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<p>Antithrombin (AT)-mediated inhibition of thrombin is important in the maintenance of hemostasis. This importance is emphasised by the fact that people with AT deficiencies are at greater risk of developing thrombophilia. AT inhibits thrombin by forming a covalent 1:1 stoichiometric thrombin-antithrombin complex (TAT) and such formed complexes rapidly are removed from the circulation by hepatic receptors. The main aim of my doctoral thesis project has been to identify and characterize these hepatic receptors. Competitive radioligand binding experiments demonstrated a low-affinity 125 I-TAT binding site on hepatic membranes. Ligand-blotting on rabbit liver plasma membranes was used to identify TAT-binding polypeptide(s). These experiments showed that 125 I-TAT interacted specifically with a 45 kDa protein which was identified as cytokeratin 18 (CK18) by amino acid sequencing. The biological relevance of this unusual interaction was verified by the presence of CK18 on the surface of rat and human hepatoma cells and the ability of anti-CK18 IgG, but not preimmune IgG, to inhibit TAT binding and internalization by these cells. Finally, the increased binding of 131 I-anti-CK18 IgG over 125 I-preimmune IgG to perfused rabbit livers supported the possibility that CK18 is expressed on the surface of hepatocytes in vivo. As a whole, these data indicate a novel biological role for cytokeratins as cellular receptors. The secondary aim of my thesis was to examine the metabolism of TAT when contained in a ternary complex with vitronectin (VN-TAT). Plasma clearance experiments revealed that VN-TAT was removed rapidly from the circulation by hepatic binding sites. Binding to these sites was found to be heparin-sensitive such that either heparin or protamine sulfate extends the VN-TAT clearance time (t½α ) ten to fifteen fold. Similarly, in vitro radioligand binding studies on hepatoma cells indicate that VN-TAT binds to low affinity heparinoid sites. Furthermore, heparin greatly reduced the internalization and degradation of VN-TAT by HepG2 cells. These data demonstrate for the first time that VN-TAT, at least partially, is cleared by hepatic sites which are most likely heparan sulfate in nature.</p> / Doctor of Philosophy (PhD)

<p>Antithrombin is a plasma serine proteinase inhibitor functioning physiologically as an anticoagulant. It inactivates thrombin and FXa by forming a 1:1 covalent complex between its P1 Arg and the catalytic serine of the proteinase. Antithrombin is a relatively inefficient inhibitor until it is activated by two specific glycosaminoglycans, heparin and heparan sulphate with the unique pentasaccharide sequence required for high affinity binding to antithrombin. Activation of antithrombin by the pentasaccharide and full-length heparin induces major conformational changes in the antithrombin molecule. The pentasaccharide binding site of antithrombin has been mapped to the D-helix, the A-helix, and the N-terminus of the molecule. Arg47 is located where the base of the A-helix is in close proximity to the amino end of the D-helix. To characterize the role of Arg47 in antithrombin binding to and its activation by the pentasaccharide, antithrombin moieties with substitutions at Arg47 were created and expressed in transfected COS-1 cells. Our data suggest that a positively charged amino acid at position 47 is essential for heparin and the pentasaccharide to bind to AT with high affinity and the pentasaccharide- and heparin-accelerated inactivation of FXa. In addition, an Arg is preferred to Lys for activation by the pentasaccharide with respect to FXa inhibition. Antithrombin interacts with target proteinases primarily through its P1-P1' residues together with flanking binding subsites in the reactive centre loop. Heparin activates antithrombin through inducing a major conformational change in antithrombin which optimizes the presentation of the reactive centre loop to the target proteinases. While the pentasaccharide is sufficient for enhancing the rate of FXa inhibition, it has little effect on thrombin inactivation. Full-length heparin, on the other hand, enhances the rates of antithrombin inhibition of both FXa and thrombin. As demonstrated by the coincident 40% increase in endogenous Trp fluorescence with plasma antithrombin, the pentasaccharide and full-length heparin were reported to induce similar conformational changes in AT. The large rate enhancement of thrombin inhibition by full-length heparin was thus attributed solely to the ability of longer chains to accommodate AT and thrombin simultaneously. Based on the fact that substrate recognition of thrombin and FXa is different, it was hypothesized that the pentasaccharide and full-length heparin induce different reactive centre loop conformations. A Pro397Trp antithrombin moiety thus was generated in transfected CHO cells. We report here, for the first time, that the pentasaccharide and full-length heparin induce different conformational changes in the reactive centre loop. It thus appears that the pentasaccharide-induced reactive centre loop conformation represents a near optimal substrate to FXa, whereas the heparin-induced RCL conformation is apparently required for near optimal interaction with thrombin.</p> / Doctor of Philosophy (PhD)

Thesis (Ph. D.)--University of North Carolina at Greensboro, 2006. / Title from PDF title page screen. Advisor: Catherine Matthews; submitted to the School of Education. Includes bibliographical references (p. 237-250).

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xi, 276 p. Includes abstract and vita. Advisor: Paul E. Post, College of Education. Includes bibliographical references (p. 263-276).

Thesis (Ph. D.)--University of Oregon, 2003. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 253-263). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--University of Oklahoma, 1996. / Includes bibliographical references.

Thesis (MEd (Mathematics & Science))--University of South Australia, 1994

Thesis (MEd)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.