Intrinsic structural and conformational mechanisms regulate the functional specificity of the coagulation system. The study of these structure-function relationships is important for understanding the strategies used in the management of clinical thrombosis. Previous studies have shown that the central enzyme in clotting, thrombin, is sequestered inside of a clot, and protected from the natural downregulator antithrombin (AT). This is problematic for anticoagulants like heparin which depend on AT. Subsequently, it was found that the key upstream propagator of thrombin, the prothrombinase enzyme complex, is also resistant to the AT-heparin. Our data show that further upstream of prothrombinase, the intrinsic tenase is only moderately protected, while there is no protection at the level of the initiator complex, extrinsic tenase. This protection phenomenon possibly reflects steric and allosteric mechanisms that ensure maximal activation of the coagulation system once a threshold stimulus is achieved. These mechanisms likely evolved as a result of conformational rearrangement, as evidenced by the proteolytic activation of thrombin activity following proteolysis of prothrombin. Indeed, subtle differences in the structural interaction of ligands with the active site can lead to substantial differences in enzyme activity. The binding of rivaroxaban and apixaban to factor Xa is nearly identical; both interact with the active site with comparable affinity. Despite this, a 3-fold faster rate of the rivaroxaban on-rate yields significantly greater prolongation of the prothrombin time (PT) and activated partial thromboplastin time (aPTT), global tests of coagulation. These small differences in ligand interaction also have allosteric consequences. Structural differences between the direct thrombin inhibitors dabigatran and argatroban yield divergent exosite-mediated thrombin binding to physiologic ligands like yA-fibrin, y'-fibrin, factor Va, and factor VIII, interactions that govern clot-mediated protection from AT inhibition, and the various functions of thrombin. These divergent effects were robust and ligand-dependent, suggesting conserved energetic scaffolds within the thrombin molecule that govern allosteric changes throughout the molecule. Because proteolysis of prothrombin yields significant allosteric and structural rearrangement that capacitates the active site for substrate recognition amd catalytic ability, we investigated the role of Ser195, a key residue in the thrombin catalytic triad in also regulating thrombin allostery. Site directed mutagenesis of Ser195 to Ala yielded a significant increase in the flexibility of the entire thrombin molecule, as evidenced by increased potency of dabigatran and argatroban in terms of their capacity to modulate exosite binding through the active site, and increased interexosite cooperative and competitive allostery. Together, these studies represent an advance in our understanding of the consequences of both small molecule ligation of coagulation proteases, as well as the consequences of subtle structural modification for overall allosteric function. / Thesis / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/19502 |
| Date | January 2016 |
| Creators | Yeh, Calvin Hsiung |
| Contributors | Weitz, Jeffrey I., Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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