Regulation of the Gene Encoding Thrombin-Activable Fibrinolysis Inhibitor in Non-Hepatic Cells By JOELLEN HSUAN-HSIEN LIN A thesis submitted to the Graduate Program in Biochemistry in the Department of Biomedical & Molecular Sciences in conformity with the requirements for the Degree of Doctor of Philosophy Queen's University Kingston, Ontario, Canada September, 2011 Copyright© Joellen Hsuan-Hsien Lin, 2011 For my wonderful family, Tom Lin, Sophie Chuang, Sandy Lin and Mathieu Garand ii Tomorrow, we continue sailing forward. iii Abstract 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. iv Taken together, this thesis provides further evidence supporting the hypothesis that platelet-derived TAFI is originated from CPB 2 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. v Acknowledgement s I would like to express my sincere gratitude to my supervisors Drs. Marlys L. Koschinsky and Michael B. Boffa for giving me the opportunity to work in their laboratories, first at Queen's University in Kingston then at the University of Windsor in Windsor, throughout the course of my almost 8 years of research training. I will always remember Dr. Koschinsky being open- minded about new ideas, embracing our individualities, encouraging creativity, and having high tolerance for differences in opinion--key ingredients in making science fun and meaningful. Drs. Colin Funk and Bruce Hill and Diane Sommerfeld have also provided advice and encouragement over the years. I would also like to thank Dr. Lev Becker for inspiration in the early stage of my career, past MLK and MBB lab members, particularly Dr. Taewoo Cho, Dr. John Tra, Steven Schadinger, Dainn Andrews, Sue Johnston, and Curtis Noordhof for fruitful discussions and great times in and outside of the lab. Furthermore, I would like to thank our assistant Branislava Zagorac for her incredible hard work, so we can go full throttle in completing projects with tight deadlines. Big thanks to other members of MLK/MBB lab, Andrew Craig, Nicole Feric, Tanya Marar, Dragana Novakovic, Rocco Romagunolo, and Corey Scipione for advice and support during difficult times, and for making MLK/MBB lab the craziest and the funniest lab my husband and I have ever been part of. Thanks to David, Skye, Kisely and Carter Barbic for their friendships. This work would be impossible without the care from my husband Dr. Mathieu Garand, who always makes sure that I am well fed, well rested and well prepared for any challenges ahead. Finally, to my parents and sister, Tom Lin, Sophie Chuang and Sandy Lin, thank you for always being there for me and Mat. vi Table of Contents Dedication........................................................................................................................................ii Abstract............................................................................................................................................iv Acknowledgements.........................................................................................................................vi Table of Contents...........................................................................................................................vii List of Figures................................................................................................................................xiv List of Tables...............................................................................................................................xvii List of Abbreviations...................................................................................................................xviii Preface.........................................................................................................................................xxiii Chapter 1: General introduction..................................................................................................1 1.1 TAFI as a molecular link between hemostasis, fibrinolysis, and inflammation.........................1 1.1.1 Cell-based model of hemostasis..................................................................................2 1.1.2 Anticoagulant and fibrinolytic systems.......................................................................4 1.2 The CPB2 gene and the properties of TAFI...............................................................................5 1.2.1 The CPB2 gene............................................................................................................5 1.2.2 The properties of TAFI................................................................................................6 vii 1.2.3 The structure of TAFI, the basis for intrinsic TAFI zymogen activity, and the TAFIa auto-inactivation mechanism......................................................................................9 1.2.4 The role of activation peptide....................................................................................11 1.3 Implication of TAFI as a risk factor for atherothrombotic diseases.........................................12 1.4 TAFI in inflammation...............................................................................................................13 1.4.1 TAFIa as an anti-inflammatory molecule..................................................................13 1.4.2 TAFI is an acute phase protein in mice.....................................................................14 1.4.3 Plasma TAFI level is associated with inflammatory disease states in humans.........15 1.4.4 TAFI in wound healing..............................................................................................16 1.5 Regulation of hepatic CPB2 expression...................................................................................17 1.6 Non-hepatic TAFI pools...........................................................................................................19 1.6.1 Brain TAFI, a potential role in Alzheimer's disease..................................................19 1.6.2 Adipocytic and endothelial TAFI..............................................................................20 1.6.3 Platelet TAFI.............................................................................................................21 1.6.4 Keratinocytic TAFI....................................................................................................22 1.7 Physiological and pathological roles of macrophages and smooth muscle cells.....................22 1.8 Rationale, hypothesis, and objectives.......................................................................................24 viii Chapter 2: Identification of human thrombin-activatable fibrinolysis inhibitor in vascular and inflammatory cells.............................................................................................29 2.1 Statement of co-authorship.......................................................................................................29 2.2 Abstract.....................................................................................................................................29 2.3 Introduction..............................................................................................................................30 2.4 Materials and methods..............................................................................................................32 2.4.1 Cell culture................................................................................................................32