Date of Award

2014

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

First Advisor

Boffa, Michael

Keywords

Cardiovascular disease, Coagulation, Fibrinolysis, Gene expression, Post-transcriptional regulation

Rights

CC BY-NC-ND 4.0

Abstract

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 a risk for thrombotic diseases. TAFI plasma concentrations vary substantially within human population, and various hormonal factors and disease states have been shown to have an impact. Regulation of 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. Our first set of studies lead to the identification of key cis-acting sequences within the 3'-untranslated region (3'-UTR) of the TAFI mRNA that specify transcript stability. Specifically, we described the presence of one stability element, followed by three instability elements. Furthermore, we identified the trans-acting factor binding to the last instability element. Tristetraprolin (TTP) is capable of binding this sequence, promoting mRNA destabilization and degradation. We also observed that another trans-acting protein factor, HuR, binds the TAFI 3'-UTR. We found that TTP and HuR play a crucial role in post-transcriptional regulation of CPB2 transcript. Pro-inflammatory mediators exerted their TAFI protein-lowering effects via TTP-mediated mRNA destabilization in human hepatocellular carcinoma (HepG2) cells. On the other hand, CPB2 mRNA and TAFI protein abundance and transcript stability were increased in THP-1 macrophages in the presence of inflammatory mediators, suggesting the possibility of tissue-specific regulation for CPB2 gene expression. We also obtained preliminary evidence that miR-124, miR-506 and miR-708 decrease endogenous TAFI mRNA and protein in HepG2 cells. Moreover, the recognition site of miR-143 includes the region containing a commonly occurring single nucleotide polymorphism that is associated with lower plasma TAFI concentrations, providing a plausible mechanistic basis for such effect. Taken together, our results provide new knowledge about the crucial role of posttranscriptional regulation in mediating TAFI protein levels. Factors that act in trans to mediate these effects include both proteins (TTP and HuR) and miRNAs (miR-124, miR-143, miR-506 and miR-708).

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