<p>Food allergies are a significant clinical problem, with symptoms including diarrhea, vomiting, or systemic anaphylaxis. To elicit allergic reactions, antigens must first cross the intestinal epithelium. The purpose of my studies was to examine macromolecular transport across intestinal epithelium, and the effect of sensitization and immune activation on transepithelial antigen transport. Rats were sensitized to a model protein antigen, horseradish peroxidase (HRP) by injection with adjuvants. Intestinal segments were removed and mounted in Ussing chambers for the study for transepithelial movement of HRP. Electon microscopy analysis of HRP transport showed that specifically sensitized rats transported HRP across the epithelium in greater amounts, and more rapidly, than naive controls or rats sensitized to an irrelevant antigen. After the hypersensitivity response, there was a significant increase in HR flux across the intestinal epithelium in HRP sensitized, but not control rats. This was accompanied by an opening of the epithelial tight junctions to allow paracellular flow of antigen. Sensitized mast cell deficient rats also had an enhanced initial uptake of antigen, but did not develop a non-specific decrease in epithelial barrier function. The role of interleukin-4 (IL-4) in the regulation of transepithelial antigen transport was examined. Treatment of human epithelial monolayers with IL-4, or with serum from atopic patients, caused a significant increase in transepithelial transport of HRP. Antibodies against IL-4 abolished the effect of atopic serum on transepithelial HRP transport. Electron microscopy analysis showed an increase in both transcellular and paracellular HRP transport. These studies show that transepithelial transport of antigen is profoundly altered by sensitization, and that mast cells and interleukin-4 enhance the delivery of antigen across the intestinal epithelium.</p> / Doctor of Philosophy (PhD)

<p>Cancer of the cervix is the second most common form of cancer in women worldwide. The natural history of cervical cancer is thought to involve sequential changes from varying grades of precursor intraepithelial lesions called CIN. However, little is known of the risk factors that can predict the oncogenic potential of a particular CIN lesion. Based on their preferential occurrence in cervical cancers and their potential oncogenic properties, the human papillomaviruses (HPV) particularly the genotypes 16, 18, 31, 33, 35 and 42 have been implicated in the etiology of invasive cervical cancer. However, these viruses could occur as either secondary pathogen of cancer or as predictor of those CIN lesions that are likely to progress to invasive disease. The hypothesis that was verified in this study was that HPV 16,33 and 18 are likely to be predictive of CIN lesions that progress to invasive cancer.</p> <p>First of all, in order to characterize the prevalent type of HPV in the target study population of B.C., a cross-sectional study was conducted and the presence of specific HPV types ascertained by the tissue in situ hybridization. The frequency of HPV types 16, or 33, was found to vary with the severity of the CIN grades, in contrast to the frequency of HPV types 6/11 and 18 that segregated independently of the CIN grades.</p> <p>Next, a case-control study was undertaken to verify the main hypothesis of HPV being predictive of CIN lesion progression to invasion. It was reasoned that the particular HPV would occur at higher frequency in CIN biopsies of cervical cancer cases than in CIN biopsies of noncases (controls). A total of 47 cases and 94 controls were enrolled from patients registered by the Cancer Control Agency of the Province of British Columbia. A case was defined as a post-pubertal woman with invasive disease and who had a CIN diagnosis at least two years prior to the invasive disease. For each case an attempt was made to enrol two control matched on grade of CIN and year of diagnosis. On each subject attempt was made to gather demographic informations that are known to be associated with cervical cancer. The HPV probes that were used included HPV 16/33 and HPV 6/11. The relative frequency of occurrence of specific HPV in the preinvasive biopsies of cases and controls were as folow: HPV 16/33 occurred in 10.6% of controls and 12.8% of cases; HPV 18 was found in 3.2% of controls and 8.7% of cases and HPV 6/11 in 2.2% of controls and 8.7% of cases. Conditional Chi-square analysis showed that the difference in the proportions of HPV positivity between cases and controls was compatible with sampling variation. Hence, with a statistical power approximately 60%, it was concluded that particular HPV could not be predictive of CIN lesions progression in the sample of population that was studied.</p> <p>However an excess risk for incurring cervical cancer, by being exposed to particular HPV at the CIN stage, was noted. The relative risk for HPV 16/33 was 2.34, [95% CI 0.70 to 7.66]; for HPV 18 was 2.45, [95% CI 0.22 to 27.80]; for HPV 6/11 2.19, [95% CI 0.39 to 12.42] or for all HPV combined was 1.87, [95% CI 0.55 to 6.28].</p> <p>Interestingly, a comparison of the frequency of HPV occurrences in the case-control study with that in the cross-sectional study revealed a lower rate of HPV positivity in the case-control component. This could possibly be due to a cohort effect.</p> / Doctor of Philosophy (PhD)

<p>Rat transferrin (RTf) was used to study the interaction of transferrin' (Tf) with the liver, with special reference to the çlycan of Tf.</p> <p>Iron uptake from Tf by cells is believed to occur by Tf receptor (TfR)-mediated endocytosis. Results from the present studies revealed that over a large range of competing diferric RTf concentrations, a constant percentage of iron is uptaken from RTf by the liver in vivo, and by hepatocytes in vitro. Hepatocytes were found to contain ~129,000 TfR/cell with ~40% expressed on the surface as estimated by both a polyclonal anti-TfR antiserum, produced as part of this thesis work, and a monoclonal anti-TfR antibody. On the basis of this estimate it was calculated that TfRs are not present in sufficient quantities to account for the observed uptake of iron. Studies of uptake and release of polyvinyl-pyrrolidone (PVP), RTf, and iron (as diferric Tf) were carried out in suspended hepatocytes. These studies demonstrated that the most likely mechanism to account for the results and to explain the iron uptake is "mixed-type" pinocytosis. The uptake of iron was found to be modulated by the type of glycan on RTf.</p> <p>On the basis of glycan microheterogeneity, at least six subforms of RTf are found to exist in rat plasma. These subforms are RTf-1, RTf-2·and RTf-3 as resolved by concanavalin A; ~20% of each is fucosylated and ~80% non-fucosylated. (The presence of fucose was found to have no measurable effect on catabolic rate, plasma iron disappearance or iron donation to liver in vivo or hepatocytes in vitro.) The slalylated subforms have different half-lives (RTf-1>RTf-2>RTf-3) with RTf-1 being significantly longer than RTf-3. Comparison of plasma iron disappearance and rates of iron donation to liver in suggested a trend (RTf-l>RTf-3>RTf-2) which was reproduced and found to be significant in studies with hepatocytes: iron uptake by hepatocytes from RTf-1 and RTf-3 could be competitively inhibited by an excess of the homologous subform. Desialylation of the subforms (RAsTfs) significantly reduced the half-lives and altered the order (RAsTf-3>RAsTf-1>RAsTf-2) with RAsTf-3 being significantly longer than RAsTf-2. The desialylated subforms were superior donors of iron to the liver in vivo. Studies to explain the enhanced rate of iron delivery by RAsTf, discounted the possibility of differing rates of iron release, but allowed postulation of a synergistic dual receptor mechanism. Results from experiments with hepatocytes in vitro supported the proposed mechanism. It is concluded that subtle differences in glycan structure can result in functional differences between Tf subforms.</p> / Doctor of Philosophy (PhD)

<p>The studies composing this thesis were designed to address the question of changes in muscle excitability during fatigue and recovery: the indirectly-evoked muscle compound action potential (M-wave) was used as an index of excitability. Earlier studies from this laboratory indicated that the rate of fatigue in human skeletal muscle depends on the frequency of excitation. The present studies have extended these findings by using a wide range of stimulating frequencies (0-30 Hz) and by comparing the change in muscle excitability in fast-versus slow-twitch muscles; the effect of ischaemia was also studied as was recovery from fatigue. Ten subjects (out of a total of fifteen) each successfully completed five experiments, spaced at least one week apart, in which intermittent tetanic trains at different frequencies were used to fatigue the ishaemic ankle dorsiflexors. The effects of ischemia were studied by repeating one experiment under non-ischaemic conditions. Five out of the ten subjects also volunteered for the experiments comparing changes in muscle excitability in soleus, versus those in tibialis anterior, in response to intermittent fatiguing stimulation.</p> <p>It was found that maintenance of excitability was possible for one minute regardless of stiumulus frequency; thereafter stimulation at the highest frequencies induced the greatest change in the amplitude of the (M-wave). The amplitude decline was also dependent on the position of the M-wave within the train of potentials: thus, at 30 Hz stimulation, the first, fourth and seventh responses within the trained decreased by 50%, 80% and 95% respectively (p<.01). The decline in M-wave amplitude was always greater than the decline in the area of the compound action potential, indicating an increase in duration due to dispersion of single fiber action potentials. At 30 Hz stimulation the areas of the first, fourth and seventh responses decreased by 33%, 56% and 82% respectively.</p> <p>On the basis of animal studies, it was hypothesized that muscle excitability would be preferentially retained in the soleus muscle; however, no significant differences emerged in M-wave changes between soleus and tibialis anterior although the onset of decline was delayed in soleus. It is proposed that this delay was due to the early potentiating mechanisms observed in soleus but not in tibialis anterior. The presence of ischaemia significantly (p<.01) accelerated the decline in both amplitude and area of the tibialis anterior M-wave. Recovery of the M-wave was limited when the tetanic stimulation ceased but progressed rapidly after the circulation was restored. M-wave failure occurred at firing rates not normally associated with neuromuscular blockade, implying propagation failure along the sarcolemmal membrane.</p> / Doctor of Philosophy (PhD)

<p>The studies that make up this thesis have been designed to describe and analyse the changes in muscle action potentials associated with muscular activity. The initial experiments were carried out in human muscle to examine changes in the muscle compound action potential (M-wave) during both stimulated and voluntary activity. It was found that the M-wave increased in amplitude either during or following either type of activity, and that this increase in M-wave amplitude could not be explained by either a movement artefact or a greater synchronization of action potential firing. It was hypothesized that the potentiation of the M-wave was due to a hyperpolarization of the muscle fibre membrane which was increasing the amplitude of the individual fibre action potentials. Experiments were then conducted on rat soleus muscle (in vivo) in order to measure changes in resting membrane and action potentials associated with contractile activity. After 5 min of intermittent tetanic stimulation at 20 Hz, the mean resting membrane potential increased from a baseline value of 79.5 ± 4.8 mV to a mean maximum of -92.6 ± 4.2 mV 3-6 min post-stimulation (p<.01). There was a corresponding increase in action potential amplitude; it rose from a resting value of 82.2 ± 10.8 mV to 98.1 ± 7.8 mV in the recovery period (p<.01). It was hypothesized that increased activity of the electrogenic Na⁺-K⁺ pump was causing the hyperpolarization. A series of experiments utilizing inhibitors of the Na⁺-K⁺ pump were then conducted; it was found that the administration of ouabain (1.25 x 10⁻⁴M), cooling the bathing medium (from 37°C to 19°C) or removal of extracellular K⁺ prevented the hyperpolarization following repetitive stimulation (p<.05). The magnitude of the electrogenic contribution of the Na⁺-K⁺ pump was then estimated by exposing stimulated muscle fibres to a high K⁺ (20 mM) medium. While in the control (unstimulated) condition this caused an immediate depolarization of the muscle fibre membrane to approximately -58 mV, stimulated fibres maintained membrane potentials of -79.5 mV (± 8.6 mV) for at least 3 min, which was approximately -30 mV greater than that predicted by the Goldman-Hodgkin-Katz (GHK) equation. It is concluded that the Na⁺-K⁺ pump plays an important role in maintaining muscle fibre excitability during muscular activity, which is additional to its role in the restoration and maintenance of ionic gradients for Na⁺ and K⁺. The temporary hyperpolarization of the muscle fibre membrane during increased Na⁺-K⁺ activity offers an explanation for the potentiation of muscle compound action potentials observed during voluntary and stimulated contractions.</p> / Doctor of Philosophy (PhD)

<p>Dexamethasone (DEX) treatment in very preterm infants has proven to facilitate earlier weaning from mechanical ventilation and supplemental oxygen, thereby lessening the severity of lung disease incurred by long-term oxygen dependency. However, DEX therapy is not without negative side-effects; studies in preterm infants and piglets have reported DEX-induced impairments in growth and bone mineral metabolism. DEX may act by altering the concentration or activity of specific components of the growth hormone (GH)/insulin-like growth factor (IGF-I) axis which are essential for regulating growth and bone mass. The first study, in preterm infants, characterized how DEX alters the circulating components of the GH-IGF-I axis and suggested potential mechanisms by which DEX delays growth and bone development as both plasma IGF-I and biochemical markers of bone metabolism were reduced during DEX. The objectives of the piglet studies were to delineate the effectiveness of adjunctive GH or GH+IGF-I to counter the detrimental effects of DEX on growth, protein turnover and bone mass. In the first studies, we administered GH, GH+IGF-I or placebo to piglets while they received a two week course of DEX. GH and GH+IGF-I partially attenuated the reductions in growth and bone mas to a similar extent. Only with respect to protein metabolism was an additional benefit observed with combined treatment (GH+IGF-I). A dose-response study revealed the minimal effective GH dose, and demonstrated that bone cell activity and weight and length gain returned to control levels during a period of rehabilitation in which no DEX or GH were administered. Currently, it is uncertain if DEX-treated infants experience similar metabolic improvements in weight and length growth or bone mineral mass post-DEX treatment or whether the metabolic insults of DEX are sustained. Longer term follow-up of DEX-treated preterm infants is required to fully comprehend to long-term consequences of DEX on growth into childhood. If there are long-term effects on growth and bone development, future studies should focus on whether GH is more effective post-DEX compared to during DEX treatment or whether adjunctive administration of other anabolic agents will counter the negative effects of DEX during development.</p> / Doctor of Philosophy (PhD)

<p>The gastrointestinal tract is particularly sensitive to stress. Stress-induced gastric ulceration and stress-induced alterations in motility have been examined. The purpose of my studies was to define stress-induced changes in the intestinal epithelium and the mechanisms involved in the epithelial responses. Wistar Kyoto rats were stressed by restraint. Jejunal and colonic tissues from stressed or control rats were removed and parametres of epithelial physiology were studied in Ussing chambers. Acute stress caused a significant increase in intestinal chloride ion secretion. In addition, tissues from stressed rats demonstrated impaired responses to neural activation. Compared to controls tissues from stressed rats were also found to have increased permeability to ions, and small inert probes, and increased permeability to a macromolecular protein. In spite of these functional abnormalities, the mucosa showed no signs of damange. Our next series of experiments showed that peripheral cholinergic nerves and corticotrophin releasing factor were responsible for mediating these epithelial responses to acute stress. Pretreatment with atropine (jejunum) or a corticotropin-releasing factor antagonist (colon) prevented the stress-induced pathophysiology, while administering corticotropin-releasing factor mimicked the colonic responses. These studies also showed that the Wistar Kyoto strain of rats developed more extreme intestinal abnormalities to stress than the parental Wistar strain, most likely due to the fact that Wistar Kyoto rats have less cholinesterase activity. In summary, my studies showed that stress impaired epithelial function along the intestinal tract. We speculate that in susceptible individuals, acute stress can cause the epithelial barrier to become leakly allowing greater uptake of small proinflammatory molecules (bacterial products) as well as larger macromolecules (antigens) from the lumen. Subsequent stimulation of immunocytes may initiate or exacerbate inflammation.</p> / Doctor of Philosophy (PhD)

<p>Roberts syndrome (RS) is a rare, recessive condition characterized by growth retardation, developmental delay and tetraphocomelia. Some RS patients (RS+), but not others (RS-), exhibit a "puffing" pf the constitutive heterochromatic regions of their chromosones (the "RS effect"). Cells from RS+ patients also show cellular hypersensitivity to DNA crosslinking agents such as mitomycin C (MMC). In the present study, correction of both the RS effect and MMC hypersensitivityin somatic cell hybrids between RS+ and normal lymphoblastoid cells (LCLs) supported the hypothesis of an association between the RS effect and mutagen sensitivity in RS+ cells. Somatic cell hybrids between two RS+ LCLs derived from patients with diverse ethnic backgrounds exhibited both the RS effect and MMC hypersensitivity indicating that these patients represent a single complementation group. Somatic cell hybrids between one of the RS+ and two different RS- LCLs demonstrated complete complementation of both the RS effect and MMC hypersensitivity indicating that these patiens represent a single complementation group. Somatic cell hybrids between one of the RS+ and two different RS- LCLs demonstrated complete complementation of both the RS effect and MMC hypersensitivity. These findings suggest that RS+ and RS- patients belong to different complementation groups.</p> <p>Fanconi anaemia (FA) is another rare, recessive disorder characterized by growth retardation, developmental delay, limb abnormalities and progressive pancytopenia. Cells from FA pateints exhibit both chromosomal and celluar hypersensitivity to DNA crosslinking agents. A study of the sensitivity and mutanility of various LCLs by ethyl methanesulphonate (EMS) indicated an increased cellular sensitivity and decreased mutability of RS+ LCLs relative to control LCLs. RS-LCLs did not exhibit these phenomena. One FA LCL from complementation group A showed a slightly increased cellular sensitivity but normal mutability. These results suggest hypomutability by EMS may be associated with the RS effect and MMC hypersensitivity.</p> <p>Somatic cell hybrids were made between one of the RS+ LCLs and an LCL from each of the four known FA complementation groups. Hybrids were examined for correction of MMC hypersensitivity, the RS effect and diepoxybutane-induced chromosome aberrations.</p> <p>Complementation was observed in hybrids with FA LCLs from complementation groups A,B and D but incomplete correction of chromosomal and cellular sensitivities to crosslinking agents in RS+ x FA C hybrids suggested a genetic association.</p> / Doctor of Philosophy (PhD)

<p>There is growing evidence that factors other than cell-surface recetors are involved in regulating the sensitivity of cells to external signals. In particular, G proteins have been implicated in the increased sensitivity of numerous receptor systems under a variety of conditions (Mishra et al., 1997). The goal of this research project was to determine the role of G proteins in mediating dopamine receptor supersensitivity in Parkinson's disease. Prelimary studies of G protein levels in human post-mortem brain tissue proved inconclusive, due to the limited availability and variability of tissue samples. Subsequent studies in the 6-hydroxydopamine (6-OHDA) rat lesion model of Parkinson's disease revealed that stimulatory G protein levels are persistently elevated following denervation (Marcotte et al., 1994). These G proteins are presumably coupled to dopamine D₁ receptors, which show clear evidence of supersensitivity despite apparently normal receptor levels. This result supports the hypothesis that G proteins are involved in the maintenance of dopamine receptor supersensitivity (Marcotte and Mishra, 1997). Stimulatory G proteins acutely following MPTP mouse model, with decreased stimulatory G proteins acutely following MPTP treatment, and increased stimulatory G proteins after long-term recovery (Marcotte et al., 1998a). Although the significance of these findings is unclear, they provide additional support for the hypothesis that G proteins are modulated in response to dopaminergic denervation. Attempts to measure functional changes in stimulatory G protein activity in the rat striatum proved unsuccessful, consistent with the available literature. Specifically, neither the GTPase nor a specific GTP binding assay was able to consistently detect stimulatory G protein activity following dopamine D₁ receptor stimulation. To provide direct evidence for the role of Golf in mediating dopamine receptor supersensitivity, Golf antisense oligonucleotides were administered to 6-OHDA lesioned rats. Intrastriatal infusion of Gold antisense, but not control sense oligonucleotides, specifically reduced apomorphine-induced rotational behaviour and Gold levels. The effects of Golf antisense infusion were at least partially reversible, supporting a specific antisense mechanism of action. However, one of the control oligonucleotides, Golf missense, consistently reduced rotational behaviour and G protein levels in a non-specific fashion. This effect was dose- and sequence-dependent, and may be due to a non-specific binding to other nucleotides or proteins (Marcotte and Mishra, 1998). Taken together, these studies support the hypothesis that stimulatory G proteins are involved in mediating dopamine D₁ receptor supersensitivity. Further characterization of the effects of in vivo antisense oligonucleotides may provide more definitive conclusions regarding the role of G proteins in mediating this phenomenon.</p> / Doctor of Philosophy (PhD)

<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)