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A dissertation entitled Role of Complement Regulatory Proteins Properdin and Factor H in Platelet/Granulocyte Aggregate Formation by Adam Z. Blatt Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences Viviana P. Ferreira, D.V.M., Ph.D., Committee Chair R. Travis Taylor, Ph.D., Committee Member Guillermo Vazquez, Ph.D., Committee Member R. Mark Wooten, Ph.D., Committee Member Randall G. Worth, Ph.D., Committee Member Amanda Bryant-Friedrich, Dr. rer. Nat , Dean College of Graduate Studies The University of Toledo August 2016 Copyright 2016, Adam Z. Blatt This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An abstract of Role of Complement Regulatory Proteins Properdin and Factor H in Platelet/Granulocyte Aggregate Formation by Adam Z. Blatt Submitted to the graduate faculty in partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences University of Toledo August 2016 Platelet binding to leukocytes is a normal response to vascular damage and inflammation, however excessive platelet/leukocyte interactions have significant pathologic potential due to the ability of platelets to directly stimulate leukocytes. Formation of stable platelet/granulocyte aggregates (PGAs) induces neutrophils to release inflammatory cytokine/chemokines and damaging proteases, produce reactive oxygen species, and upregulate adhesion molecule and tissue factor expression. High levels of circulating PGAs are found in patients with diverse inflammatory vascular diseases, and animal models of vascular injury have shown benefits to limiting PGA formation in vivo. The complement system enhances PGA formation in human blood stimulated with thrombin receptor-activating peptide (TRAP), however precise mechanisms for the effects of complement are unknown. Here, we utilized ex vivo and in vitro flow cytometry assays to show that properdin, a positive regulator, and Factor H (FH), a negative regulator, of the alternative complement pathway (AP) have key roles in controlling the extent of TRAP-mediated PGA formation in human whole blood. Physiological properdin oligomers added exogenously to TRAP-stimulated whole blood iii enhance PGA formation and AP activity, while blocking endogenous properdin function with inhibitory antibodies limits PGA formation. Blocking C5a-C5a receptor interactions limits PGA formation to similar levels seen with inhibition of properdin and abrogates exogenous properdin-mediated increases in PGA formation. C5a directly enhances TRAP-mediated PGA formation and CD11b expression on granulocytes, indicating C5a is the key complement effector molecule for PGA formation. The effects of properdin on PGA formation, are tightly controlled by FH cell-surface protection. FH C-terminal domains 19-20 are key for regulating AP activity on the surface of platelets and neutrophils, and for controlling C5a generation at the platelet/granulocyte interface in TRAP-stimulated blood. Finally, mutations in domains 19-20 associated with atypical hemolytic uremic syndrome (aHUS) have differential effects on the ability of FH to control PGA formation and limit AP activity on platelets and neutrophils, suggesting that increased PGA formation contributes to thrombosis seen in patients with some, but not all, aHUS-related FH mutations. Our work has elucidated critical mechanisms governing PGA formation, and could potentially be used to develop novel therapeutics to limit thromboinflammation in inflammatory diseases. iv For my family, who set me on a path toward my dreams and gave me the courage to follow it v Acknowledgements First and foremost, I would like to thank my family. You have continuously given me the love, support, and guidance that has made me the man I am today and pushed me to pursue the M.D./Ph.D. program. You gave me the strength to realize that nothing in this life is impossible if you set your mind to it. And when life throws me curveballs, you are there to pick me up and get me back on course. To my mentor, Dr. Viviana Ferreira, I give you my deepest thanks for molding me into the scientist I am, today. For all the long hours you spent training me, mentoring me, and ultimately pushing me to strive for excellence in everything I do, I am gracious. For countless hours training me when I joined the lab, I would like to thank Dr. Gurpanna Saggu. Your patience and dedication in training me influenced the entire outlook of my time in the lab. I would also like to thank the other lab members who provided assistance, stimulating conversations, and an enjoyable work atmosphere, especially Heather Emch, Koustubh Kulkarni, and Sean Ehinger. I would like to thank my committee members for their critical feedback and support throughout the duration of my project. Finally, I would like to express my utmost appreciation to all the members of the Department of Medical Microbiology and Immunology, who made it a great place to come to work for the past 3 years, and who helped me to relax and decompress outside the lab. vi Contents Abstract………………………………………………………………………………... iii Acknowledgements……………………………………………………………………. vi Contents……………………………………………………………………………….. vii List of Figures………..……………………………………………………………….. xii List of Abbreviations………………………………………………………………… xvi 1 Introduction…………………………………………………………………………. 1 1.1 The complement system………………………………………………………. 1 1.1.1 Background……………………………………………………………. 1 1.1.2 Mechanisms of activation…………………………………………….. 1 1.1.2.1 The classical pathway………………………………………… 2 1.1.2.2 The lectin pathway…………………………………………… 4 1.1.2.3 The alternative pathway……………………………………… 5 1.1.2.4 The terminal pathway………………………………………… 7 1.1.2.5 The extrinsic pathway………………………………………... 8 1.1.3 Effector functions……………………………………………………... 8 1.1.3.1 The C3 opsonins……………………………………………… 8 1.1.3.2 The anaphylatoxins…………………………………………… 9 1.1.3.3 The MAC……………………………………………………...11 1.1.4 Regulatory proteins………………………………………………….. 11 1.1.4.1 Membrane bound regulatory proteins……………………..... 12 vii 1.1.4.2 Fluid phase regulatory molecules…………………………… 12 1.1.4.2.1 Factor H………………………………………... 13 1.1.4.2.2 Properdin…………………………………………. 17 1.2 Platelets…………………………………………………………...…………... 20 1.2.1 Introduction to platelets…………………………………………………. 20 1.2.2 Complement and platelets………………………………………………. 23 1.3 Neutrophils…………………………………………………………................ 25 1.3.1 Introduction to neutrophils……………………………………………. 25 1.3.2 Complement and neutrophils………………………………………….. 27 1.4 Platelet/granulocyte aggregates……………………………………………... 28 1.4.1 Introduction to platelet/granulocyte aggregates………………………. 28 1.4.2 Physical contacts……………………………………………………… 29 1.4.3 Communication through soluble mediators…………………………... 31 1.4.4 Complement and PGA………………………………………………… 31 2 Hypothesis…………………………………………………………...……………... 32 3 Specific aims…………………………………………………………...…………… 33 4 Materials and Methods…………...……………………………………………...... 35 4.1 Materials…………………………………………………………...…………. 35 4.1.1 Buffers…………………………………………………………...……. 35 4.1.2 Antibodies…………………………………………………………...... 36 4.1.3 Other reagents…………………………………………………………. 37 viii 4.2 Methods…………………………………………………………...………….. 39 4.2.1 Generation of platelet-rich and platelet-poor plasma from half-tube of blood anticoagulated with different direct thrombin inhibitors......... 39 4.2.2 Clotting assay…………………………………………………………. 41 4.2.3 Assessment of effects of direct thrombin inhibitors on platelet aggregation…………………………………………………………..... 41 4.2.4 Assessment of effects of reagents on AP-mediated lysis of rabbit erythrocytes by normal human sera…………………………………… 42 4.2.5 Assessment of effects of anticoagulants on AP-mediated lysis of rabbit erythrocytes by human plasma………………………………...... 43 4.2.6 Separation of physiological properdin forms…………………………. 43 4.2.7 Measurement of TRAP-mediated PLA formation, C3 fragment deposition, and CD11b expression……………………………………. 43 4.2.8 Analysis of C5a levels in PLA reaction supernatants………………… 45 4.2.9 Platelet isolation………………………………………………………. 46 4.2.10 Platelet activation……………………………………………………... 47 4.2.11 Mapping regions of FH that contribute to the regulation of the AP on thrombin-activated platelets…………………………………………... 48 4.2.12 Mapping regions of FH that contribute to the regulation of the AP on arachidonic acid-activated platelets…………………………………… 49 4.2.13 Measurement of effects of rH19-20 mutants on C3 fragment ix deposition on thrombin-activated platelets……………………………. 49 4.2.14 Isolation of neutrophils………………………………………………... 51 4.2.15 Measurement of AP activation on neutrophils………………………... 52 4.3 Statistics…………………………………………………………...………….. 53 5 Results…………………………………………………………...………………….. 54 5.1 Specific aim 1: To determine molecular mechanisms by which properdin enhances PGA formation………………………………………... 54 5.1.1 To assess whether recombinant hirudin is a suitable alternative for lepirudin in whole blood PGA assays……………………………... 54 5.1.2 To determine whether physiological properdin forms increase PGA formation………………………………………………………... 59 5.1.3 To determine whether inhibition of endogenous properdin reduces TRAP-induced PGA formation………………………………. 62 5.1.4 To determine which complement pathways contribute to PGA formation…………………………………………………………...…. 68 5.1.5 To determine how properdin-mediated C5a generation enhances TRAP-mediated PGA formation……………………………………… 70 5.2 Specific aim 2: To determine the role of Factor H in the interaction between platelets and neutrophils…………………………………………..
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