BIOLOGICALLY ACTIVE ASSEMBLIES THAT ATTENUATE THROMBOSIS ON BLOOD-CONTACTING SURFACES A Dissertation Presented to The Academic Faculty by Zheng Qu In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Bioengineering Georgia Institute of Technology December 2012 BIOLOGICALLY ACTIVE ASSEMBLIES THAT ATTENUATE THROMBOSIS ON BLOOD-CONTACTING SURFACES Approved by: Dr. Elliot L. Chaikof, Advisor Dr. Larry V. McIntire The Wallace H. Coulter Department of The Wallace H. Coulter Department of Biomedical Engineering Biomedical Engineering Georgia Institute of Technology Georgia Institute of Technology Department of Surgery Beth Israel Deaconess Medical Center Dr. Julia E. Babensee Dr. W. Robert Taylor The Wallace H.Coulter Department of Division of Cardiology Biomedical Engineering Emory University School of Medicine Georgia Institute of Technology Dr. Stephen R. Hanson Department of Biomedical Engineering Oregon Health and Science University Date Approved: November 2, 2012 Dedicated to my parents, for their unconditional love. ACKNOWLEDGEMENTS The progress and advances made in this research were enabled by contributions from many colleagues and peers, as well as support from family and friends. It is these relationships that were forged during the course of my career that I cherish the most. First and foremost, I want to express my sincerest gratitude for Dr. Elliot Chaikof, for guiding me through the many challenges of scientific research with innovative ideas and patient advice, and above all, by always keeping the “big picture” in perspective. Dr. Chaikof has taken the time to personally support every step along the way of my career as a PhD candidate despite his huge commitments to the clinic. He has fostered my growth as an independent scientist, and facilitated many of the exciting collaborations that were instrumental in shaping the development of this dissertation. It has been an honor to be mentored by a towering figure in both the field of vascular surgery and biomedical engineering. Dr. Chaikof’s enthusiasm and drive to achieve tangible impact, through translating innovative science to clinically effective therapies that improve patient care, has fired me with a vision that will shape my professional growth for years to come. I want to thank my committee members, Dr. Julia Babensee, Dr. W. Robert Taylor, Dr. Larry McIntire, and Dr. Stephen Hanson, for their multidisciplinary guidance, insight, and support. As a chemical engineer by training, I sincerely appreciate the time and assistance from leaders in the fields of material science, thrombosis, and cardiovascular biology to steer my ventures into the field of biomaterial blood- compatibility. In particular, the collaborative efforts with Dr. Hanson and his talented group of engineers and surgeons at the Oregon Primate Research Center to develop non- human primate models of thrombosis were essential in the testing and validation of the iv research presented here. I want to thank Ms. Ulla Marzec, Mr. Sawan Hurst, and Dr. Monica Hinds for their creativity, patience, and rigor in the iterative development and testing of clinically relevant animal models. I wish all the best for Dr. Hanson, as well as Dr. McIntire, in their retirement! I also would like to thank Dr. Abraham Stroock and Dr. Mario Cabodi from Cornell University, who instilled in me a passion for scientific discovery by taking me on as an undergraduate research assistant to apply principles of transport phenomena from the classroom in the development of new medical therapies. Every member of the Chaikof Lab has been involved in my dissertation in some form, and they have made my graduate school life much more enjoyable beyond the bench experience. I will fondly remember the many classes we have taken together, the group outings, and the birthday celebrations. I am forever indebted to Dr. Carolyn Haller, who has taught me nearly every assay and protocol needed to complete this work since the day I joined the lab. By drawing on her extensive background, Dr. Haller has provided me countless detailed and candid feedback which helped me gain the skills and intuition for experimental design and implementation. I would also like to acknowledge Dr. John Wilson, whose scientific rigor and enthusiasm for high impact research have forever influenced my approach towards science. It has been an honor working with Dr. Wilson in the field of pancreatic islet transplantation. Moreover, I appreciate the collaborative learning environment fostered through working with Dr. Sumanas Jordan, Dr. Murali Urlam, Dr. Sharmila Muthukrishnan, Dr. Venkat Krishnamurthy, Dr. Wookhyun Kim, Dr. Wanxing Cui, Dr. Adam Martinez, Dr. Jeff Caves, and Dr. Vivek Kumar, who contributed time, materials, and advice from drawing on their unique expertise and skills. v Since the Chaikof Lab transition to Boston, I had the fortunate opportunity to build an ongoing collaboration with Dr. David Liu’s lab at the Department of Chemistry and Chemical Biology at Harvard. A major portion of this dissertation would not have been possible without reagents provided by Mr. Brent Dorr, a graduate student in Dr. Liu’s lab. This collaboration will undoubtedly influence the long term approach by both the Chaikof and Liu labs to achieve their research missions. Beyond the exchange of scientific ideas and reagents, the contagious enthusiasm, optimism, and passion for new discovery from Brent and the Liu lab members were equally important in shaping the concluding years of my thesis. I want to also highlight Dr. Erbin Dai’s patience and skill in operating on small animal models to validate my designs. Dr. Dai’s can-do attitude, combined with his rare expertise in replicating the most complex clinical procedures in small animals, will undoubtedly be a key driver in the development of new therapies from the Chaikof Lab in the future. It has been fortunate that the list of colleagues in the Georgia Tech, Emory, Cornell, and Harvard academic and hospital communities, the Wyss Institute, and Genzyme, as well as friends from Ithaca, Atlanta, and Boston, is too long to list individually here, and I express my sincerest thanks for their time and contributions to my personal and professional growth. I am also indebted to the National Institute of Health, Georgia Tech, as well as Medtronic, for their financial aid during my research career. Above all else, I’m forever grateful to my parents, who were always willing to lend an ear to my troubles, encourage me when everything seems hopeless, and cheer me on when I succeed. I could not have made it this far without their unconditional love and support. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ………………………………………………………..... iv LIST OF TABLES …………………………………………………………................xii LIST OF FIGURES …………………………………………………………………xiii LIST OF SYMBOLS AND ABBREVIATIONS ………………………………….xxiv SUMMARY ………………………………………………………………………...xxvi CHAPTER 1 Introduction …………………………………………………………….............1 1.1 Significance …………………………………………………………1 1.2 Rationale …………………………………………………………….2 1.3 Central Hypothesis and Specific Aims ……………………………….3 2 Background …………………………………………………………………5 2.1 Current Synthetic Arterial Substitutes ………………………………….5 2.1.1 Dacron Prosthesis ……………………………………………….6 2.1.2 ePTFE Prosthesis ………………………………………………….8 2.1.3 Polyurethane Prosthesis ……………………………………….10 2.2 Interface between Thrombosis and Inflammation ………………….14 2.2.1 Platelets and Coagulation in the Pas de Deux of Hemostasis …15 2.2.2 Protease Activated Receptors at the Nexus between Coagulation and Inflammation ……………………………………17 2.2.3 Coagulation-activated Mediators in Inflammatory Disorders ….20 2.2.4 Platelet Activation Strengthens Antigen Presentation …………...22 vii 2.2.5 Clinical Perspectives …………………………………………25 2.3 Passive Thromboresistant Surface Engineering Strategies …………28 2.3.1 Poly(ethylene glycol) …………………………………………..28 2.3.2 Albumin Coating ……………………………………………….29 2.3.3 Carbon Coating …………………………………………………29 2.3.4 Phosphorylcholine Coating ……………………………………30 2.3.5 Elastin-inspired Surfaces ………………………………………31 2.4 Inhibition of Thrombin and Fibrin Formation ……………………..32 2.4.1 Heparin ………………………………………………………32 2.4.2 Thrombomodulin ………………………………………………34 2.4.3 Direct Thrombin Inhibitors and Fibrinolytic Agents …………35 2.5 Inhibition of Platelet Adsorption ……………………………………35 2.5.1 Antiplatelet Drugs ……………………………………………35 2.5.2 Nitric Oxide ……………………………………………………36 2.6 Biohybrid Vascular Grafts ………………………………………….37 2.6.1 Endothelial Cell Seeding ………………………………………..37 2.6.2 Promoting In Vivo Reendothelialization ………………………38 2.7 Summary ……………………………………………………………..39 3 Layer-by-layer Assembly of Polyelectrolyte Multilayers as Substrate for Covalent Immobilization of Biomolecules ……………………………….41 3.1 Introduction ……………………………………………………….41 3.2 Results and Discussion ……………………………………………44 3.2.1 Assembly of PEM Films on Quartz …………………………..44 viii 3.2.2 Assembly of PEM Films on ePTFE grafts ……………………..53 3.3 Conclusion …………………………………………………………58 3.4 Methods ……………………………………………………………58 3.4.1 Synthesis and Characterization of Oxidized Alginate …………..58 3.4.2 Synthesis and Characterization of FITC labeled PLL and PEI …59 3.4.3 Assembly of PEM Films on Quartz and ePTFE ………………60 3.4.4 PEM Film Stability and Crosslinking ……………………………61 4 A Biologically Active Surface Enzyme Assembly That Attenuates Thrombus Formation …………………………………………………………62 4.1 Introduction …………………………………………………………..62 4.2 Results and Discussion …………………………………………….67 4.2.1 Coating of ePTFE Grafts with a Polyurethane Film …………..67 4.2.2 Chemical