Effects of Mutations Introduced Inside Or Outside the Reactive Centre Loops of Heparin Cofactor II and Alpha-1-proteinase Inhibitor on Function and Specificity
Library & Archives Canada, 2008 - 194 pages
Heparin cofactor II (HCII) and alpha-1-proteinase inhibitor (alpha 1-PI) are important inhibitors of plasma proteases, such as thrombin and neutrophil elastase. Both proteins belong to the family of serine protease inhibitors (serpins), and consist of 8-9 alpha-helices, three beta-sheets and a distinctive reactive centre loop (RCL) protruding from the serpin body. The RCL of serpins is an important determinant of protease specificity, particularly the P1-P1' reactive site bond. HCII is a specific thrombin inhibitor, even though thrombin prefers cleaving substrates with a P1 Arg and HCII has a Leu residue at this position. Rapid inhibition of thrombin by HCII is mainly due to allosteric activation of the serpin by glycosaminoglycans such as heparin and dermatan sulphate, leading to the release of its unique 75 residue N-terminal tail that is subsequently able to bind to thrombin exosite I. Unlike HCII, alpha 1-PI is a glycosaminoglycan-independent serpin that becomes an efficient thrombin inhibitor when its natural PI Met residue is replaced with an Arg. However, this mutation also increases the rate of activated protein C (APC) inhibition. Designing a glycosaminoglycan-independent serpin specific for thrombin has been suggested to be a desirable goal. Previously, researchers have attempted to increase the rate of thrombin inhibition by HCII by exchanging its P1 Leu residue with a P1 Arg. Although the variant exhibited an increased rate of thrombin inhibition in the absence of glycosaminoglycans, the thrombin-HCII complex was unstable in the presence of heparin. In this thesis, the effects of single and multiple residue RCL mutations were characterized in HCII, and the possibility of stabilizing the HCII L444R-IIa complex was examined by neutralizing the residues of the glycosaminoglycan-binding domain of the variant and therefore removing the need for glycosaminoglycan activation. The results with the twelve novel HCII variants described here suggest that replacing the RCL of HCII with that of either AT or alpha1-PI M358R converts the inhibitor into a substrate for thrombin, that replacing the P1 residue of HCII with a positively charged residue leads to serpin-enzyme complex instability in the presence of heparin and that replacing RCL residues adjacent to the P1 Arg leads to complex instability even in the absence of heparin, as does neutralizing the charged amino acids of the glycosaminoglycan-binding domain of HCII L444R. This latter finding suggests that converting HCII into an efficient thrombin inhibitor in the absence of heparin is a difficult goal to achieve. In order to build a glycosaminoglycan-independent serpin that is specific for thrombin, the 75 amino acid tail of HCII was appended to the N-terminus of alpha1-PI M358R. The rate of thrombin inhibition by the chimeric molecule (HAPI M358R) increased 21-fold relative to alpha1-PI M358R, whereas the rate of APC inhibition remained essentially unchanged. The thrombin specificity of HAPI M358R was further increased by exchanging its RCL with that of HCII or parts of AT. The resulting chimeric proteins were 2,000 to over 10,000 times more reactive with thrombin than with APC. Furthermore, they maintained their ability to inhibit thrombin in the presence of soluble fibrin or fibrin clots, they reduced thrombin clotting times more efficiently than unfused alpha1-PI variants, and they remained stable in mouse plasma both in vitro and in vivo. Together with the favourable kinetic data, these results suggest that further testing of these molecules in a thrombosis animal model is appropriate.
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