The regulation of coagulation is central to many diseases, including heart disease and stroke. The objective of this research is to better understand the different mechanisms by which the serpins antithrombin (AT) and protein C inhibitor (PCI) inhibit free thrombin and thrombin bound to thrombomodulin (TM). Specifically the role of the H-helix, and other predicted contact points between the serpin and TM will be investigated. Previously we made the observation that, unlike AT, PCI is a potent inhibitor of thrombin bound to TM. The heparin binding domains of PCI and AT also differ; in AT the D-helix is a major part of the heparin-binding domain, while the H-helix is the heparin-binding domain in PCI. Alignment of the sequences of PCI, AT and heparin cofactor II (HCII) suggests that AT is unique in having a negatively charged H-helix, while the other serpins have positively charged helices. In recent work we demonstrated that changing the charge of the H-helix of AT makes it behave more like PCI in inhibiting thrombin bound to either heparin or thrombomodulin. The crystal structure of thrombin complexed with TM has recently been solved. This structure was used to generate a molecular model of AT complexed with thrombin and TM to explain kinetic data design new experiments.
The first aim of this proposal is to continue to explore the roles of the D and H helices of AT in the inhibition of thrombin in the presence and absence of TM or heparin. The molecular model of a complex between thrombin, TM and AT revealed several other potential contact points between AT and TM, which would not be present between PCI and TM. In addition, the amino terminus of AT contains several more amino acids than does PCI, forming a loop which appears to sterically interfere with TM bound to thrombin.
The second aim i s to explore the importance of this loop by removing amino acid residues from the center of the loop and assaying the ability of these mutants to inhibit thrombin bound to TM. Another contact point in the complex is between AT residues R259 to R262 and three negatively charged amino acids on TM (E357, D398 and EH00).
The third aim i s to change these residues and measure the impact on inhibition of thrombin bound to TM The outcome of these experiments will provide a clearer understanding of the different mechanisms by which PCI and AT inhibit thrombin complexed with TM.
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| Fortenberry, Y M; Whinna, H C; Cooper, S T et al. (2007) Essential thrombin residues for inhibition by protein C inhibitor with the cofactors heparin and thrombomodulin. J Thromb Haemost 5:1486-92 |
| Rehault, Sophie M; Zechmeister-Machhart, Margareta; Fortenberry, Yolanda M et al. (2005) Characterization of recombinant human protein C inhibitor expressed in Escherichia coli. Biochim Biophys Acta 1748:57-65 |
| Yang, Likui; Manithody, Chandrashekhara; Walston, Timothy D et al. (2003) Thrombomodulin enhances the reactivity of thrombin with protein C inhibitor by providing both a binding site for the serpin and allosterically modulating the activity of thrombin. J Biol Chem 278:37465-70 |
