Date

March 2010

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Molecular and Medical Genetics

Institution

Oregon Health & Science University

Abstract

Fibrinogen is the zymogen precursor of fibrin, the protein that makes up blood clots. Fibrinogen is synthesized in the liver and circulates in plasma at a concentration of approximately 3 mg/mL. Fibrinogen is comprised of two sets of three polypeptide chains, which are encoded by the fibrinogen α (FGA), β (FGB) and γ (FGG) genes. Plasma fibrinogen concentration is an independent predictor of cardiovascular disease (CVD) including myocardial infarction (MI), coronary artery disease (CAD) and stroke. Recently, a gamma chain variant of fibrinogen, γ’ fibrinogen, was discovered as an additional independent risk factor for the development of MI and CAD. However, the mechanisms that regulate the production of this variant isoform have not been elucidated. The aim of this dissertation was to examine novel mechanisms that regulate the production of total and γ’ fibrinogen. Additionally, we sought to examine the molecular defect in a patient that presented with hemorrhage and a diagnosed dysfibrinogenemia. Interferon-γ (IFN-γ) was identified as a novel regulator of total fibrinogen synthesis. We identified an IFN-γ activated sequence (GAS) within the promoter of FGG. We have shown that IFN-γ signals to activate signal transducer and activator of transcription-1 (STAT1), which dimerizes and binds to the novel GAS element within the FGG promoter to decrease transcription. We also show that IFN-γ is able to inhibit interleukin-6 (IL-6) induced fibrinogen synthesis by interfering with the ability of IL-6 induced STAT3 to bind to known response elements within the FGG promoter. We have also identified IL-6, IFN-γ and tumor necrosis factor-α (TNF-α) as molecules that differentially regulate the production of the fibrinogen γ chain isoforms. IL-6 acts by increasing total fibrinogen while decreasing γ’ fibrinogen production, thereby lowering the ratio of γ’/total fibrinogen. TNF-α decreases γ’ fibrinogen while not affecting the levels of total fibrinogen produced, again decreasing the γ’/total fibrinogen ratio. IFN-γ decreases total fibrinogen synthesis, but not γ’ synthesis, to increase the γ’/total fibrinogen ratio. Additionally, we have discovered that the fibrin degradation product D-dimer is able to enter HepG2 cells and travel to the perinuclear space. Subsequently, we see a decrease in the amount of γ’ fibrinogen produced, suggesting that D-dimer is a negative regulator of γ’ fibrinogen synthesis. While we were not able to identify a receptor for D-dimer on the cell surface, we found ample evidence that D-dimers are taken up via endocytosis. Finally, we identified an R275C mutation in the γ chain of fibrinogen in a 54-year-old female. Most patients with this mutation are asymptomatic, and those that do exhibit symptoms usually present with thrombosis. Our patient was rather unique in that their presentation was hemorrhagic, and this work shows that the γR275C mutation is enough to sustain a hemorrhagic phenotype.

Identifier

doi:10.6083/M4Q81B2H

School

School of Medicine

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