<p>Thrombin is a multifunctional serine protease that plays a central role in hemostasis. Unlike related serine proteases of the hemostatic system, thrombin is unique in that is has both procoagulant and anticoagulant activities. Structural features defined by X-ray crystallographic studies of thrombin provide a molecular basis for the enzyme's specificity. These features include the active site cleft and two anion-binding electropositive exosites located on opposite poles of the thrombin molecule. What is less evident from crystallographic studies is the thrombin's flexibility and its capacity to undergo conformational changes upon ligand binding to the exosites. These studies were undertaken to explore different but interrelated aspects of thrombin regulation. The first goal of this thesis was to determine how prothrombin fragment 2 (F2), a prothrombin activation fragment, binds to thrombin and modulates its activity. Cocrystallographic studies have shown that the interaction F2 with thrombin involves the formation of salt bridges between the kringle inner loop of F2 and anion-binding exosite II of thrombin. When F2 binds to thrombin, it has been shown to evoke conformational changes at the active site and at exosite I of the enzyme. Using plasma, recombinant, and synthetic F2 peptides (F2, rF2, and sF2, respectively) we have further localized the thrombin binding domain on F2. F2, rF2(1-116), rF2(55-116), and sF2(63-116), all of which contain the kringle inner loop (residues 64-93) and the acidic C-terminal connecting peptide (residues 94-116), bind to thrombin-agarose. In contrast, analogues of the kringle inner loop, sF2(63-90), or the C-terminal connecting peptide, sF2(92-116), do not bind. Thus, contrary to predictions from the crystal structure, the C-terminal connecting peptide as well as the kringle inner loop are involved in the interaction of F2 with thrombin. F2 and sF2(63-116) bind saturably to fluorescently labelled-active-site-blocked-thrombin with Kd values of 4.1 and 51.3 μM, respectively. The affinity of sF2(63-116) for thrombin increases about 5-fold (kd=10 μM) when Val at position 78 is substituted with Glu. F2 and sF2(63-116) bind to exosite II on thrombin because both reduce the heparin-caalyzed rate of thrombin inhibition by antithrombin - 4-fold. In contrast, only F2 slows the uncatalyzed rate of thrombin inactivation by antithrombin. Like F2, sF2(63-116) induces allosteric changes in the active site and exosite I of thrombin because it alters the rates of thrombin-mediated hydrolysis of chromogenic substrates and displaces fluorescently-labelled hirudin₅₄₋₆₅ from active-site-blocked thrombin, respectively. Both peptides also prolong the thrombin clotting time of fibrinogen in a concentration-dependent fashion reflecting their effects on the active site and/or exosite I. The different functional changes evoked by F2 and sF2(63-116) likely reflect additional contacts if F2 relative t the smaller sF2(63-116) and suggest that ligand binding to various subdomainds within exosite II may have different effects on thrombin function. The important implication of these findings is that distinct allosteric effects evoked by ligand binding to subdomainds of exosites may contribute to the diversity of thrombin function at the molecular level. The activity of thrombin is also regulated by blood-borne protease inhibitors. The second goal of this work was to gain insight into the mechanism by which thrombin is inactivated by heparin cofactor II (HCII), a serine protease inhibitor (serpin) in plasma that selectively inhibits thrombin in a reaction that is accelerated ≥1000-fold by glycosaminoglycans (GAGs) such as dermatan sulfate (DS) and heparin. Current thinking is that GAG binding to HCII disrupts ionic bonds between the amino-terminal acidic domain and the GAG-binding domain of HCII, thereby permitting the acidic domain to interact with exosite I on the thrombin. Based on this allosteric activation model, we predicted that substitution of basic residues in the GAG-binding domain of HCII with neutral ones would mimic the catalytic effect of GAGs. Compared with wild-type recombinant HCII expressed in BHK cells (wt rHCII), mutation of Arg¹⁸⁴, Lys¹⁸⁵, Arg¹⁹², Arg¹⁹³ (Mut C) or Arg¹⁸⁴, Lys¹⁸⁵, Arg¹⁸⁹, Arg¹⁹², Arg¹⁹³ (Mut D) reduced the affinity for heparin-Sepharose and increased the uncatalyzed rate of thrombin inactivation ~130-fold (from 4.6 x 10⁴ M⁻¹ min⁻¹ to 6.2 x 10⁶ and 6.0 x 10⁶ M⁻¹ min ⁻¹, respectively). Furthermore, unlike wt rHCII or plasma-derived HCII (pHCII), neither heparin nor dermatan sulfate increased the rate of thrombin inhibition by Mut C or Mut D. The increased basal rate of thrombin inhibition by these mutants reflects displacement of their amino-terminal acidic domainds because (a) they inhibit ϒ-thrombin at a 65-fold slower rate than α-thrombin, (b) the exosite 1-binding fragment hirudin-(54-65) decreases the rate of thrombin inhibition, and (c) deletion of the amino-terminal acidic domain (-del74) of Mut D reduces the rate of thrombin inhibition ~ 100-fold. To determine whether GAG-mediated bridging of thrombin to HCII contributes to accelerated thrombin inhibition, we compared the catalytic effects of longer heparin or dermatan sulfate chains with those of shorter chains. Heparin chains comprised of 30 or more saccharide units produced an ~5-fold greater increase in the rate of thrombin inhibition by pHCII, wt rHCII, and wt-del74 than heparin chains comprised of 20 or fewer saccharide units. In contrast, dermatan sulfate and a low molecular weight fragment of dermatan sulfate stimulated thrombin inhibition by pHCII and wt rHCII to the same extent, and neither agent affected the rate of thrombin inhibition by wt-del74. Our findings support the concept that heparin and dermatan sulfate activate HCII by releasing the acidic amino-terminal domain from intramolecular connections with the GAG-binding domain. Since both GAGs produce ≥ 1000-fold increase in the rate of thrombin inhibition by HCII, our observation that only heparin serves as a template raises the possibility that dermatan sulfate induces more extensive allosteric changes than heparin. In addition to regulation by serpins, thrombin function is also modulated by its incorporation into forming thrombi. Despite being catalytically active, fibrin-bound thrombin is protected from inactivation by inhibitors, notably antithrombin (AT)/heparin. The resistance of fibrin-bound thrombin to inactivation by AT is thought to reflect formation of a productive ternary thrombin-fibrin-heparin complex in which thrombin is protected from inactivation by AT. The anchoring of thrombin in a productive ternary complex is mediated by thrombin's exosites, fibrin via exosites I and heparin via exosite II. It has been proposed that productive ternary complex assembly is dependent on binary interactions between thrombin-heparin, thrombin-fibrin, and heparin-fibrin. Unlike heparin, DS inhibitors soluble and fibrin-bound thrombin equally well, however the explanation for this phenomenon is unclear. The third goal of this work was to determine why fibrin-bound thrombin is susceptible to inactivation by the HCII/DS complex but not by the AT/heparin complex. The results of this study indicate that, unlike heparin, DS does not promote the formation of a productive ternary thrombin-fibrin-DS complex. This concept is supported by three lines of evidence. First, in the presence of fibrin monomer (Fm), thrombin is protected from inhibition by HCII/heparin, but not by HCII/DS as quantified by protease inhibition assays under pseudo first-order conditions. Second, DS does not promote the binding of radiolabeled active site-blocked thrombin (¹²⁵I-FPR-thrombin) to fibrin. In contrast, heparin augments ¹²⁵I-FPR-thrombin binding to fibrin in a concentration-dependent manner. Third, DS does not interact with fibrin and binds to thrombin with a 22-fold lower affinity than heparin (Kd values of 2.6 μM and 117 nM, respectively). These results reveal that, although exosite I and exosite II of thrombin can be ligated by fibrin and DS, respectively, productive ternary complex does not occur because DS is unable to bridge thrombin to fibrin. These findings indicate that all three binary interactions are essential for productive ternary complex formation. We also examined the protective effect of the thrombin-fibrin-heparin complex on thrombin inhibition by Mut D. Whereas Fm alone has little effect on the uncatalyzed rate of thrombin inhibition by Mut D, addition of heparin decreases the rate of thrombin inhibition by Mut D ~ 30 fold (from 6.0 x 10⁶ M⁻¹ min⁻¹ to 2.1 M⁻¹ min⁻¹). Furthermore, in the presence of Fm, heparin causes a dose-dependent decrease in the DS-catalyzed rate of thrombin inhibition by HCII. These observations reveal that the protective effect of heparin results from the anchorin of thrombin in a productive thrombin-fibrin-heparin complex in which exosite I is inaccessible to the amino-terminus of HCII. Collectively, these studies illustrate different modes of regulating thrombin function, all of which are intricately interrelated. The remarkable diversity of thrombin activity allows thrombin to serve multiple functions in highly controlled processes in hemostasis.</p> / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/6840 |
| Date | 09 1900 |
| Creators | Liaw, Patricia C. Y. |
| Contributors | Weitz, Jeffrey I., Medical Sciences |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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