ENGINEERING OF SERPINS FOR VASCULAR APPLICATION Jill Camille Rau A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pathology and Laboratory Medicine. Chapel Hill 2008 Approved by: Frank C. Church, Ph. D. Herbert C. Whinna, M. D., Ph. D. Alisa S. Wolberg, Ph. D. Maureane R. Hoffman, M. D., Ph. D. David R. Clemmons, M.D. ©2008 JILL CAMILLE RAU ALL RIGHTS RESERVED ii ABSTRACT Jill Camille Rau: The Engineering of Serpins for Vascular Application (Under the direction of Frank C. Church, Ph.D.) “The Engineering of Serpins for Vascular Applications” contributes to the understanding of serine protease inhibitors (serpins) in cardiovascular disease, the relationship between their structures and activities, and the manipulations of those structures for the benefit of medicine. Chapter 1 presents a comprehensive overview of serpins’ structure-activity relationships, pathophysiology and their roles in thrombosis, hemostasis and fibrinolysis. Chapter 2 establishes the co-localization of (pro)thrombin with heparin cofactor II (HCII) in the core of atherosclerotic plaques and demonstrates a positive correlation between the presence of HCII, (pro)thrombin or antithrombin (AT) and lesion severity. Additionally, these results indicate that atheromas may act as a non-specific watershed for plasma proteins and should serve as a warning against assumptions that (co-)localization of protein in atheromas implies a pathophysiologic role in atherosclerosis. Chapter 3 utilizes a gain-of-function approach to examine glycosaminoglycan (GAG)-binding in serpins. An α1-protease inhibitor (α1PI) mutant, α1PIPitt-GAG, containing five basic residues homologous to the HCII D-helix was shown to have HCII-like GAG- accelerated thrombin inhibition. Results confirm the benefit of using a gain-of-function approach to the study of serpins. The finding that GAG-binding and associated functionality iii can be added to a non-GAG binding serpin has exciting implications for the engineering of serpins for biomedical purposes. In Chapter 4, a chimeric serpin, HATpin, was engineered with intent to create a potent anti-thrombotic, anti-inflammatory, anti-atherogenic protein. HATpin was designed to comprise 1) the reactive center loop of AT for specificity of thrombin and factor Xa over activated protein C; 2) the N-terminal acidic domain of HCII to utilize thrombin exosite 1 for specific thrombin inhibition; 3) the GAG-binding region of HCII to target it to areas of vascular injury; 4) on an α1PI backbone. Although HATpin was successfully created, its inhibitory profile failed to meet expectations. The results from Chapter 4 illustrate our need for a better understanding of serpin structural domain interactions and challenge the concept that serpin domains are strictly modular building blocks that can be exchanged without a contextual effect. Finally, Chapter 5 outlines additional future studies that can be pursued based on questions that arose during the completion of this dissertation. iv ACKNOWLEDGEMENTS This endeavor took almost five years to achieve. I started, even before my graduate school training, as a technician in Frank Church’s lab four years prior with virtually no understanding of basic laboratory science. In the nine years since I began learning in this field, I’ve come a very long way. This hasn’t been on my own. There have been many, many people and groups who have supported me, encouraged me, taught me, challenged me, kept me sane, paid for me and contributed in some way, big and small to the completion of this dissertation. I would like to recognize those people and put down in black and white that they have contributed to my growth and to this dissertation. I owe them much and am very appreciative of their help. First, this dissertation project could not be undertaken without funding. I appreciate those institutions who have believed in me and my science. The Holderness Foundation provided me funds for a summer research fellowship during the summer after my first year of medical school. The Integrative Vascular Biology Training Program funded by the National Institutes of Health, National Heart Lung and Blood Institute (NIH-NHLB 5T32HL069768- 04) and directed by Nobuyo Maeda, Ph. D. took me on as a trainee for my first several years of graduate school. The Triangle Community Foundation selected me to receive the 2006- 2007 Gertrude B. Elion Mentored Medical Student Award which provided me funds for educational purposes that I have put to wonderful use and has greatly enhanced my v education and the completion of this dissertation. The American Heart Association then took a chance on me and provided me with a Pre-Doctoral Fellowship in 2007 (AHA 0715191U ) . Finally, the National Institutes of Health, Institute on Aging have granted me a Ruth L. Kirschstein National Research Service Award (NIH-NIA 1F30AG029053 - 01A2) to pay for both the completion of my Ph.D. and my M.D. degrees. Additionally, I am grateful to The Department of Pathology and Laboratory Medicine and to Frank Church for providing additional funds for lapses in financial support and for funding the laboratory research I performed. Second, I would like to acknowledge and thank my committee members for their diligence, support, and mentoring. David Clemmons, M.D. initially came on board because I was going to devote some of my dissertation to a topic related to his own research. That did not pan out, however, Dr. Clemmons cheerfully remained on my committee to support me and my research. Maureane Hoffman, M.D., Ph.D. is an amazing woman and a wonderful role model for me. She’s acted as a friend, mentor and scientific consultant. She provides calm and insightful advice and always makes the time to work and meet with me. Alisa Wolberg, Ph. D. is an inspiration. Not much older than myself, Alisa is running her own laboratory. She is also always willing to go the extra mile to meet with me to review my work. She is very thoughtful and level headed and has kept my Ph.D. on track. Herb Whinna, M.D., Ph.D. is combination friend, mentor and boss. I’ve worked with Herb for 9 years and enjoyed doing so. As a committee member Herb is superb. He cuts to the chase, understands the science very well and gives the bottom line. As a friend and a person, I have the utmost respect for Herb. He has helped to shape my laboratory and Ph.D. experience into vi an enjoyable one and I appreciate his passion and cynicism both. My final committee member, Frank Church will be discussed later. Next, I would like to acknowledge all those individuals (of which there are many) who provided a scientific contribution to my dissertation and or training. Peter Gettins, Ph. D. of the Department of Biochemistry and Molecular Genetics at the University of Illinois at Chicago provided me with the cDNA for both a1PI and a1PIPittsburgh. Steve Olson, Ph.D. of the Center for Molecular Biology of Oral Diseases, College of Dentistry at the University of Illinois at Chicago helped me understand, optimize and interpret the extrinsic fluorescence assays. James A. Huntington, Ph.D. and the members of his laboratory, Denis O’Keeffe, Daniel Johnson, Jonathan Langdown, Wendy Carter, Ph.D. , Stefan Luis, Aiwo Zhou, Li Wei, Ph. D. and Professor Robin Carrell all welcomed me with open arms into their lab for a 4 month long project in the Cambridge Institute for Medical Research, U.K.. There, in addition to having a wonderful time, I learned a considerable amount about serpins and science in general. I am extremely grateful to have had the opportunity to work with them. I am thankful the following individuals in the UNC School of Public Health: to Bob Schoonhoven from the Center for Environmental Health and Suceptibility for teaching me to perform immunohistochemistry; and the members of the Biometric Consulting Laboratory, Carolyn Deans, Brian Armstrong and Kelly Hunnigan under the guidance of Gary Koch, Ph. D. for performing my statistical analysis. In the UNC School of Medicine, I would like to thank John Sondek, Ph.D. and his laboratory in the Department of Biochemistry and Biophysics for letting me use their fluorometer; James Faber, Ph.D. in the Department of Cell and Molecular Physiology for acting as a collaborator on one of my grants what was funded. I would like to thank David vii Siderovski, Ph. D. and Lee Graves, Ph. D. and their laboratories in the Department of Physics for lending me pretty much anything I asked for, including their advice and guidance. In the Department of Pathology and Laboratory, my home department, there are many individuals who contributed to my research and training and deserve recognition. First, the graduate students, they are fun and welcoming and always helpful when in need of scientific help, a shoulder to cry on or a beer. Next, Lawrence Kiss, M. D. ranked some of my immunohistochemistry slide sets. David T. Thomas, M.D., served on my committee before moving to New York and guided me through much of the pathology in my dissertation. Donna Thompson trained me to section paraffin embedded tissue and let me use her facilities. Similarly, Lisa Cremeans taught me to formalyn-fix and paraffin embed and also to snap freeze fresh tissue. Howard Reisner, Ph. D. and Gayle McGhee trained me to use the Aperio Scanning Scope so that I would have easily used and attractive images of my slide sets. I am thankful to all the individuals associated with the MD/PhD Training Program here at UNC. They have been incredibly supportive during this very long endeavor. The students in the program all experience similar experiences and they are invaluable assets and friends.