<p>Antithrombin (AT) and heparin cofactor II (HCII) are the predominant inhibitors of thrombin in plasma. They belong to the se rine p[barbelow]rotease in hibitor, or serpin, family of proteins and they inhibit their target proteases through a mechanism that is unique to this family of molecules. AT and HCII provide an ideal substrate on the reactive centre loop and subsequently form 1:1 stoichiometric inhibitory complexes with their target proteases through a mechanism that results in major conformational changes in these serpins. While thrombin acts as a target protease with these serpins, neutrophil elastase (NE), another serine proteases, reacts with AT and HCII within the reactive centre loop. Unlike thrombin, however, the interaction of NE with AT and HCII results in cleavage and inactivation of these serpins, without forming inhibitory complexes with NE. The aim of this thesis was to analyze the effects of mutagenesis of the NE cleavage sites within AT and HCII to determine the limits of amino acid substitutions that would permit AT and HCII to retain function but be less susceptible to inactivation by NE. Analyses of proteins expressed in a cell-free expression system and then in COS-1 mammalian cell culture were used to study the effects of mutagenesis at P4 and then at P4 and P5 in rabbit and human AT. While charged and polar amino acid substitutions severely reduced the function of AT, substitution of the bulkiest residue, tryptophan, at P4 and then at P5 and P5 had minimal effects on the thrombin-inhibitory activity of AT. However, the susceptibility to NE cleavage did not appear to be affected by any substitutions that were made in AT. Analysis of amino acid substitutions in bacterially-derived HCII P6 variants demonstrated a similar flexibility in amino acid composition at the site of NE cleavage, with respect to the ability to inhibit thrombin, although substitution of polar and bulky residues within the reactive centre loop of this serpin increased its resistance to NE inactivation. These results demonstrate that the amino acid composition of the reactive centre loop of AT and HCII is flexible to allow the maintenance of thrombin-inhibitory activity, although some limitations do exist. The effects of residue substitutions within the reactive centre loop on the susceptibility to NE cleavage are less clear, but the presence of bulky amino acids at the primary NE cleavage site, at least in HCII, appears to reduce the proteolytic activity of this protease. The production of an NE-resistant thrombininhibitory serpin provides the basis for the possible development of "hardened serpins" which might be of therapeutic importance in the future.</p> / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/7392 |
| Date | 07 1900 |
| Creators | Cunningham, Andrew Michael |
| Contributors | Sheffield, W.P., Medical Sciences |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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