagenesis and revealed that cysteines at positions 94, 97, 184, and 187 were critical. Mutation of these cysteines resulted in secretion of monomeric FGL2. Computer modeling of FGL2 tetramers predicted an asymmetric arrangement that was similar to the structure of multimeric ficolin. The data presented in this thesis provide mechanistic insights into the immunosuppressive activity of soluble FGL2, and a foundation for the development of a novel and potentially highly effective immunosuppressive therapy.

Thesis (Ph. D.)--Auburn University. / Abstract. Vita. Includes bibliographical references (p. 76-102).

Thesis (M.Sc. (Tropical Medicine))--Mahidol University, 1971.

Thesis (Ph. D.)--University of Nevada, Reno, 2005. / Includes bibliographical references. Online version available on the World Wide Web.

Bacteroides fragilis is a member of the normal microbiota that resides in the human lower gastrointestinal tract. This bacterium is of clinical significance because it is the most frequently isolated Gram-negative obligate anaerobe from peritoneal abscesses and bloodstream infections. Human fibrinogen is a hexameric-glycoprotein that is important for fibrin-mediated abscess formation and limiting the spread of infection. B. fragilis can bind and degrade fibrinogen which may aid in its escape from abscesses into the bloodstream, thereby promoting bacteraemia. In addition to fibrinogen, binding of B. fragilis to fibronectin, a component of the extracellular matrix, found in association with fibrinogen at wound sites, has also been reported. An outer membrane protein, BF1705, expressed by B. fragilis was found to share homology with BspA from Tannerella forsythia which is known to bind fibrinogen. The gene encoding BF1705 was deleted from the B. fragilis NCTC 9343 genome in the present work using a markerless gene deletion technology. Proteins derived from the outer membranes of wild-type B. fragilis were able to bind fibronectin and fibrinogen in far-western blots. Similar protein extracts from the ΔBF1705 strain did not bind fibrinogen and fibronectin, which confirms the role of BF1705 in adhesive interactions with proteins of the host extracellular matrix. The possible involvement of BF1705 in fibrinogen degradation was ruled out because the ΔBF1705 strain still degraded fibrinogen. To identify the proteases involved in degradation of fibrinogen, four genes encoding putative extracellular metallo- and serine proteases in the size range 45-50 kDa were deleted from the NCTC 9343 genome. All of the single and multiple mutants defective in these selected proteases were still capable of degrading fibrinogen as determined by zymography. Expression of eight B. fragilis proteases in E. coli did not lead to detectable degradation of fibrinogen. These observations suggest that these proteases alone cannot degrade fibrinogen and either that an unidentified protease is responsible for degradation or that there is redundancy in the proteases involved. Under conditions of nitrogen limitation bacteria resort to scavenging nitrogen from the environment to replenish the depleting intracellular nitrogen content. By examining the differential regulation of the B. fragilis transcriptome under nitrogen replete and depleting conditions, a potential role for BF1705 and secreted proteases in nutrient binding and assimilation were studied. Growth on conventional glucose defined medium with ammonia as the nitrogen source was compared to growth in defined medium with glutamine as nitrogen source. A reduced doubling time and diauxic growth in the medium containing glutamine indicated nitrogen limitation. Comparison of the transcriptome derived from cultures of B. fragilis grown on either ammonia or glutamine by RNA-Seq did not reveal a significant upregulation of BF1705 in response to nitrogen limitation. This observation in conjunction with its inability to degrade fibrinogen suggests that the primary role of BF1705 might be as an adhesin and does not act directly in nutrient binding and degradation. Nevertheless, nitrogen limitation was found to induce the expression of four protease-encoding genes by over a 2-fold (adjusted p value < 0.05). The molecular weight of three of these proteases were identified to be within the size range of 45-55 kDa which corresponded to the lysis bands detected by fibrinogen zymography with wild-type B. fragilis protein extracts. Therefore the possible involvement of these three proteases in fibrinogen degradation could be assessed. A 155-fold upregulation (adjusted p value < 0.05) in asnB, encoding a homologue of asparagine synthetase B, under conditions of nitrogen limitation suggest a previously uncharacterised aspartate metabolism pathway for ammonia generation via arginine catabolism in B. fragilis. Ammonia thus formed might aid in sustaining B. fragilis growth under nitrogen deprived conditions. In addition to nitrogen assimilation, significant upregulation was observed in the expression of genes involved in regulation of oxidative stress and metronidazole resistance. The observed changes in the transcriptome will add to our understanding of the B. fragilis metabolism and potential assist with unravelling the mechanisms of infection mediated by this important opportunistic pathogen.

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