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A Dissertation Entitled Role of Complement Regulatory Protein A Dissertation entitled Role of Complement Regulatory Protein Properdin in Complement Activation on Platelets and in the Formation of Platelet-Leukocyte Aggregates by Gurpanna Saggu Submitted to the Graduate Faculty in partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences ___________________________________________________ Viviana P. Ferreira, D.V.M., Ph.D., Committee Chair ___________________________________________________ Stanislaw Stepkowski, D.V.M., Ph.D., Committee Member ___________________________________________________ R. Mark Wooten, Ph.D., Committee Member ___________________________________________________ Z. Kevin Pan, M.D., Ph.D., Committee Member ___________________________________________________ Guillermo Vazquez, Ph.D., Committee Member ___________________________________________________ Patricia R. Komuniecki, Ph.D., Dean College of Graduate Studies The University of Toledo May, 2014 Copyright 2014, Gurpanna Saggu 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 Protein Properdin in Complement Activation on Platelets and in the Formation of Platelet-Leukocyte Aggregates by Gurpanna Saggu Submitted to the Graduate Faculty in partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences University of Toledo, May 2014 Patients with inflammatory cardiovascular disease have an increased number of circulating activated platelets and platelet-leukocyte aggregates (PLAs), both of which play a central role in the initiation and progression of disease. Activated platelets can activate the complement system on their surface, with potential consequences in vascular inflammation and thrombosis. Properdin, a positive regulator of the alternative pathway (AP) of complement, is produced mainly by stimulated leukocytes. The mechanisms by which properdin participates in complement activation on platelets and in PLA formation remain unknown. We have shown that the physiological forms of human properdin bind directly to activated, but not resting, platelets. The binding of properdin promotes AP complement activation on activated platelets, as measured by C3b and C9 deposition on their surface, by forming novel C3 convertases on the platelet [C3(H2O),Bb]. Removal of surface proteins by treating platelets with a low dose of proteinase K, leads to reduced properdin binding to activated platelets. On the other hand, chondroitin sulfate-A (a glycosaminoglycan that is released by platelets upon activation) iii increases the binding of properdin to activated platelets by ~4 fold. These results suggest that interaction of properdin with activated platelets may depend on a protein receptor as well as glycosaminoglycans (i.e. proteoglycans). We have also determined that properdin released by PMA-stimulated neutrophils binds to activated platelets. Since activated neutrophils and platelets directly interact with one another in pro-inflammatory microenvironments, the fresh properdin produced by neutrophils would be available to platelets at high concentrations. Using ex- vivo whole blood assays, we show that properdin leads to an increase in PLA formation in TRAP (thrombin receptor activating peptide)-stimulated whole blood and inhibition of properdin leads to decrease in PLA formation. Our data also show that properdin-mediated PLA formation is controlled by complement regulatory protein factor H. Altogether, the results support a role for properdin in the cellular microenvironment, contributing to complement activation on activated platelets and PLA formation, with potential consequences in inflammation pathophysiology. iv To my father who encouraged me to take this path, and my mother who assured me that I was capable of it. To my brother who has been my strength throughout. v Acknowledgements First and foremost I want to thank my major advisor Dr. Viviana P. Ferreira. It has been an honor to be her first Ph.D. student. I appreciate all her contributions to make my Ph.D. experience productive and stimulating. The dedication she has for her work was contagious and motivational for me, even in the tough times during the pursuit of my Ph.D. I would like to thank to my committee members, Dr. Mark Wooten, Dr. Stanislaw Stepkowski, Dr. Kevin Pan and Dr. Guillermo Vazquez, for all their time, constructive criticism and advice they have offered. I would like to thank Dr. Claudio Cortes who spent long hours training me during the initial phase. I would also like to thank Heather Emch, Dr. Galia Ramirez, Laci Bloomfield and Adam Blatt for all their support. I would also like to acknowledge all the faculty, staff and students in the Dept. of Medical Microbiology and Immunology. They have created a very professional and friendly work environment. Lastly, I want to thank my parents and brother for supporting me in all my pursuits; and I want to thank my loving, supportive, encouraging, and (not so) patient husband Mithun, whose unconditional support during this Ph.D. is so appreciated. vi Contents Abstract………………………………………………………………………………………………………. iii Acknowledgements……………………………………………………………………………............ vi Contents……………………………………………………………………………………………………... vii List of Tables…………………………………………………………………………………………….... xvi List of Figures…………………………………………………………………………………………….. xvii List of Abbreviations………………………………………………………………………………….. xxi 1 Introduction…………………………………………………………………………………............ 1 1.1 The Complement System……………………………………………………………….... 1 1.1.1 Background…………………………………………………………………………….. 1 1.1.2 Activation of the complement system……………………………………. 4 1.1.2.1 Classical Pathway………….……………………………………………... 4 1.1.2.2 Lectin pathway……………………………………………………….......... 6 1.1.2.3 Alternative pathway…………………………………………………….. 8 1.1.2.3.1 Complement component C3…………………..………….... 8 1.1.2.3.2 Initiation of the alternative pathway: Formation of the initial fluid phase C3 convertase……………………………... 9 1.1.2.3.3 Initial C3b deposition and formation of C3b,Bb convertase………………………………………………………………………. 10 vii 1.1.2.3.4 Role of properdin in amplification of the alternative pathway………………………………………………………... 11 1.1.2.4 C5 convertase and the terminal complement pathway………………………………………………………………………………….... 12 1.1.2.5 Regulatory proteins that allow the alternative pathway to distinguish between host/self cells (non- activating surfaces) and activating/pathogenic surfaces……….. 14 1.1.2.5.1 Soluble negative regulators of the alternative pathway…………………………………………………………………………. 14 (a) Factor I……………………..…………………………………. 14 (b) Factor H………………………………………………………. 14 1.1.2.5.2 Membrane-bound negative regulators of the alternative pathway………………………………………………………… 17 (a) Decay accelerating factor……………………………… 17 (b) CR1……………………………………………………………... 17 (c) Membrane cofactor protein…………………………... 17 (d) CD59……………………………………………………………. 17 1.1.2.6 Properdin: a positive regulatory protein with functions in complement initiation……………………………………........ 18 1.1.2.6.1 Properdin sources…………………………………………....... 18 1.1.2.6.2 Properdin structure…………………………………………... 19 1.1.2.6.3 Role of properdin as an initiator versus stabilizer of the alternative pathway convertase……………………………... 20 viii 1.1.2.6.4 Binding of properdin to a variety of cell surfaces and potential binding ligands for properdin……………………... 21 1.1.2.6.5 Importance of separating physiological forms of properdin (P2-P4) from aggregated (“activated”) properdin to study the specificity of properdin-target interactions.……………………………………………………………………. 22 1.1.2.6.6 The role of properdin in the local microenvironment………………………………………………………….. 24 1.2 Platelets…………………………………………………………………………………………... 27 1.2.1 Background…………………………………………………………………………….. 27 1.2.1.1 Platelet origin………………………………………………………………. 27 1.2.1.2 Platelet structure and function……………………………………. 27 1.2.2 Platelet activation…………………………………………………………………... 30 1.2.2.1 Platelet agonists…………………………………………………………... 31 1.2.2.1.1 Thrombin and thrombin receptor-activating peptide (TRAP)………..…………………………………………………....... 31 1.2.2.1.2 Arachidonic acid………………………………………………... 32 1.2.2.1.3 Adenosine Diphosphate…………………………………….. 33 1.2.2.2 Platelet granules………………………………………………………….. 34 1.2.3 Interactions between platelets and the complement system… 36 1.3 Platelet-leukocyte aggregates………………………………………………………… 39 1.3.1 Background…………………………………………………………………………….. 39 1.3.2 Mechanisms of platelet-leukocyte aggregate formation……….. 40 ix 1.3.3 Activation of complement on platelets and on leukocytes…….. 41 2 Hypothesis…………………………………………………………………………………………….. 44 3 Specific aims………………………………………………………………………………………….. 45 4 Materials and Methods…………………………………………………………………………. 47 4.1 General Methods……………………………………………………………………………... 47 4.1.1 Buffers…………………………………………………………………………………….. 47 4.1.2 Antibodies………………………………………………………………………………. 48 4.1.3 Other reagents………………………………………………………………………... 50 4.1.4 Serum and additional complement proteins…………..……………... 51 4.1.5 Separation of physiological forms of properdin…………………..... 53 4.1.6 Platelet isolation and activation…………………………………………….. 54 4.2 Specific Methods (separated by the Aim involved)………………………… 56 4.2.1 Methods Aim 1: To determine the molecular mechanisms involved in alternative pathway activation on activated platelets.... 56 4.2.1.1 Measurement of properdin binding to platelets………………... 56 4.2.1.2 Measurement of platelet activation by properdin……………... 57 4.2.1.3 Measurement of presence of properdin
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