<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)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/7236 |
| Date | 12 1900 |
| Creators | Wells, John Michael |
| Contributors | Blajchman, Morris A., Science |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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