A Quantitative Investigation of Selected Reactions in the Fibrinolytic Cascade

Previous work has shown that thrombin activatable fibrinolysis inhibitor (TAFI) was unable to prolong lysis of purified clots in the presence of Lys-plasminogen (Lys-Pg), indicating a possible mechanism for fibrinolysis to circumvent prolongation mediated by activated TAFI (TAFIa). Therefore, the effects of TAFIa on Lys-Pg activation and Lys-plasmin (Lys-Pn) inhibition by antiplasmin (AP) were quantitatively investigated using a fluorescently labeled recombinant Pg mutant which does not produce active Pn. High molecular weight fibrin degradation products (HMW-FDPs), a soluble fibrin surrogate that models Pn modified fibrin, treated with TAFIa decreased the catalytic efficiency (kcat/Km) of 5IAF-Glu-Pg cleavage by 417-fold and of 5IAF-Lys-Pg cleavage by 55-fold. A previously devised intact clot system was used to measure the apparent second order rate constant (k2) for Pn inhibition by AP over time. While TAFIa was able to abolish the protection associated with Pn modified fibrin in clots formed with Glu-Pg, it was not able to abolish the protection in clots formed with Lys-Pg. However, TAFIa was still able to prolong the lysis of clots formed with Lys-Pg. TAFIa prolongs clot lysis by removing the positive feedback loop for Pn generation. The effect of TAFIa modification of the HMW-FDPs on the rate of tissue type plasminogen activator (tPA) inhibition by plasminogen activator inhibitor type 1 (PAI-1) was investigated using a previously devised end point assay. HMW-FDPs decreased the k2 for tPA inhibition rate by 3-fold. Thus, HMW-FDPs protect tPA from PAI-1. TAFIa treatment of the HMW-FDPs resulted in no change in protection. Vitronectin also did not appreciably affect tPA inhibition by PAI-1. Pg, in conjunction with HMW-FDPs, decreased the k2 for tPA inhibition by 30-fold. Hence, Pg, when bound to HMW-FDPs, protects tPA by an additional 10-fold. TAFIa treatment of the HMW-FDPs completely removed this additional protection provided by Pg. In conclusion, an additional mechanism was identified whereby TAFIa can prolong clot lysis by increasing the rate of tPA inhibition by PAI-1 by eliminating the protective effects of Pn-modified fibrin and Pg. Because TAFIa can suppress Lys-Pg activation but cannot attenuate Lys-Pn inhibition by AP, the Glu- to Lys-Pg/Pn conversion is able to act as a fibrinolytic switch to ultimately lyse the clot.

Regulation of the Gene Encoding Thrombin-Activable Fibrinolysis Inhibitor

Disequilibrium between coagulation and fibrinolysis can lead to severe haemostatic disorders such as thrombosis and hemophilia. Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like pro-enzyme that, once activated, attenuates fibrinolysis. TAFI may also mediate connections between coagulation and inflammation. Studies have associated high plasma TAFI levels with risk for thrombotic diseases. Interestingly, steroid hormones, such as estrogen and progestogens used in hormone replacement therapy or oral contraceptive preparations, have been shown to affect plasma TAFI levels. Regulation of the expression of the gene encoding TAFI, CBP2, is likely an important determinant of the role of the TAFI pathway in vivo; this concept motivated the investigations described in this thesis. In Chapter 2, the results of my research lead to the identification of key transcription factors regulating CPB2. Specifically, we described the binding of NF-Y and HNF-1 to the CPB2 promoter. NF-Y was shown to be an important factor for the basal CPB2 promoter activity. Binding of HNF-1 is essential for the activity of the promoter and is potentially responsible for the liver specific expression of CPB2. In Chapter 3, we set to investigate the effect of female sex hormone on hepatic expression of CPB2. We demonstrated that the levels of TAFI protein secreted from cultured hepatoma cells (HepG2) are decreased by 17beta-estradiol and progesterone. The change in protein expression was paralleled by decreases in CPB2 mRNA abundance and promoter activity. Deletion analysis of the CPB2 promoter indicated that the genomic effects of estrogen and progesterone are likely mediated via a non-classical mechanism. In Chapter 4, we evaluated the effects of various inflammatory mediators on expression of the gene encoding mouse TAFI (Cpb2). Our results showed that Cpb2 mRNA abundance and promoter activity are up-regulated by inflammatory mediators IL-1beta, IL-6, and TNFalpha. We also showed that TNFalpha mediates its effect via the binding of NFkB. Additionally, our results suggest that TNFalpha promotes the binding of NFkB to the promoter by increasing its translocation to the nucleus. The NFkB site is not conserved between human and mouse and may explained the different responses to inflammation observed in vivo.

A Quantitative and Mechanistic Assessment of Activated Thrombin-Activatable Fibrinolysis Inhibitor and its Role in Pathological Bleeding and Thrombosis

The coagulation and fibrinolytic systems are linked by the thrombin-thrombomodulin complex which regulates each system through activation of protein C and TAFI, respectively. We have used novel assays and techniques to study the enzymology and biochemistry of TAFI and TAFIa, to measure TAFI activation in hemophilia A and protein C deficiency and to determine if enhancing TAFI activation can improve hemostasis in hemophilic plasma and whole blood. We show that TAFIa not TAFI attenuates fibrinolysis in vitro and this is supported by a relatively high catalytic efficiency (16.41μM-1s-1) of plasminogen binding site removal from fibrin degradation products (FDPs) by TAFIa. Since the catalytic efficiency of TAFIa in removing these sites is ~60-fold higher than that for inflammatory mediators such as bradykinin it is likely that FDPs are a physiological substrate of TAFIa. The high catalytic efficiency is primarily a result of a low Km which can be explained by a novel mechanism where TAFIa forms a binary complex with plasminogen and is recruited to the surface of FDPs. The low Km also suggests that TAFIa would effectively cleave lysines from FDPs during the early stages of fibrinolysis (i.e. at low concentrations of FDPs). Since individuals with hemophilia suffer from premature fibrinolysis as a result of insufficient TAFI activation we quantified TAFI activation in whole blood from hemophilic subjects. Both the rate of activation and the area under the TAFI activation time course (termed TAFIa potential) was determined to be reduced in hemophilia A and the TAFIa potential was significantly and inversely correlated with the clinical bleeding iii phenotype. Using a novel therapeutic strategy, we used soluble thrombomodulin to increase TAFI activation which improved the clot lysis time in factor VIII deficient human plasma and hemophilic dog plasma as well as hemophilic dog blood. Finally, we briefly show in a biochemical case study that TAFI activation is enhanced in protein C deficiency and when afflicted individuals are placed on Warfarin anticoagulant therapy, TAFI activation is reduced. Since TAFIa stabilizes blood clots, this suggests that reducing TAFI activation or inhibiting TAFIa may help restore blood flow in vessels with pathological thrombosis.

Identification of Genes Involved in the C. elegans VAB-1 Eph Receptor Tyrosine Kinase Signaling Pathway

The generation of a functional nervous system requires that neuronal cells and axons navigate precisely to their appropriate targets. The Eph Receptor Tyrosine Kinases (RTKs) and their ephrin ligands have emerged as one of the important guidance cues for neuronal and axon navigation. However, the molecular mechanisms of how Eph RTKs regulate these processes are still incomplete. The purpose of this work was to contribute to the understanding of how Eph receptors regulate axon guidance by identifying and characterizing components of the Caenorhabditis elegans Eph RTK (VAB-1) signaling pathway. To achieve this objective I utilized a hyper active form of the VAB-1 Eph RTK (MYR-VAB-1) that caused penetrant axon guidance defects in the PLM mechanosensory neurons, and screened for suppressors of the MYR-VAB-1 phenotype. Through a candidate gene approach, I identified the adaptor NCK-1 as a downstream effector of VAB-1. Molecular and genetic analysis revealed that the nck-1 gene encodes for two isoforms (NCK-1A and NCK-1B) that share similar expression patterns in parts of the nervous system, but also have independent expression patterns in other tissues. Genetic rescue experiments showed that both NCK-1 isoforms can function in axon guidance, but each isoform also has specific functions. In vitro binding assays showed that NCK-1 binds to VAB-1 in a kinase dependent manner. In addition to NCK-1, WSP-1/N-WASP was also identified as an effector of VAB-1 signaling. Phenotypic analysis showed that nck-1 and wsp-1 mutants had PLM axon over extension defects similar to vab-1 animals. Furthermore, VAB-1, NCK-1 and WSP-1 formed a complex in vitro. Intriguingly, protein binding assays showed that NCK-1 can also bind to the actin regulator UNC-34/Ena, but genetic experiments suggest that unc-34 is an inhibitor of nck-1 function. Through various genetic and biochemical experiments, I provide evidence that VAB-1 can disrupt the NCK-1/UNC-34 complex, and negatively regulate UNC-34. Taken together, my work provides a model of how VAB-1 RTK signaling can inhibit axon extension. I propose that activated VAB-1 can prevent axon extension by inhibiting growth cone filopodia formation. This is accomplished by inhibiting UNC-34/Ena activity, and simultaneously activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex.

Regulation of the Gene Encoding Thrombin-Activable Fibrinolysis Inhibitor in Non-Hepatic Cells

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like pro-enzyme that, once activated, attenuates fibrinolysis. TAFIa also possesses anti-inflammatory properties. Although liver is the main source of plasma TAFI, platelet-derived TAFI has also been reported. An alternatively spliced TAFI variant resulted from the skipping of exon 6 and a 52-base deletion in exon 10 of CPB2 mRNA (∆6+10) was described to be brain specific. This TAFI variant is reputed to possess a secretase-like activity that cleaves β-amyloid precursor protein to form β-amyloid, a process involved in the onset of Alzheimer's disease. In this thesis, we report the identification of CPB2 mRNA and TAFI protein in various vascular and inflammatory cells. Specifically, we describe the expression of CPB2 mRNA in the megakaryocytic cell lines MEG-01 and Dami, the monocytic cell line THP-1, and peripheral blood mononuclear cells. TAFI protein was detected in differentiated Dami and THP-1 cells. We next describe the effect of external stimuli such as phorbol myristate acetate (PMA) on CPB2 expression in Dami and THP-1 cells. We found that PMA treatment increases both CPB2 mRNA abundance and promoter activity in Dami cells, and decreases both CPB2 mRNA abundance and promoter activity in THP-1 cells. Deletion analysis of the CPB2 promoter indicated cell-type specific regulation of CPB2 gene expression. Finally, we evaluated the expression of alternatively spliced CPB2 mRNA variants in hepatic and non hepatic cells. We found that exon 6 skipping variants are expressed in all cell types of interest. The variant previously reported to be brain specific was also found to be expressed in platelets. We found that the alternatively spliced TAFI variants accumulated inside the cells in a non-secretable, hypoglycosylated form and showed no carboxypeptidase activity. Taken together, this thesis provides further evidence supporting the hypothesis that platelet-derived TAFI is originated from CPB2 gene expression in megakaryocytes. Moreover, our data imply a potential for site-specific anti-inflammatory control provided by macrophage-derived TAFI. Alternative splicing of the CPB2 mRNA may give rise to variants with an intracellular role, perhaps as a peptidase chaperone, and may modulate the synthesis of secretable TAFI.

Hormonal factors and insulin-like growth factor-1 in North American postmenopausal women

Background: Mechanisms underlying the effect of estrogen exposure on breast cancer risk remain unclear. Insulin-like growth factor-1 (IGF-1) levels have been positively associated with breast cancer and are a potential mechanism. Objectives: The objectives of this thesis are: 1) to explore whether the reproductive risk factors and the lifetime cumulative number of menstrual cycles (LCMC), as measures for long-term estrogen exposure, are associated with IGF-1 levels, and 2) to examine the effect of an aromatase inhibitor (AI) on IGF-1 levels, and the potential interaction with BMI. Methods: A cross sectional study and a randomized controlled trial nested with the MAP.3 chemoprevention trial were used to address objective 1 and 2, respectively. 567 postmenopausal women were selected. Anthropometric measurements, lifestyle factors, reproductive characteristics and serum IGF-1 concentrations were collected at baseline and one year. Objective 1. The LCMC was computed as a composite measure of the reproductive characteristics. Multivariable linear regression models were used to assess the association between IGF-1 levels and LCMC and the hormonal risk factors, while adjusting for potential covariates. Objective 2. Changes in IGF-1 were compared between the exemestane and placebo, and effect modification by BMI was tested with an interaction term. Results: Objective 1. Women aged 55 years or older at menopause had 16.26 ng/mL (95% CI: 1.76, 30.75) higher IGF-1 compared to women aged less than 50 years at menopause. Women in the highest category of menstrual cycles (≥500 cycles) had an average 19.00 ng/mL (95%CI: 5.86, 32.14) higher concentration of IGF-1 compared to women in the lowest category (<350). Exogenous hormones had no effect on postmenopausal IGF-1 levels. Objective 2. Exemestane significantly increased IGF-1 levels by 18% (95% CI: 14%-22%); while, placebo had no effect on IGF-1. The changes in IGF-1 were significantly different between the treatment arms (P<0.0001) and no significant interaction was observed between treatment and BMI on IGF-1 changes (P=0.1327). Conclusion: Objective 1. Larger number of menstrual cycles and a later age at menopause are positively associated with IGF-1. IGF-1 may be one mechanism by which prolonged estrogen exposure increases cancer risk. Objective 2. We conclude that the reduced cancer risk observed with AI therapy likely occurs in an IGF-1 independent mechanism. Further studies exploring the clinical consequences of increased IGF-1 on AI therapy are needed.