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Polyphosphate as a modulator of the complement system Wat, Jovian Ming Wai 2013

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    POLYPHOSPHATE AS A MODULATOR OF  THE COMPLEMENT SYSTEM  by  Jovian Ming Wai Wat  B.Sc., Queen?s University, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2013  ? Jovian Ming Wai Wat, 2013    ii  Abstract  Coagulation and complement are evolutionarily related, with several well-described mechanisms of cross-talk. Recently, it was established that polyphosphate (polyP) is a physiologic activator and promoter of coagulation. I hypothesized that polyP also plays a role in regulating complement, and thereby acts as an additional molecular bridge between coagulation and complement. Evidence to support this was provided by studies in bacteria, where defects in polyP synthesis and degradation alter its resistance to serum-mediated killing. In this thesis, I show that polyP suppresses total complement-mediated lytic activity and the terminal pathway, resulting in decreased lysis of foreign erythrocytes by the membrane attack complex (MAC). In contrast, monophosphate exhibits 10-fold less inhibition in total hemolytic activity, and has no effect on the terminal pathway. I also provide evidence that polyP destabilizes the C5b,6 complex to prevent functional MAC formation. Implications of the role of polyP in complement modulation are discussed.     iii  Preface  A version of Chapter 2 has been submitted for publication and pending review. Wat, J., Foley, J.H., Krisinger, M.J., Lei, V., Ocariza, L.M., Wasney, G.A., Lameignere, E., Strynadka, N.C., Smith, S.A., Morrissey, J.H., and Conway, E.M. Polyphosphate suppresses complement activity via the terminal pathway. I designed, performed, and analyzed all experiments (with the exception of gel filtration) and wrote the first draft of the manuscript. All other authors contributed to the design and analysis of experiments, and helped write the manuscript.     iv  Table of Contents Abstract ........................................................................................................................................................ ii Preface ........................................................................................................................................................ iii Table of Contents ....................................................................................................................................... iv List of Tables .............................................................................................................................................. vi List of Figures ........................................................................................................................................... vii List of Abbreviations ............................................................................................................................... viii Acknowledgements .................................................................................................................................... x Dedication .................................................................................................................................................. xii 1. Introduction ......................................................................................................................................... 1 1.1. Coagulation ................................................................................................................................... 1 1.1.1. Overview of Coagulation Activation ....................................................................................... 1 1.1.1.1. Initiation ......................................................................................................................... 1 1.1.1.2. Amplification .................................................................................................................. 2 1.1.1.3. Propagation ................................................................................................................... 2 1.1.1.4. Factor XII ....................................................................................................................... 3 1.1.2. Examples of Negative Regulators of Coagulation ................................................................. 3 1.1.2.1. Antithrombin/Heparin..................................................................................................... 3 1.1.2.2. Tissue Factor Pathway Inhibitor .................................................................................... 5 1.1.2.3. Thrombomodulin/Activated Protein C ........................................................................... 6 1.1.3. Fibrinolysis ............................................................................................................................. 6 1.1.4. Thrombin Activatable Fibrinolysis Inhibitor ............................................................................ 7 1.2. Complement System ..................................................................................................................... 7 1.2.1. Overview of Complement Activation ..................................................................................... 8 1.2.1.1. Classical Pathway ......................................................................................................... 8 1.2.1.2. Lectin Pathway .............................................................................................................. 9 1.2.1.3. Alternative Pathway..................................................................................................... 10 1.2.1.4. Terminal Pathway ........................................................................................................ 11 1.2.2. Functional Roles of Complement ........................................................................................ 11 1.2.2.1. Extracellular Destruction of Cells ................................................................................ 11 1.2.2.2. Enhancement of Inflammation .................................................................................... 13 1.2.2.3. Opsonisation of Target Cells ....................................................................................... 13 1.2.3. Negative Regulators of Complement .................................................................................. 14 1.2.3.1. Soluble Inhibitors of the Classical and Lectin Pathways ............................................. 14 1.2.3.2. Soluble Inhibitors of the Alternative Pathway .............................................................. 14 1.2.3.3. Membrane Inhibitors of Complement .......................................................................... 15   v  1.2.4. Properdin ............................................................................................................................. 16 1.2.5. Polyion Effects on Complement .......................................................................................... 17 1.2.5.1. Polycations .................................................................................................................. 17 1.2.5.2. Polyanions ................................................................................................................... 17 1.3. Coagulation and Complement Cross-Talk .................................................................................. 18 1.3.1. Coagulation Factors Modulate Complement Activity .......................................................... 18 1.3.2. Complement Components Modulate Coagulation .............................................................. 19 1.4. Polyphosphate............................................................................................................................. 19 1.4.1. Biochemistry, Synthesis, and Degradation .......................................................................... 19 1.4.2. Cellular and Tissue Localization .......................................................................................... 23 1.4.3. Function ............................................................................................................................... 24 1.4.3.1. Prokaryotes ................................................................................................................. 24 1.4.3.2. Eukaryotes .................................................................................................................. 25 1.5. Hypothesis, Goal and Objectives ................................................................................................ 27 2. Polyphosphate suppresses complement via the terminal pathway ............................................ 28 2.1. Introduction .................................................................................................................................. 28 2.2. Materials and Methods ................................................................................................................ 30 2.3. Results ........................................................................................................................................ 32 2.4. Summary ..................................................................................................................................... 44 2.5. Supplemental Materials and Methods ......................................................................................... 52 2.6. Supplemental Figures ................................................................................................................. 56 3. General Discussion ........................................................................................................................... 59 3.1. Role of PolyP in Suppression of Complement by Bacteria ......................................................... 59 3.2. Role of PolyP in Complement Suppression at Site of Injury ....................................................... 63 3.3. Comparing the Roles of PolyP and Other Polyanions on Complement Suppression ................ 64 3.4. Role of PolyP in Modulation of Other Complement Activities ..................................................... 68 4. Conclusion ......................................................................................................................................... 70 Bibliography .............................................................................................................................................. 72      vi  List of Tables Table 3.1 Average molecular weights of different heparins. .......................................................... 65     vii  List of Figures Figure 1.1 Schematic representation of the cell-based model of coagulation. ............................................. 4 Figure 1.2 Schematic representation of the complement cascade. ............................................................ 12 Figure 1.3 Thrombin-mediated interplay between coagulation and complement. ...................................... 20 Figure 1.4. Chemical structure of polyphosphate. ...................................................................................... 22 Figure 1.5 Effects of polyphosphate on coagulation. .................................................................................. 26 Figure 2.1 PolyP suppresses total hemolytic activity. ................................................................................. 33 Figure 2.2 PolyP suppresses terminal pathway hemolytic activity in serum. ............................................. 37 Figure 2.3 PolyP suppresses the TP in a size-dependent manner............................................................. 38 Figure 2.4 PolyP directly affects the function of terminal pathway components. ........................................ 40 Figure 2.5 PolyP reduces the mobility of terminal pathway proteins in native gels. ................................... 42 Figure 2.6 Gel filtration to assess interactions of C5b,6 and C7 with polyP. .............................................. 43 Figure 2.7 PolyP alters the thermal stability of C5b,6. ................................................................................ 45 Supplemental Figure 2.1 Standard curve for the total complement-mediated hemolytic assay. ................ 56 Supplemental Figure 2.2 Standard curve for the terminal pathway assay using serum as the source of complement. ................................................................................................................................................ 57 Supplemental Figure 2.3 Standard curve for the terminal pathway assay using purified complement components. ................................................................................................................................................ 58 Figure 3.1 PolyP reduces the mobility of factor H in native gels................................................................. 61 Figure 3.2 Fluid phase inactivation of C3b by factor H and factor I. ........................................................... 62 Figure 3.3 Comparison of the effects of polyP and other polyanions on terminal pathway activity............ 66    viii  List of Abbreviations Activated protein C APC Alternative pathway AP Antithrombin III AT C1 esterase inhibitor C1-INH C4-binding protein C4BP Calf intestinal alkaline phosphatase CIAP Classical pathway CP Complement receptor 1/2/3 CR1/2/3 Decay accelerating factor DAF/CD55 Differential scanning fluorimetry DSF Endopolyphosphatase PPN Exopolyphosphatase PPX Factor B FB Factor D FD Factor H FH Factor I FI Fibrin Fn Fibrinogen Fg Fibroblast growth factor FGF Gelatin veronal buffer GVB Glycosaminoglycans GAG   ix  Glycosylphosphatidylinositol GPI Lectin pathway LP Low molecular weight heparins LMWHs Mannose-binding lectins MBL MBL-associated serine proteases MASP Membrane attack complex MAC Membrane cofactor protein MCP/CD46 Normal human serum NHS Paroxysmal nocturnal hemoglobinuria PNH Polyphosphate PolyP Polyphosphate kinase PPK Protease activated receptor PAR Rabbit/chicken erythrocytes r/cRBC Relative fluorescence units RFU Soluble MAC sMAC Terminal pathway TP Thrombin activatable fibrinolysis inhibitor TAFI Thrombomodulin TM Tissue factor TF Tissue factor pathway inhibitor TFPI Tissue plasminogen activator tPA Unfractionated heparin UFH    x  Acknowledgements  First and foremost, I would like to thank my supervisor Dr. Edward M. Conway. I cannot thank you enough for all the opportunities you have given me ? CIHR Master?s Award, ISTH oral presentation, scholarships for my Ph.D., just to name a few. Your guidance and support also kept me going these past two years. You are always just minutes or hours away from replying to my email cries, even in the middle of the night as I submit my drafts to you at 2 in the morning. I don?t believe I will find anyone else with that kind of response efficiency, ever. You were also always willing to listen and help with personal issues, like that time I contacted you for advice before I even joined the lab. For all these reasons and more, thank you.  Thank you to my committee members, Dr. Edward L.G. Pryzdial and Dr. Ross T.A. MacGillivray for their supervision and making sure I am on the right track to finishing my degree as well as being a better scientist.  Of course, there is no way I could have ever made it without losing my sanity if it weren?t for my awesome lab mates, former and present (in no particular order):  Yane, thanks for putting up with me when I was stumbling around the lab like a blind mouse pup. I learned most from my degree when I was under your wing, from techniques to just how to be a better scientist. If it weren?t for your encouragement, I don?t think I would?ve mustered up the courage to just go ahead and try things out on my own, and be as confident in myself as I am today.  Verena, thanks for all your technical help, support, and friendliness. You?re incredibly talented and skilled, and you probably have no idea how much I look up to you. I?m going to miss our coffee breaks!  Mike, thanks for your mentorship when I dived into complement blindly after my first year. I actually never imagined myself working on complement before that ? it felt so daunting, so complicated, and almost boring. My eyes have truly opened since then, and I could not be more wrong about complement being boring.  Jonathan, I also can?t thank you enough for the guidance and tremendous support you?ve given me. Somehow I ended up being under your care as well even though you were managing so many projects simultaneously already, but you were always there to answer my (dumb) questions. Of course, I also couldn?t have asked for a better roommate at ISTH ? I probably would?ve gone insane over my presentation if it weren?t for your encouragement and support. The words you said to me the night after my presentation were really inspirational and encouraging, and I am more motivated than ever to work harder and strive to be better. I will never forget those words, whether you want me to or not. Once again, my sincere thanks.   xi   Erica, thanks for always being there to support me (like a mother!). Thanks for putting up with my shameless whining and whimpering prior to and after my presentation at ISTH. Also love that sweet photo you took of me at my talk!  Sahana, thanks for being there for me like a sister (don?t forget you?re not that much older than me!). Thanks for putting up with what little brothers do, like taking over your bench space, pulling pranks on you, making dumb jokes, all that fun stuff. It?s been great having you sitting across from my work bench, always there to hear me out with my work and personal issues. ?Almost there?almost there??   Victor, thanks for teaching me so much these past two years, such as how there can?t be 200% methanol, how to fill out the numerous pages of forms over a mouse bite, how PBS tastes like, how not to defrost a freezer, or how expensive the metal cart in the lab is. I am certain the knowledge you have instilled in me will be an asset to my success in the future. Being serious now, thanks for being there to help me with all things big and small, and being the comedic relief in the lab. I can?t imagine how it would be like if you weren?t the lab manager of the Conway lab  Alice, Jesi, Josh, Linnette: I?ve known you all for a relatively short time, but they have all been fun times. I?m not kidding you, the picture you (with Sahana and Victor) took and sent to me before my ISTH presentation made my day. Every time I got nervous I thought of the picture, and I instantly calmed down, not even exaggerating. Thanks for all your support, encouragement, and friendliness.  To all my friends outside of the lab, thanks for all your support and for believing in me. Special thanks to my best friend Cindy who always listened to me and had to put up with my pseudo-debates with her, and also to Grace who always supported me, cheered me up, and encouraged me to do what I love.  To my family, thanks for always being so supportive in every way possible, and for tolerating me as I hermit in my room completing my thesis. Without your encouragement, I might be doing something else right now with my life instead of something I love. Thank you for your guidance in life, and giving me the freedom to decide which path to take at the fork in the road.  Thank you, reader, for reading this long list of acknowledgements. Now, sit back and enjoy reading the rest of my thesis. It has truly been enjoyable working on this, and I hope you will find my project as interesting as I did.      xii  Dedication To my family, who has never ending support for me.     1  1. Introduction  Evolution has provided higher order organisms ways to protect themselves from acquiring infections through wounds that open to the environment. The coagulation system closes the wound, while the immune system such as complement wards off any invading pathogens. For an even more effective and rapid response, effectors of coagulation and complement engage in cross-talk to support the cause. However, mechanisms of the interplay have not been well characterized. In this thesis, I describe a molecule that may be a novel molecular bridge that connects coagulation and complement. 1.1. Coagulation 1.1.1. Overview of Coagulation Activation The coagulation system consists of a tightly regulated cascade of proteolytic activation events that ensure rapid response and repair to a localized area of vascular damage. It involves the sequential activation of a series of zymogens by proteases to yield a fibrin clot that is cross-linked by the transglutaminase FXIIIa. While the classical waterfall cascade of coagulation describes two distinct and independent pathways,1,2 the current cell-based model of coagulation is divided into three phases: initiation, amplification, and propagation (Figure 1.1).3 1.1.1.1. Initiation  During this phase, low amounts of active coagulant factors are generated. This phase begins with injury to the endothelial cell layer lining the inside of the blood vessel, exposing blood to subendothelial cells bearing tissue factor (TF). TF binds to FVIIa,   2  found at low concentrations in the blood, or to FVII and promotes its autoactivation.4 The TF/FVIIa complex can then activate trace amounts of FIX5 and FX. FXa complexes with FVa (derived from activated platelets6 or from plasma FV activated by thrombin or FXa7), forming the prothrombinase complex that cleaves prothrombin to thrombin.  1.1.1.2. Amplification The low amounts of thrombin generated on the TF-bearing cells participate in the amplification phase, which sets the stage for a burst in thrombin generation during the propagation phase. Thrombin activates platelets, causing the release of procoagulant contents from granules and flipping of anionic phospholipids to the platelet exterior, which is essential for the efficient assembly and activity of the prothrombinase and tenase complexes.8 Furthermore, thrombin activates the procoagulant cofactors FV and FVIII, as well as the FXI zymogen.9,10 These events allow for enhanced thrombin generation required for clot formation that cannot be sufficiently met by the initiation phase alone. 1.1.1.3. Propagation  The last phase in the model, the propagation phase, occurs on the activated platelet surface expressing anionic phospholipids. FXIa, activated by thrombin on the platelet surface,9,10 activates FIX; FIXa, activated by FXIa or by TF/FVIIa, complexes with FVIIIa (activated by thrombin) to form a tenase complex that activates FX, and finally, FXa complexes with FVa (from activation by thrombin or FXa) to form the prothrombinase that generates additional thrombin. The generated thrombin feeds back into the amplification and propagation phases and results in even more thrombin.   3  Ultimately, this leads to thrombin-mediated cleavage of fibrinogen to fibrin, and thrombin-mediated activation of the transglutaminase FXIIIa which cross-links fibrin to yield a stable clot. 1.1.1.4. Factor XII  Although not included in the cell-based model of hemostasis,3 the serine protease FXIIa can also activate FXI. In the original waterfall cascade model of coagulation, FXII is the initiator of the intrinsic pathway.1,2 However, the protease appears to be dispensable for hemostasis, as patients deficient in FXII exhibit prolonged activated partial thromboplastin time but no bleeding abnormalities.11 Despite this, it appears to be important for thrombosis since mice deficient in FXII are protected from arterial thrombus formation and ischemic brain injury.12,13 The conundrum becomes more complicated by the elusiveness of a known physiologic activator of FXII. It was only recently that polyphosphate (polyP) and extracellular RNA were proposed to be physiologic activators of FXII;14?16 the status of collagen remains disputed.17?20 1.1.2. Examples of Negative Regulators of Coagulation 1.1.2.1. Antithrombin/Heparin  Antithrombin III (AT) is a key regulator of coagulation as it potently suppresses thrombin, FXa, and FIXa activity.21 As a serpin, it inhibits the proteases through an irreversible covalent interaction, forming a 1:1 AT-protease complex. Its activity is potentiated by the polyanionic molecules heparan sulfate, a glycosaminoglycan (GAG) that can be found on cell surfaces, or heparin, a soluble form of the molecule.22 Both   4   Figure 1.1 Schematic representation of the cell-based model of coagulation. Coagulation is initiated by the exposure of tissue factor to blood, leading to the generation of low amounts of thrombin. Thrombin amplifies the response by activating platelets, cofactors and proteases required for more robust thrombin generation in the propagation phase, resulting in the formation of a stable fibrin clot. TF, tissue factor; FII, prothrombin; FIIa, thrombin; Fg, fibrinogen; Fn, fibrin.   5  unfractionated heparin (UFH) and low molecular weight heparins (LMWHs) are used clinically as anticoagulants.  The mechanisms by which AT inhibits thrombin and FXa are not identical. In both cases, AT binds heparin (or heparan sulfate) through ionic interactions. The GAGs also bind thrombin and FXa and act as bridges, bringing AT together with the proteases. This is the sole mechanism by which thrombin is inhibited by AT.23 However, the additional requirement for FXa and FIXa inhibition is that AT must specifically bind to a pentasaccharide moiety on the GAGs to induce a conformational change that makes it receptive to their binding.23,24 Therefore, long-chain UFH that can form a better bridge can effectively inhibit both thrombin and FXa, while LMWHs with shorter lengths are best at inhibiting FXa.25 Fondaparinux, a synthetic heparin pentasaccharide, can only inhibit FXa.26 1.1.2.2. Tissue Factor Pathway Inhibitor  Tissue factor pathway inhibitor (TFPI) is the physiological regulator of the initiation phase of coagulation.27 It is constitutively expressed by microvascular endothelial cells and released as a plasma protein,28 but a cell-associated form has also been reported.29 Its reversible inhibitory effects on coagulation are two-fold: it can directly inhibit FXa,30 which is enhanced by the presence of protein S,31 and it can inhibit the TF/FVIIa/FXa complex, preventing both FXa activity and additional activation of FX.32    6  1.1.2.3. Thrombomodulin/Activated Protein C  Thrombomodulin (TM) is a membrane glycoprotein on endothelial cells that serves to protect the vasculature from the basal activation of coagulation.33 It primarily acts as a cofactor for thrombin, allowing the protease to bind and activate the zymogen protein C to activated protein C (APC).34 APC, with its cofactor protein S, negatively regulates coagulation by proteolytically inactivating the cofactors required for intrinsic tenase and prothrombinase complex function, namely FVIIIa and FVa, consequently putting a stop to thrombin generation.35 As such, TM effectively changes the effector function of thrombin from procoagulant to anticoagulant. TM also promotes the inhibition of thrombin by serpins.36?38 1.1.3. Fibrinolysis  The resolution of the coagulation response is the breakdown of the fibrin clot through a process called fibrinolysis. The processes can be distinguished into two phases, the initial slow phase and then a fast phase. In the slow phase, tissue plasminogen activator (tPA) released from endothelial cells binds to fibrin with low affinity and activates low quantities of plasminogen to plasmin. Plasmin cleaves fibrin at the carboxyl end of basic residues, weakening the fibrin structure and in some cases exposing C-terminal lysines. C-terminal lysines are positive regulators of fibrinolysis by being cofactors for tPA and plasminogen binding to fibrin, resulting in up to 30-fold accumulation of plasminogen on the clot surface.39 Therefore, during the fast phase, the partially degraded fibrin greatly accelerates the rate at which plasmin is formed. Plasmin, as part of a feed-forward mechanism, also cleaves tPA and enhances its ability to activate plasminogen not bound to fibrin.40 Moreover, plasmin can convert Glu-  7  plasminogen that is bound to C-terminal lysines to Lys-plasminogen, which has higher affinity for tPA.41 The purpose of the fast phase is to generate a burst of plasmin for effective degradation of the fibrin clot. 1.1.4. Thrombin Activatable Fibrinolysis Inhibitor  Thrombin activatable fibrinolysis inhibitor (TAFI) is a zymogen produced by the liver that can be proteolytically activated to TAFIa, a process most efficient when thrombin is complexed with TM.42 TAFIa has carboxypeptidase activity, allowing it to cleave C-terminal arginine and lysine residues.43 As mentioned above, C-terminal lysine residues on fibrin generated by plasmin act as cofactors for tPA and plasminogen binding on the partially degraded fibrin surface. TAFIa removes these lysine and arginine residues, resulting in 1) reduced plasminogen binding to fibrin,44 2) reduced tPA-mediated activation of plasminogen,45 3) reduced generation of Lys-plasminogen,46 and 4) prolonged clot lysis time. In fact, above a certain threshold of TAFIa activity determined by the rate of plasminogen activation and plasmin inhibition, TAFIa can keep lysis at the slow phase and stop fibrinolysis altogether.47?49 However, TAFIa undergoes spontaneous thermodynamic decay and its function is self-limiting. 1.2. Complement System  Complement is also a blood-borne serine-protease cascade of reactions that is evolutionarily related to coagulation, probably derived from a common ancestral cascade. As such, they engage in several cross-talk pathways that will be discussed in a later section.   8  1.2.1. Overview of Complement Activation In contrast to coagulation, the complement system, comprised of over 30 different soluble and cell surface proteins, is a component of innate immunity that primarily modulates the inflammatory response. In addition to enhancing inflammation via signalling molecules, one of the unique consequences of complement activation is the destruction of target cells in the absence of immune cells. This is achieved by a cascade of serine protease activation steps that leads to the formation of a pore-like structure called the membrane attack complexes (MAC) on the target cell surface, allowing water influx and resulting in the lysis of the cell. Target cells may include foreign cells such as bacteria, damaged host cells, or even otherwise healthy host cells that lack appropriate protective mechanisms. Circulating complement proteins are mostly synthesized by the liver as pro-proteins and become activated upon encountering an activating surface. These complement initiating factors can activate complement primarily via three pathways: classical, lectin, and alternative pathways. All three pathways converge first at the transformation of C3 by the C3-convertase, to C3b with the release of the anaphylatoxin, C3a. This leads to the terminal pathway, which involves the non-enzymatic assembly of the MAC. 1.2.1.1. Classical Pathway The classical pathway (CP) is primarily activated by immune complexes (e.g. antibodies) on the surface of a target cell, and thus represents a bridge between the innate and the adaptive immune system. However, some molecules such as lipopolysaccharide, DNA, lipoproteins, and chondroitin sulfate may also activate this   9  pathway, and C-reactive protein instead of immunoglobulins can recognize the antigen to activate complement.50 The initiating molecule of the CP is C1q, which binds to the cell surface target that has been opsonised by immunoglobulins (specifically IgG or IgM). Binding of C1q to a ligand leads to the recruitment of 2 molecules each of the serine proteases C1r and C1s. C1r bound to C1q on the surface autoactivates, and activated C1r in turn proteolytically activates C1s. This fully active pentameric structure is called the C1 enzyme complex. C1s of the active C1 enzyme complex first cleaves C4 into C4a and C4b. C4b contains a highly reactive thioester bond that allows it to covalently attach onto the nearby activating surface. Surface-bound C4b then fixes C2, facilitating its cleavage to C2a and C2b by the adjacent C1s-containing C1 enzyme complex. C2a in association with C4b is referred to as the classical/lectin pathway C3-convertase (C4b2a) which is critical for subsequent activation steps in the pathway. The CP is important for the elimination of several bacteria (e.g. Streptococcus pneumoniae)51 and apoptotic cells.52,53 Deficient activation of the CP is associated with autoimmune diseases, such as systemic lupus erythematosus.54 1.2.1.2. Lectin Pathway Similar to the classical pathway, the lectin pathway (LP) leads to the activation of C2 and C4 to produce the same C3-convertase. However, rather than an antibody-dependent pathway, mannose-binding lectins (MBL) produced by the liver or ficolins bind to exposed mannose residues on pathogen surfaces. This leads to activation of MBL-associated serine proteases (structurally similar to C1r and C1s) including MASP-1 and MASP-2. These serine proteases can then activate C2 and C4 to form the C4b2a   10  C3-convertase. The LP is particularly important for resistance to pneumococcal and parasitic infections.55,56  1.2.1.3. Alternative Pathway The alternative pathway (AP) is arguably the most important activation pathway of complement due to it being constitutively active, and because it is responsible for the majority of C3a and C3b generation through an amplification loop. Constitutive activation of C3 is the result of the so-called ?tickover?, the spontaneous hydrolysis of the reactive thioester on C3 in solution, yielding a C3b-like product, C3(H2O). Most is rapidly inactivated by factor I, using factor H (FH) as a cofactor. However, it can function similarly to C3b, to which the serum protein factor B (FB) can bind. This causes FB to undergo a conformational change that exposes a cleavage site for factor D (FD), a constitutively active circulating serine protease. Cleavage by FB by FD generates two fragments of FB ? Ba and Bb. The Bb fragment, in complex with C3(H2O), cleaves C3 into C3a and C3b. The newly generated C3b exposes the reactive thioester bond so that it can covalently bind to amino and hydroxyl groups on cell surfaces. C3b complexed with Bb (C3bBb) is the C3-convertase of the AP. By this mechanism, in the presence of an appropriate surface for the C3b binding, the amount of C3b generated is greatly amplified. Any C3b generated through the action of the C4b2a C3-convertase from the CP and LP additionally feeds into this pathway, further augmenting complement activation, i.e. cleavage of more C3.   11  1.2.1.4. Terminal Pathway The three pathways converge at the formation of the C3-convertases, whether it is the C4b2a or C3bBb complex. Both enzyme complexes lead to the cleavage of C3 and formation of C3a and C3b. Beyond a certain density of C3b generated, the substrate specificity of the C3-convertases change and the enzyme complexes become C5-convertases. The C5-convertase is the master switch for the formation of the MAC complex in the terminal pathway (TP). C5-convertases cleave C5 into C5a and C5b. C5b immediately interacts with C6 to form a stable C5b,6 complex. Recruitment of C7 to the complex results in the exposure of a hydrophobic tail on C7 that can insert into the outer leaflet of a nearby cell membrane. C8 then binds to the C5b-7 complex, which anchors the complex firmly to the lipid bilayer. Finally, the addition and polymerization of C9 on the complex leads to functional MAC formation that can destroy the target cell (Figure 1.2). The number of C9 molecules incorporated into each functional MAC is variable and ranges from 3-18 C9 molecules.57?59 1.2.2. Functional Roles of Complement 1.2.2.1.  Extracellular Destruction of Cells  A major function of complement is to eliminate invading pathogens and damaged host cells, without the necessity of immune cells by inserting multiple pore-like MACs through the target cell membrane. This leads to osmotic lysis of the target cell. Sufficient  MAC deposition on the target cell ? estimated to be approximately 850 complexes for erythrocytes60 ? results in cell lysis.   12   Figure 1.2 Schematic representation of the complement cascade. Complement is activated via the classical, lectin, or the alternative pathways. All three pathways converge at the formation of the C3 convertases which cleave C3, yielding C3a and C3b. C3b further participates in the formation of the C5 convertases which similarly cleaves C5 into C5a and C5b. C5b immediately complexes with C6 to form a stable C5b,6 complex. The addition of C7 allows the complex to attach on to a nearby membrane, and the incorporation of C8 anchors the complex to the lipid bilayer. The polymerization of C9 on the C5b-8 complex forms the functional pore-like membrane attack complex (MAC), which allows for the osmotic lysis of the target cell.     13  1.2.2.2. Enhancement of Inflammation  Cleavage fragments throughout the complement cascade may not participate in the formation of the MAC but do exhibit pro-inflammatory properties. C3a, C4a, and C5a are anaphylatoxins that modulate the innate immune response by binding to cell surface receptors such as the C3a and C5a receptors, which belong to the family of G-protein coupled receptors. Anaphylatoxins increase the release of pro-inflammatory cytokines, chemotaxis and activation of leukocytes, vascular permeability, contraction of smooth muscle cells, and degranulation of inflammatory cells.61 Potency of these anaphylatoxins is ranked C5a > C3a > C4a,62 and are at least partly inactivated by serum carboxypeptidase N, which cleaves C-terminal arginine residues.63  The pathophysiologic relevance of C4a as an anaphylatoxin in humans is controversial. Human C4a is active in guinea pig models,64?67 but its role in humans is unclear.64 There are conflicting reports regarding C4a signalling, with some suggesting the involvement of the C3a receptor and others suggesting the presence of a C4a-specific receptor.65?68 Considering its poor potency and its rapid deactivation by carboxypeptidase N, some investigators prefer to exclude C4a from the list of anaphylatoxins.69 1.2.2.3. Opsonisation of Target Cells  Complement directly promotes cell-mediated removal of target cells by enhancing phagocytosis by leukocytes. C1q, C3b, iC3b, C3d, C4b, and C4d are all capable of opsonising the target surface and promoting phagocytosis.70 Three complement receptors are present on cell surfaces that can bind to these molecules. Complement receptor 1 (CR1), expressed by all leukocytes and B-lymphocytes,   14  recognizes primarily C3b and C4b and, to some extent, iC3b; complement receptor 2 (CR2) is primarily expressed by B-lymphocytes and can recognize iC3b, C3d, and C3b; and complement receptor 3 (CR3) is present on all leukocytes and natural killer cells, and binds iC3b.70  1.2.3. Negative Regulators of Complement Without negative regulators, the assembly of the MAC will occur on all cell surfaces, including host cells. Thus, to protect the host cell from complement-mediated destruction, host cells require mechanisms to prevent complement activation on its own surfaces. Various soluble and membrane proteins are known regulators of complement. 1.2.3.1. Soluble Inhibitors of the Classical and Lectin Pathways C1 esterase inhibitor (C1-INH) is a plasma serine protease inhibitor (serpin) that inhibits the C1 complex. It covalently interacts with C1r and C1s and prevents their ability to catalyze the activation of C2 and C4 to propagate complement activation through the CP. It can also similarly inactivate MASP-2 to prevent LP activation.71 C4-binding protein (C4BP) has ?decay accelerating? activity by increasing the rate at which the short-lived C3- and C5-convertases of the CP and LP are uncoupled. It binds C4b and promotes its dissociation from C2a. 1.2.3.2. Soluble Inhibitors of the Alternative Pathway Factor H (FH) is a serum protein that has no enzymatic activity but serves as a cofactor for the inhibitory serine protease factor I (FI). FH binds C3b and exerts three functions: 1) it prevents FB from binding to C3b, thereby interfering with formation of the C3-convertase; 2) it binds preformed C3- and C5-convertases and facilitates the release   15  of Bb from the complex, which effectively destroys convertase function, and 3) it acts as a cofactor for FI-mediated cleavage of C3b to an inactive product known as iC3b, which cannot participate in convertase formation.72 Because of its potent ability to affect both C3- and C5-convertase formation, FH is recognized as the main fluid-phase negative regulator of the AP. FH also participates in recognizing between self and non-self. The tickover mechanism of the alternative pathway indiscriminately deposits C3b onto host and foreign cells, and whether C3b is inactivated or continues on to amplify the alternative pathway depends on the affinity of C3b and the particular cell surface for FH.73,74 C3b bound to activators of the alternative pathway exhibit reduced affinity for FH,73?75 while C3b deposited near polyanions on host cells such as glycosaminoglycans or sialic acids ? generally absent from microbial surfaces ? reverses this inhibition in a FH-dependent manner.76 In addition to FH, FI can also bind other cofactors such as complement receptor 1 (CR1), membrane cofactor protein (MCP/CD46), and C4BP. FI uses these cofactors to markedly enhance its ability to cleave and inactivate its substrates C3b and/or C4b.77 Its involvement in the inactivation of both C3b and C4b indicate its ability to hinder all the complement pathways. 1.2.3.3. Membrane Inhibitors of Complement Decay accelerating factor (DAF or CD55) is a glycosylphosphatidylinositol (GPI)-linked plasma membrane-bound protein that prevents the assembly of the convertases. It recognizes cell-surface C3b and C4b and interferes with the binding and activation of FB and C2,78 thus inhibiting complement activation via all pathways.   16  CD59 (also referred to as homologous restriction factor) is a GPI-linked membrane protein that forms one of the last lines of defense against lysis of self cells (termed homologous lysis).79 It inhibits MAC assembly and function by preventing the binding or polymerization of C9 to C5b-8.79?81  Clusterin and vitronectin are serum proteins that also target the terminal pathway of complement. Following the assembly of C5b,6 with C7, clusterin and vitronectin can prevent the C5b-7 complex from attaching to the cell membrane, leading to aggregation of complexes and loss of complement activity. They can also prevent C9 interaction with C8 as well as prevent the polymerization of C9.82?84 1.2.4. Properdin  Properdin, or factor P, stabilizes the C3 convertase of the AP (C3bBb). Native C3bBb (with Mg2+ as its cofactor) has a half-life of approximately 90 seconds,85 but properdin can stabilize this complex by 5- to 10-fold.86 In addition, unlike the majority of other complement proteins, properdin is primarily synthesized and secreted by activated leukocytes (monocytes/macrophages, neutrophils)87,88 and T cells89 rather than hepatocytes. This ensures that properdin does not enhance the convertase activity on host cells at rest, but only at local areas of infection and inflammation.  Recently, properdin has been shown to be a pattern recognition molecule that can bind to microbial surfaces, apoptotic and malignant host cells directly, and in the absence of other complement proteins.90,91 Properdin can also recruit fluid phase C3b (e.g. generated by the tickover mechanism) and provide a scaffold for convertase formation.91 Properdin can therefore enhance alternative pathway function by stabilizing the C3 convertase and recruiting C3b to the target surface for convertase assembly.   17  1.2.5. Polyion Effects on Complement 1.2.5.1. Polycations The effects of polycations on complement have not been well characterized. Several reports have demonstrated that they can potentiate terminal pathway activity, while others have shown that they can inhibit complement activity in general. Synthetic polycations such as poly-L-lysine and protamine sulfate, as well as naturally-derived lysine-rich histones and myelin basic proteins appear to antagonize the inhibitory effects of serum inhibitors of reactive lysis (designated C567-INH) and thus potentiate lytic activity.92?94 Properdin is also a cationic protein that stabilizes the C3 convertase of the alternative pathway and thus promote complement activation.86 However, several studies also reported that poly-L-lysine and polycationic proteins can inhibit complement-mediated lysis95 through modulation of C3 convertase formation or activity.96?98 It has been suggested that polycations are primarily responsible for modulating the activity of the classical pathway.95 1.2.5.2. Polyanions Not only is heparin an anticoagulant, but it has long been known to inhibit complement activity as well through various mechanisms, including inhibition of the C1 enzyme complex,99?105 C3 convertases of the classical and alternative pathways,76,106?110 and the assembly of the MAC.94,111 Its suppressive effect has also been observed in vivo.112?115 In contrast to polycations, polyanions such as heparin primarily suppress the alternative pathway.95   18  1.3. Coagulation and Complement Cross-Talk The complement and coagulation pathways are both serine protease cascades. Phylogenetic studies support the notion that they are evolutionarily related, probably derived from a common ancestral cascade.116 The coagulation system and the innate immune system are often activated simultaneously by similar stimuli. This is thought to have an evolutionary purpose ? the simultaneous activation of both systems results in local thrombus formation which can trap and prevent invading pathogens from entering the systemic circulation, while the immune system removes the threat. It would therefore not be a surprise that proteases from one cascade might affect the activity of proteins from the other, thus blurring the boundaries between complement and coagulation. 1.3.1. Coagulation Factors Modulate Complement Activity  Several coagulation-related proteases influence the activity of complement. For example, in vitro, FIXa, FXa, FXIa, thrombin, and plasmin cleave C3 and C5, releasing their activation fragments.117 In vivo, C3 knockout mice can still generate C5a in the absence of the C3b-dependent C5 convertases via thrombin-mediated cleavage of C5.118 Moreover, C5 that has been cleaved by thrombin can participate in MAC assembly and displays enhanced lytic activity.119 FXIIa activates C1 and the classical pathway,120,121 and activated platelets promote classical and alternative pathway activities.122,123  There is also cross-talk in negative regulators. For example, thrombin induces CD55 expression on endothelial cells in a PAR-1-dependent manner,124 which in turn   19  prevents complement activation against host cells. TAFIa inactivates the anaphylatoxins, C3a and C5a (Figure 1.3).125 1.3.2. Complement Components Modulate Coagulation  Complement components can also influence coagulation.  MASP-2 and MASP-1 can cleave and activate thrombin and FXIIIa, respectively.126,127 Platelets can be activated by C1q,128 C3a,129,130 or by sublytic MAC deposition on the surface,131 causing granule secretion, microparticle formation, thrombin generation, and consequently, clotting.132?134 In addition, the anaphylatoxin C5a, as well as the cytolytically inactive form of the MAC (i.e. soluble MAC) can induce TF expression on endothelial cells and neutrophils.135?137 C5a can also induce the expression of plasminogen activator inhibitor-1 on mast cells and basophils,138 thereby suppressing fibrinolysis.  1.4. Polyphosphate  Polyphosphate (polyP) is an ancient molecule that pre-dates the first organisms, produced by the high temperatures of volcanoes and deep oceanic steam vents. As life on earth developed, polyP also followed the cells and organisms throughout evolution. Organisms have utilized polyP for various functions, from simple metabolism to even cell signalling in higher organisms. 1.4.1. Biochemistry, Synthesis, and Degradation PolyP is an evolutionarily conserved molecule that is found in all cells, including bacteria, fungi, insects, plants, and mammals. It consists of a linear chain of inorganic orthophosphates linked by high energy phosphoanhydride bonds. Lengths vary between and within organisms, ranging from tens to many hundreds of orthophosphates   20   Figure 1.3 Thrombin-mediated interplay between coagulation and complement. Thrombin is a key player in coagulation-complement cross-talk. Thrombin can cleave C5 into C5a and C5b. C5a in turn can induce endothelial cells to express tissue factor and enhance the coagulation response. C5b generation leads to formation of the membrane attack complex, where it can combat invading pathogens as well as activate platelets, leading to classical and alternative pathway activation. Thrombin also protects the host by inducing endothelial cells to express CD55, and by activating thrombin-activatable fibrinolysis inhibitor to reduce anaphylatoxin activity. FIIa, thrombin; TAFI, thrombin-activatable fibrinolysis inhibitor; TM, thrombomodulin; TF, tissue factor; MAC, membrane attack complex; CP, classical pathway; AP, alternative pathway.   21  per chain of polyP. Prokaryotic polyP comprises chains that range from hundreds to thousands of orthophosphate units per chain, while eukaryotic polyP has chains that are shorter, i.e., tens to low hundreds of units long (Figure 1.4). In bacteria and unicellular eukaryotes (e.g. protozoans), the biosynthesis of polyP occurs through enzymes called polyphosphate kinases (PPK). The most widely conserved and best characterized PPK is PPK1. PPK1, first found in Echerichia coli and  then in other bacteria, including Neisseria meningitidis and Pseudomonas aeruginosa, is a membrane bound enzyme which catalyses the polymerization of polyP from ATP in a reversible manner, favouring polyP synthesis.139?142 However, while PPK1 is the enzyme responsible for the majority of polyP synthesis in P. aeruginosa, mutants that lack PPK1 still retain approximately 20% of polyP compared to wildtype.143 PPK2 has since been identified to be another highly conserved polyphosphate kinase in bacteria that can still reversibly synthesize polyP from both ATP and GTP, but preferentially catalyzes the reverse reaction.142,144 A recently discovered third polyphosphate kinase found in the bacterium Silicibacter pomeroyi, PPK3, primarily uses polyP for the phosphorylation of pyrimidine nucleotide disphosphates such as CDP.145 Despite being well characterized in bacteria and unicellular eukaryotes, the biosynthesis of polyP in higher order eukaryotes has remained elusive. Yeast and animal cells can synthesize polyP from phosphate in the media, but the enzymatic activity is not found in whole-cell lysates.146 Interestingly, a Ca2+-ATPase purified from human erythrocytes has polyphosphate kinase- and phosphotransferase-like activities.147 Consistent with this, polyP production in mammalian cells has been linked     22          Figure 1.4. Chemical structure of polyphosphate. Polyphosphate is a polyanionic string of inorganic phosphates that is found in all organisms and can range from tens to hundreds of units long. In bacteria, it is synthesized by polyphosphate kinases and degraded by polyphosphatases.   23  to mitochondrial respiration, as inhibition of the mitochondrial ATP-synthase also blocked polyP synthesis.148 PolyP can also be targeted by enzymes for unidirectional degradation. The two primary enzymes responsible for its degradation are exopolyphosphatase (PPX) and endopolyphosphatase (PPN). PPX degrades polyP by liberating a terminal orthophosphate,149 while PPN cleaves internal phosphoanhydride bonds to yield smaller polyP chains that are approximately 60 units long.150 Mammalian intestinal alkaline phosphatases are also potent exopolyphosphatases capable of cleaving terminal phosphates from polyP chains.151 1.4.2. Cellular and Tissue Localization  In prokaryotes, polyP is primarily stored within intracytoplasmic volutin granules (also called polyP granules), so-called due to its original discovery in the bacterium Spirillum volutans.152 They can also be found on the cell surface where they may strengthen capsule function, as seen in N. meningitidis.153   Volutin-like granules are also observed in eukaryotic cells, but referred to as acidocalcisomes for its membrane-bound structure and their high intra-organellar calcium and proton levels.154 Acidocalcisomes are present in algae (e.g., Chlamydomonas reinhardtii),155 protists (e.g., Plasmodium berghei),156 bacteria (e.g., Agrobacterium tumefaciens),157 and even human platelets (as dense granules),158 indicating that these organelles have been conserved through evolution from prokaryotes to multicellular eukaryotes. Other subcellular locations of polyP include lysosomes,159 nuclei, vacuoles, and the mitochondria, each possessing characteristic chain lengths.160   24   PolyP in the rat has been localized to the brain, heart, kidneys, liver, and lungs.161 Human bone is also an abundant source of polyP.162 Human platelets also store polyP within their acidocalcisome-like dense granules.158 Human primary myeloma cells also contain polyP in the nucleoli, which can translocate to the cytoplasm following treatment with an RNA polymerase inhibitor.163  Several fibroblast and mast cell lines also express polyP.161,164 PolyP has also reportedly been found in plasma and serum,165 and has a circulation half-life of approximately 90 minutes.16 1.4.3. Function 1.4.3.1. Prokaryotes The functions of polyP in prokaryotes have been well-defined. As a phosphate-based molecule, polyP chelates metal ions and thus can reduce the toxicity of heavy metal ions such as Zn2+, Fe3+, Cu2+, and Cd2+. PolyP participates in the regulation of intracellular Ca2+ in yeast by acting as a Ca2+ sink in the vacuoles.166 Exogenous polyP has antibacterial properties, again by chelating Ca2+ and Mg2+ that are essential for the integrity of Gram-positive bacterial cell walls.167?169 PolyP stores inorganic phosphate that can later be hydrolyzed for cellular functions such as energy production (e.g. in the form of ATP)170 or substrate phosphorylation via polyphosphate kinases or phosphotransferases.171 Hydrolyzed polyP can also be an effective buffer against alkali stress.172 Several additional functions of polyP have been described. Transformation competent E. coli rely on a ?-polyhydroxybutyrate-Ca2+-polyP complex for the uptake of DNA from the environment into the cell for genetic recombination.173?175 A similar complex has been found on human erythrocytes and implicated in polyP synthesis and   25  transferase activity. PolyP is also involved in gene regulation as it is implicated in growth and survival,176?178 quorum sensing, biofilm formation, and virulence.143,179 Enzymatic degradation of polyP also reduces mitochondrial metabolism,180 highlighting its importance for normal function of the respiratory chain. PolyP on the cell capsule of Neisseria bacteria is important for resistance against human serum-mediated killing.181,182 1.4.3.2. Eukaryotes  The role of polyP in higher organisms has been explored to a lesser extent. Isolated reports indicate that polyP promotes the proliferation of cancer cells and human fibroblasts.183,184 Somewhat conflicting, others have shown that polyP promotes apoptosis of plasma cells and suppresses metastasis and angiogenesis.185,186 In these situations, the mechanisms by which polyP functions are entirely unknown.  The roles of polyP in bone formation and in coagulation have been better characterized. PolyP is found in bone tissue,162 where it promotes the differentiation of mesenchymal stem cells into osteoblasts,187,188 regulates calcification and hydroxyapatite deposition,189?192 and enhances bone formation and regeneration.193,194 In coagulation, polyP is secreted from the dense granules of activated platelets and promotes clotting in a chain length-dependent manner195 by directly activating FXII and the contact pathway;15,16 acting as a cofactor for the activation of FV and FXI by thrombin and FXa,16,196,197 and strengthening fibrin (Figure 1.5).198     26   Figure 1.5 Effects of polyphosphate on coagulation. Polyphosphate (polyP) promotes clotting in a chain length-dependent manner. (A) Long-chain polyP (hundreds to thousands of phosphate units long) such as those found in bacteria is very potent at (1) activating FXII, (2) accelerating FV activation by thrombin and FXa, (3) strengthening fibrin, and (4) promoting FXI back activation by thrombin. (B) Platelet-size polyP (60-100 phosphate units long) is best at accelerating FV and FXI activation (2 and 4). Figure adapted from Morrissey et al. 2012, Blood.199     27  1.5. Hypothesis, Goal and Objectives  Neisseria mutants that lack functional PPK or PPX have increased and decreased sensitivity to serum-mediated killing, respectively,181,182 suggesting a role for polyP in suppressing complement activity. PolyP promotes coagulation through various means.15,16,195?198 Since coagulation and complement are connected evolutionarily and functionally,200 it is possible that polyP can represent another mechanism of cross-talk between the systems. Therefore, it is hypothesized that polyP can modulate complement activity. The overall goal of this thesis was to evaluate the role of polyP in complement.  Specific objectives in this thesis include: 1. Evaluate and compare the effects of polyP and monophosphate on global complement activation and resulting hemolytic activity 2. Evaluate and compare the effects of polyP and monophosphate on terminal pathway activity, which is an ion-independent process 3. Elucidate mechanism(s) by which polyP affects terminal pathway activity     28  2. Polyphosphate suppresses complement via the terminal pathway1 2.1. Introduction Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate, linked by phosphoanhydride bonds201?203. It is found in all mammalian cells and lower organisms and localized in lysosomes, dense granules, mitochondria and nuclei. The polymer varies in length from cell to cell and in different organisms, ranging from 60-100 in human platelets, to up to thousands of phosphate units in some bacteria146,171,204. In platelets, there is abundant polyP that is localized in dense granules158 and released upon activation, whereupon it is found in platelet-rich thrombi at concentrations of 1-3 ?M15. At physiologic pH, each internal unit has a monovalent negative charge, and thus the polymers are highly anionic. This property led to the finding that polyP is a physiologic anionic surface on which factor XII, prekallikrein and high molecular weight kininogen, assemble for contact activation of coagulation16. Subsequent studies confirmed that polyP is prothrombotic and pro-inflammatory in in vivo mouse models15,205 and there are multiple steps in the coagulation cascade at which polyP acts to achieve this end15,195,196,198,206?209. The effects of polyP on coagulation are concentration- and size-dependent195. Thus, platelet-sized polyP (P60-100) primarily accelerates thrombin-mediated activation of factor XI and factor V, while larger size                                             1 A version of this chapter is in preparation for publication. Jovian Wat, Jonathan H. Foley, Michael J. Krisinger, Victor Lei, Linnette Mae Ocariza, Gregory A. Wasney, Emilie Lameignere, Natalie C. Strynadka, Stephanie A. Smith, James H. Morrissey, and Edward M. Conway. Polyphosphate suppresses complement activity via the terminal pathway.    29  polyP triggers coagulation via contact activation of factor XII and enhances fibrin polymerization. The observation that polyP modulates coagulation raised the question as to whether polyP also regulates the evolutionarily-related, blood-borne proteolytic cascade, complement. The complement system comprises over 30 soluble and membrane-bound proteins, contributing to innate and adaptive immunity, aiding in the disposal of danger-associated molecular patterns (for reviews210,211). Complement activation, which often occurs in concert with coagulation, is achieved via three pathways ? the lectin pathway (LP), the classical pathway (CP) and the alternative pathway (AP). These converge with C3 convertase-mediated transformation of C3 into C3a and C3b. The C3a anaphylatoxin recruits leukocytes and activates platelets123. C3b deposition on bacteria promotes opsonization by leukocytes and is required for formation of the C5 convertase that cleaves C5 into C5a and C5b. C5b rapidly binds to C6, forming a tight C5b,6 complex, which then binds to C7, yielding the C5b-7 complex. This attaches to the outer leaflet of a target membrane. The subsequent addition of the heterotrimeric C8??? further stabilizes and anchors the now C5b-8 complex to the cell by inducing a conformational change in C8 and burying a hydrophobic tail through the lipid bilayer. Multiple C9 subunits finally join for assembly of the C5b-9 pore-like, lytic membrane attack complex (MAC)212.  Coagulation and complement are tightly regulated and coordinated to limit blood loss, eliminate pathogens and damaged cells, and promote healing. Although often viewed as distinct, they are highly integrated, with several recently identified cross-talk pathways213?221. For example, thrombin activates C5 and enhances formation of the   30  MAC119. Conversely, C5a promotes expression of tissue factor and plays a key role in the pathogenesis of the fetal loss syndome associated with the anti-phospholipid antibody222. Several other biochemical connections have also been documented127,220,223.  In spite of evidence of links between coagulation and complement, the possibility that polyP regulates complement in mammalian systems is unexplored. It has, however, been reported that a Neisseria meningitidis mutant that lacks the polyphosphatase that normally degrades polyP, is resistant to complement-mediated death via the alternative pathway, indicating that polyP facilitates evasion from complement-mediated killing181. I therefore tested the hypothesis that polyP provides a bridge between complement and coagulation224. Using sensitive hemolytic assays, I show that polyP significantly suppresses complement via the terminal pathway and that this occurs in a concentration-dependent, size-dependent, and ion chelation-independent manner. My data indicate that polyP binds to and destablizes C5b,6, thereby interfering with optimal assembly and/or membrane binding of the lytic MAC. The findings reveal a novel mechanism by which complement is regulated, and underline the complex relationship between coagulation and complement. 2.2. Materials and Methods Reagents Chemicals were purchased from Sigma-Aldrich (St. Louis, Missouri, USA) unless otherwise stated. PolyP preparations were synthesized, size-fractionated and quantified by the malachite green assay as described195, with concentrations expressed in terms of phosphate monomers (monomer formula: NaPO3). Mean number or range of   31  phosphate units comprising polyP are indicated in the subscript. Gelatin veronal buffer (GVB, pH7.4), normal human serum (NHS), and human plasma-derived C5b,6, C7, C8, and C9 were from Complement Technology, Inc. (Tyler, Texas, USA). Rabbit erythrocytes (rRBC) were freshly obtained from citrated venous blood following venipuncture performed by Animal Care Services technicians at the University of British Columbia, with approval by the local Animal Ethics committee. Chicken erythrocytes (cRBC) were from Colorado Serum Company (Denver, Colorado, USA). Hemolytic assays were performed in 96-well non-treated microplates from Corning (Amsterdam, Netherlands). SYPRO Orange was from Invitrogen (Burlington, ON, Canada).   Hemolytic Assays Erythrocyte lysis assays210,225 were used to measure complement-mediated hemolytic activity. Complete details are provided in the Supplemental Data section. Lysis was first determined relative to the control of 100% lysis with H2O. For total hemolytic and terminal pathway (TP) assays, the concentrations of NHS and C5b,6 to initiate complement activation were established from pilot studies to obtain ~70-80% erythrocyte lysis at 30 min (see Supplemental Data Figures S1, S2 and S3). Results were normalized to this amount of lysis, and shown in figures as ?relative RBC lysis?. All results are representative of experiments performed in triplicate, a minimum of 3 times. Analytic Gel Filtration, Differential Scanning Fluorimetry  (DSF), and Native Polyacrylamide Gel Electrophoresis (PAGE) Native PAGE, DSF (also referred to as thermal shift assay)226, and analytic gel filtration were used to assess polyP-protein interactions. Methods for the first two are described in Supplemental Data. For DSF, 25 ?L reaction mixtures consisting of 0.4   32  mg/mL protein (0.14 mg/mL for C5b,6), varying concentrations of polyP diluted in HBS (20 mM HEPES, 150 mM NaCl, pH 7.4), and 5x SYPRO Orange were loaded into MicroAmp Fast Optical 96-well Reaction Plates (Applied Biosystems). Samples were heat denatured in an Applied Biosystems StepOnePlus Real-Time PCR System using a ramp configuration starting at 25oC and increasing at 1 Co min-1 to 95oC. Fluorescence (in relative fluorescence units, RFU) was measured every 30 seconds226. Data for each curve were normalized to the maximum and the minimum of the curve. Statistical Analyses Analyses were performed with GraphPad Prism version 5.0 (San Diego, California, USA). Where indicated, one-way ANOVA with Bonferroni?s multiple comparison tests were performed. Results shown are means ? standard error of the mean. Statistical significance refers to p < 0.05. 2.3. Results PolyP suppresses total hemolytic activity in a concentration-dependent manner I first assessed the effects of polyP on total complement-mediated hemolytic activity. NHS diluted to yield 70-80% lysis (see Methods) was pre-incubated with varying concentrations of polyP that comprises more than 1000 orthophosphate units (NaPO3) per chain (polyP>1000), and that optimally promotes coagulation195. Orthophosphate (P1), the monomeric unit of polyP, was used as a control. Reactions were initiated by the addition of rRBC and hemolysis was measured after 30 min (Figure 2.1). P1 and polyP>1000 suppressed hemolysis in a concentration-dependent manner. However, at equivalent molar concentrations of the monomeric form, polyP>1000 was strikingly more effective at suppressing hemolysis. The IC50 (the concentration    33        Figure 2.1 PolyP suppresses total hemolytic activity. rRBC were incubated with 4.5% serum in the presence of increasing concentrations of P1 (?) or polyP>1000 (?). Values were normalized to baseline lysis in the absence of phosphate. Curves were fitted to a nonlinear regression inhibitory dose-response model to determine IC50 for P1 and polyP, shown by dotted lines. Results are representative of 3 experiments, each performed in triplicate.   34  required to achieve half maximal inhibition of lysis) was ~3.5 mM  for P1 and ~0.35 mM for polyP>1000. Since complement activation via the alternative and classical pathways is dependent in part on the presence of ionic calcium and magnesium, it is likely that the observed suppression of total hemolytic activity by monophosphate concentrations exceeding ~5 mM was induced primarily by chelation of those ions. However, chelation would not account for the enhanced activity of polyP as compared to P1, since the ionic strengths of the polyP and P1 at equivalent monomeric concentrations are similar. Nonetheless, to unequivocally determine whether polyP suppresses complement via ion chelation-independent mechanisms, I assessed its effects on the TP.  PolyP but not monophosphate, suppresses complement-mediated lysis via the TP in serum The TP of complement is initiated by the rapid binding of C5b to C6, and the subsequent and sequential binding of C7, C8 and several C9 molecules to form the pore-like C5b-9 MAC. This pathway is ion-independent and thus any effects of polyP would also be independent of its capacity to chelate cations.  I first studied the effects of polyP on the TP of complement in NHS. TP activity in NHS, measured by lysis of cRBC, was initiated by adding a limiting amount of exogenous purified C5b,6 to achieve 70-80% lysis after 30 min. A molar excess of EDTA was used to prevent upstream activation of complement and generation of endogenous C5b,6. In the absence of C5b,6, there was no detectable complement-mediated cRBC lysis (data not shown). P1 at concentrations ranging from 0.1 to 1000 ?M, had no effect on complement-mediated hemolysis in NHS via the TP (Figure 2.2A). In contrast, polyP>1000 inhibited the TP in serum in a concentration-dependent manner,   35  with an IC50 of ~10 ?M polyP>1000 (calculated based on monomeric phosphate) (Figure 2.2A). To verify that the effects observed were dependent on the integrity of the polymers, I pre-treated polyP with calf intestinal alkaline phosphatase (CIAP), an exopolyphosphatase that cleaves polyP into monomeric units151. CIAP alone had no effect on the TP hemolytic activity, whereas CIAP treatment of the polyP>1000 completely abrogated its ability to dampen hemolytic activity via the TP (Figure 2.2B). In addition, co-incubation of up to 2 mM P1 in combination with polyP>1000 at a concentration of 200 ?M had no effect on the suppressive properties of the polyP>1000 (Figure 2.2C). Suppression of TP hemolytic activity is dependent on the chain length of polyP   The effects of polyP on coagulation are dependent on chain length. I therefore tested whether polyP of different chain lengths could suppress TP hemolytic activity. Medium chain length polyP (polyP40-160) had a similar concentration-dependent suppressive effect as polyP>1000 on TP mediated hemolysis (Figure 2.3A). However, shorter length polyP (polyP<30) was less potent, and an IC50 could not be achieved even with 5 mM polyP<30. A wider range of different polyP chain lengths was examined (Figure 2.3B) for effects on the TP. At equivalent 100 ?M concentrations (based on the monomeric form), P1, diphosphate (Na4P2O7) (P2) and triphosphate (Na5P3O10)  (P3) had no effect on TP hemolytic activity. However, polyP with a mean length of 22 orthophosphate units (polyP22), significantly dampened TP hemolytic activity, and the extent of suppression by longer-chain polyP increased in a size-dependent manner.  PolyP depends on early TP components to suppress lytic activity I next examined whether polyP suppresses TP hemolytic activity through direct interaction(s) with TP complement components (C5b,6, C7, C8, or C9) or if other serum  36    37  Figure 2.2 PolyP suppresses terminal pathway hemolytic activity in serum. (A) P1 (?) or polyP>1000 (?) were titrated into the terminal pathway assay in the presence of 2% serum and 250 pM C5b,6. The IC50 for polyP is shown by dotted lines and was determined as in Figure 1. Results are representative of more than 5 experiments, each performed in triplicate. (B) 600 ?M polyP>1000 was incubated overnight with 400 U/mL calf intestinal alkaline phosphatase (CIAP), and then added to the terminal pathway assay at a final polyP>1000 concentration of 100 ?M. The control represents lysis in the absence of CIAP and polyP, but with an equivalent concentration of CIAP digestion buffer. ?CIAP? represents lysis in the presence of CIAP but absence of polyphosphate. The findings indicate that suppression of the TP by polyP requires the integrity of the polymer. Values were normalized to baseline lysis from the control condition. Each column represents quadruplicate data points. (C) 2 mM P1 and 200 ?M polyP>1000 were added singly or in combination in the terminal pathway assay. Values were normalized to baseline lysis from the control condition in which no phosphate was added. Excess monomer could not overcome suppressive properties of polyP. n= 3 independent experiments, each performed in triplicate.     38   Figure 2.3 PolyP suppresses the TP in a size-dependent manner. (A) P1 (?), polyP<30 (?), polyP40-160 (?), and polyP>1000 (?) were titrated into the terminal pathway assay in the presence of 2% serum and 250 pM C5b,6. The IC50 is shown only for polyP>1000. The TP was suppressed in a size-dependent manner. Results are representative of 3 experiments, each performed in triplicate. (B) (Left) 100 ?M of different size polyphosphates were added to the TP assay as above. * reflects comparisons to Control without phosphate (? < 0.001, n=4). (Right) TBE-urea gel, stained with toluidine blue for polyP, shows the relative size distributions of the fractionated polyP.   39  factors are also required. This was achieved by replacing NHS with purified TP components C7, C8 and C9 as the source of complement. TP hemolytic activity in this purified system was measured by sequentially adding cRBC, varying concentrations of polyP, and then purified C5b,6, C7, C8, and C9. Similar to the findings with serum, polyP dose-dependently suppressed erythrocyte lysis, while P1 had no effect (Figure 2.4A). The IC50 of polyP>1000 in this assay system was ~1 ?M. The effect of polyP was not specific to the species of the target erythrocytes, because TP hemolytic activity could also be suppressed by polyP when human erythrocytes were used (data not shown). Overall, the findings indicate that polyP interferes with TP complement-mediated lysis of RBC by binding directly to one or more of the TP complement components or to the target red cell membrane.  PolyP interferes with TP hemolytic activity at early steps in assembly of the MAC I delineated the step(s) in the TP at which polyP interferes with the function of the MAC to lyse erythrocytes. This was achieved by performing the TP assay in which polyP was added at different steps of the reaction, i.e., before C5b,6, after C5b,6, after C7, after C8, or simultaneously with C9 (Figure 2.4B). PolyP>1000 at a concentration of 200 ?M, suppressed lysis to <5% of maximal lysis (i.e. without the addition of polyP), when added prior to or immediately after C5b,6. In contrast, polyP had no effect on cRBC lysis in the TP assay when added after C7, after C8 or with C9. Equivalent results were obtained with polyP60-100 (data not shown). The findings indicate that polyP interferes with optimal MAC function/assembly by destabilizing C5b,6, preventing C5b,6 from interacting with C7, or by causing further downstream TP complexes (C5b-7, C5b-8 or C5b-9) to be unstable or incapable of attaching or inserting into the RBC   40   Figure 2.4 PolyP directly affects the function of terminal pathway components. (A) In a purified system, P1 (?) or polyP>1000 (?) were titrated into the terminal pathway assay in the presence of 20 pM C5b,6 and excess C7, C8, and C9. PolyP suppresses the TP in a similar manner as in serum (Figure 2A). Results are representative of 3 experiments, each performed in triplicate. (B) The terminal pathway assay was performed by sequentially adding cRBC, C5b,6, C7, C8, and C9, with 200 ?M polyP>1000 added at different steps as indicated by arrows below the figure. Values were normalized to baseline lysis in the control condition without the addition of polyP. Each column represents quadruplicate data points. Relative lysis between control, after C7, and after C8 conditions were not statistically significant (? > 0.05).   41  membrane. Once C5b-7 forms, however, polyP can no longer modulate the function of the MAC. PolyP alters the stability of C5b,6 and C6 but not C5 or C7 Using several independent approaches, I investigated whether polyP interacts directly with different components of the TP and whether it alters the stability and thus, the function of these components. In the first, native PAGE was used to assess polyP-protein binding. Platelet derived polyP was previously shown to bind to thrombin (Kd ~ 5 nM) but not to prothrombin196, findings that are evident on native gels (Figure 2.5). Similar to thrombin?s interaction with polyP, I showed that C5b,6, C6 and C7 all exhibited a band shift in the presence of polyP, consistent with a physical interaction under these experimental conditions. In a second approach, with the assistance from Grey Wasney and Linnette Mae Ocariza, we performed gel filtration studies with either C5b,6 or C7 in the presence or absence of polyP>1000. PolyP had no effect on the chromatogram for C7 (Figure 2.6A), but caused a dramatic shift to a higher oligomerized state in the C5b,6 elution profile (Figure 2.6B), indicating a direct interaction of C5b,6 with polyP. The broader C5b,6-polyP peak is consistent with polyP destabilizing and/or inducing aggregation of C5b,6. In the third approach, differential scanning fluorimetry (DSF) was used to determine the thermal stability of the complement proteins in the presence and absence of polyP. This technique involves heat-denaturing proteins and exposing internal hydrophobic regions to the aqueous environment, which are detected by the fluorescent dye SYPRO Orange226. Interactions between a protein and a binding partner change the thermal stability of the protein, observed as a shift in the melting (denaturation)    42          Figure 2.5 PolyP reduces the mobility of terminal pathway proteins in native gels. 2 ?g of protein were incubated with or without 6 ?g polyP>1000 and resolved by native PAGE. The gel was stained with Coomassie blue for detection. PolyP binds to and causes a shift in the migration of thrombin (IIa), but does not bind to or affect migration of prothrombin (II). PolyP causes a gel shift in C5b,6, C7 and C8. Results are representative of 4 independent experiments.     43   Figure 2.6 Gel filtration to assess interactions of C5b,6 and C7 with polyP. The effect of polyP on the gel filtration elution profiles of C5b,6 and C7 was evaluated. (A) The presence of polyP>1000 (10mM) does not affect the retention time or elution profile of C7. (B) P1 (monoP) (10mM) does not affect the retention time or elution profile of C5b,6. However, the presence of polyP (10mM) binds to and shifts C5b,6 to a more highly oligomerized or aggregated state.   44  curve. In the absence of polyP, the melting curve of C5b,6 (Figure 2.7A) is characterized by three transition phases (arrows), likely due to differential thermal stabilities between C5b,6 domains. When C5b,6 was incubated with polyP>1000, a dose-dependent leftward shift in the second transition phase at 59.5 oC was observed (expanded in Figure 2.7B), strong evidence that polyP binds to a distinct domain on C5b,6. PolyP had no effect on the melting curve of C5, but did cause a dose-dependent destabilization shift in the C6 melting curve (Figures 2.7C and 2.7D, respectively), suggesting that polyP interacts with C5b,6 via C6. A minimal shift in the C7 curve was observed only in the presence of a high concentration (1 mM) of polyP (Figure 2.7E). PolyP60-100 similarly affected the thermal stability of each of the complement proteins (not shown). The addition of 1 mM monophosphate (P1) had no effect on the thermal stability of any of the proteins. 2.4. Summary The key findings in this thesis are that polyP dampens complement via the terminal pathway, that this occurs in a concentration- and polymer size-dependent manner, and that the mechanisms involve, at least in part, destabilization of C5b,6, thereby interfering with optimal lytic function of the C5b-9 MAC. This is the first account of a complement-inhibitory function for polyP in a eukaryotic system. Previously, Zhang et al.181 used mutant forms of Neisseria meningitidis that lack an exopolyphosphatase responsible for cleaving cellular polyP, and showed that these bacteria were resistant to complement-mediated killing. They demonstrated that exogenous polyP could protect wild-type bacteria from complement-mediated death. Thus, in these bacteria, polyP is a weapon of survival, utilized to overcome host innate immunity182. My findings are in line    45   Figure 2.7 PolyP alters the thermal stability of C5b,6. C5b,6 (A, B), C5 (C), C6 (D), and C7 (E) were individually incubated with a range of concentrations of polyP>1000 and relative fluorescence (RFU) was measured as the proteins were thermally denatured. An increase in RFU indicates protein denaturation. Controls are protein without phosphate. For C5b,6 (A), arrows with numbers indicate the three transition phases exhibited during denaturation in the absence of polyP. B focuses on the 2nd transition phase of C5b,6 (boxed region from A). PolyP induces concentration-dependent denaturation of C5b,6 and C6, but not C5 or C7. Results were similar with polyP60-100 (not shown). Results are representative of 3 independent experiments.   46  with theirs, supporting the notion that polyP in humans may dampen the innate immune response via suppression of complement.  Previous studies established that polyP initiates activation of coagulation and inflammation via the contact pathway, accelerates the generation of FVa by FXa and thrombin, and enhances the integrity and stability of the fibrin clot16,198. The biochemical studies were validated in vivo in mice, where injections of polyP caused a thrombotic and pro-inflammatory response, the latter mediated by increased generation of FXIIa, kallikrein, and bradykinin15,205. That polyP can inhibit complement and limit the innate immune and inflammatory response, appears at odds with its profound and well-characterized procoagulant and pro-inflammatory properties. How can this be explained? It is not unprecedented, at least in the coagulation system, for a molecule to have dual and functionally opposing activities that are delicately balanced in time and space to maintain homeostasis. A perfect example is thrombin. Through dynamic allosteric changes and interactions with key cofactors, receptors and substrates, thrombin elicits procoagulant and anticoagulant properties that are orchestrated to ensure a rapid and localized response to injury and bleeding, while preventing widespread and prolonged thrombosis (reviewed in227,228). Thrombin is also promiscuous in terms of its role in regulating inflammation and innate immunity. It induces the release of pro-inflammatory cytokines229?231, is chemotactic and activates complement factor C5118,119. Conversely, at low concentrations, thrombin elicits vascular endothelial barrier protective properties232 and generates TAFIa which in turn, inactivates pro-inflammatory mediators bradykinin, osteopontin, C3a and C5a233. It therefore should not be surprising that polyP has diverse functions. Indeed, polyP may provide local protection to host cells (e.g.,   47  platelets, endothelial cells, leukocytes), while inducing pro-inflammatory/procoagulant effects at other sites. These properties may be differentially exhibited based on the pathophysiologic situation, the cellular source of the polyP, the presence of key ions in the target pathways, the extracellular concentration of the polyP, and/or the size of the polymer at the site of action.  Polymer size is indeed important in the function of polyP in the coagulation system, where it dictates the specific steps in the cascade at which it optimally performs195. More extensive study of the role of polyP in the multiple pathways involved in complement activation may similarly reveal differential regulation based on size. My studies do suggest that polymer size may distinguish the effects of polyP in coagulation and complement. PolyP preparations with polymer lengths less than 30 orthophosphate units had no effect on the plasma clotting time195. Previous in vivo studies that revealed the prothrombotic and pro-inflammatory properties of polyP, were performed using polymers with chain lengths that exceeded 45 orthophosphate units15,205, and thus, it is not known whether shorter length polymers would have the same effect. The shortest length polymers used in my in vitro studies were polyP22 and polyP31, sizes which would be expected to have minimal effects on coagulation. Notably, these were still able to effectively suppress the TP. Since I have not yet evaluated the role of polyP in vivo in animal models of human disease associated with excess complement activation, I can only speculate that using shorter forms of polyP might selectively reduce activation of complement through the TP. It is also possible that lower concentrations than those used in vivo in previously reported studies15,205, might spare the prothrombotic and pro-inflammatory effects of polyP and favour complement inhibition and dampening of   48  inflammation. These studies, using a range of sizes and concentrations of polyP in different models of disease, will be important to perform, as therapies targeting polyP might ultimately be tailored to independently modulate coagulation and complement to meet patients' needs.  Platelets play a central role in maintaining hemostasis and promoting thrombus growth, and are also critical in immune surveillance (reviewed in234). In addition to being a major source of polyP, they express several complement receptors, store complement regulatory and activating factors, and provide a surface for generation of C3a, C5a and for assembly of the MAC123,132,235. Although platelets participate in the innate immune response by promoting complement activation, this is tightly regulated by platelet-derived negative regulators of complement, such as factor H (FH), clusterin, CD55 and CD59. Indeed, the platelet must protect itself from excess activation and complement-mediated destruction. This is accomplished in part by the release from activated platelets of the major fluid phase negative regulator of complement, FH236. PolyP, released from activated platelets, is believed to participate in initiation and activation of coagulation, but might also modulate the local inflammatory response by suppressing complement activation. In humans, low platelet levels of polyP are found in patients with dense granule storage pool diseases237,238, such as Hermansky Pudlak Syndrome239 and Chediak Higashi Disease240. Patients with these disorders exhibit a bleeding diathesis, but interestingly, often suffer from largely unexplained recurrent, life-threatening infections. These may be due to leukocyte defects. Alternatively, the immune defects may be due partly to alterations in complement that could be partially   49  attributed to reduced polyP release. It will be interesting to evaluate complement activation in these patients. I explored the mechanisms by which polyP suppresses complement activation. As a highly anionic polymer, polyP chelates divalent metal ions241. Initiation and activation of complement until formation of the C5 convertases, is highly dependent on Mg2+ and Ca2+. In my studies using the total hemolytic activity assay, increasing concentrations of monophosphate (P1) suppressed cell lysis, but only at molar concentrations equivalent to or greater than the concentrations of ionic calcium and magnesium in the assay system and required for complement activation. PolyP>1000 at equivalent molar concentrations was much more potent in suppressing complement-mediated cell lysis, suggesting that chelation of calcium and magnesium ions was not the only mechanism. I subsequently focused on examining the effect of polyP on the terminal pathway, which proceeds without the participation of enzymes and is entirely ion-independent.  The TP of complement is in effect, a point of no return that without intervention by negative regulators, inevitably results in formation of the multiprotein MAC. Three major negative regulators of the TP that prevent cell lysis have been described, all of which variably bind to TP components to prevent formation and integration of a stable and functional MAC. CD59 is a glycosylphosphatidylinositol (GPI)-linked protein that binds to C8 and C9 and prevents C9 from polymerizing242. Deficiencies of CD59 are associated with paroxysmal nocturnal hemoglobinuria (PNH)243. Clusterin binds to C7, C8 and C9, inducing a conformational change that reduces the capacity of the C5b-9 complex to integrate into the membrane244,245. Vitronectin binds to C5b-7 in the fluid   50  phase (but not to C5b,6), allowing formation of a soluble MAC that cannot bind to the membrane246. PolyP is a fourth negative regulator of the terminal pathway that also binds to TP components to reduce cell lysis. Several lines of physico-chemical evidence led to the conclusion that polyP binds to and destablizes the earliest component of the TP, such that formation of a functional MAC is reduced. Electromobility shift assays in native gels showed that polyP directly interacts with several complement components. These findings, which do not necessarily indicate a functional relationship, prompted us to utilize other approaches to establish the mechanisms. By sequentially adding the TP components to the cRBC in the TP hemolytic assay, I determined that polyP could significantly suppress lysis when added before or after C5b,6, but not after C7. This indicated that polyP could 1. inhibit C5b,6-C7 interactions, 2. interfere with C5b-7 anchoring to the membrane, 3. limit C5b-7 interactions with C8, and/or 4. reduce C5b-8 or C5b-9 integration into the membrane. In gel filtration studies, polyP binds directly to C5b,6, supporting my native gel studies, while DSF clearly shows that polyP destabilizes C5b,6 in a concentration-dependent manner, findings that are consistent with altered assembly of the MAC.  In summary, I have uncovered a novel mechanism by which complement is negatively regulated. Future studies will focus on delineating the in vivo relevance using mouse models of human disease, and examining whether other steps in the complement system are also regulated by polyP. The interplay between polyP, complement and coagulation is important to understand, as alterations in their cross-talk may manifest as disease. Currently, the only available drug that effectively suppresses complement activation, eculizumab, is expensive247 and its efficacy beyond PNH and   51  atypical hemolytic uremic syndrome is not established. Alternative therapies that may be more broadly and possibly safely used for many diseases are urgently needed.     52  2.5. Supplemental Materials and Methods Hemolytic Assays Erythrocyte (RBC) lysis assays as previously described210,225 were used to measure complement activity. Erythrocytes were washed three times in GVB with the reaction buffers as indicated, and counted using the Advia 120 Hematology System from Siemens (Siemens, Erlangen, Germany). Reactions in 300 ?L were allowed to proceed  for 30 min at 37?C, after which unlysed cells were pelleted by centrifugation at 600 x g. Erythrocyte lysis, reflected by the released hemoglobin in the supernatant, was quantified by measuring the absorbance at 405 nm using the Mithras LB 940 microplate reader from Berthold Technologies (Bad Wildbad, Germany). Percent lysis is relative to the control of 100% lysis with H2O. For total hemolytic activity assays, normal human serum (NHS) (4.5%), polyP, and rabbit erythrocytes (rRBC) (6.0x107 cells/mL) were added sequentially to initiate lysis. Reagents were diluted in GVB. Reactions were stopped by the addition of 30 mM EDTA in GVB. Lysis in the absence of NHS was subtracted as background.  For serum-based terminal pathway (TP) hemolytic assays, components were diluted in GVB containing 10 mM EDTA (GVB-E), the latter added to prevent upstream complement activation and generation of endogenous C5b,6. 2% NHS was used as the source of C7, C8, and C9, and hemolysis of chicken erythrocytes (cRBC) (3.3x108 cells/mL) was initiated by addition of exogenous purified C5b,6, the concentration of which was determined from pilot studies to yield ~70-80% lysis at 30 min. No C5b6 activity was detected in the NHS prior to initiating the reaction. For some experiments, instead of NHS, purified terminal pathway complement components were used in the   53  TP assays. In these assays, in a final volume of 300 ul GVB, lysis was achieved by the sequential addition of the following components: cRBC (3.3x108 cells/mL), C5b,6 (20 pM), C7 (15 nM), C8 (10 nM), and C9 (25 nM). The unlysed cell pellet was removed by centrifugation and lysis was measured immediately after the 30-minute reaction. Calf Intestinal Alkaline Phosphatase (CIAP) Digestion of PolyP 600 ?M polyP was incubated with 400 U/mL calf intestinal alkaline phosphatase (CIAP; Invitrogen Life Technologies Inc., Burlington, ON, Canada)  for 18 hrs in TBS (20 mM tris, 150 mM NaCl, pH 8.4) in the presence of 2 mM MgCl2 and 0.2 mM ZnCl2. Complete digestion of polyP into monomers was confirmed by TBE-urea polyacrylamide gel electrophoresis followed by staining with 0.05% toluidine blue and by the malachite green assay248. CIAP-treated polyP was added to the TP assay at a final concentration of 100 ?M. Visualization of PolyP in TBE-Urea Gels PolyP can be visualized in both native and TBE-urea gels using the metachromatic stain toluidine blue which, upon binding to polyP, shifts the absorption peak from 630 nm to 530 nm. 10 nmol of phosphate were mixed with 5x sample buffer (15% Ficoll 400, 0.25% xylene cyanol FF, 0.25% bromophenol blue, 5x TBE) and loaded into 10% TBE-urea Precast Ready Gels (8.6 x 6.8) from Bio-Rad. Running buffer was 1x TBE containing 90 mM Tris, 90 mM borate, 2.7 mM EDTA, pH 8.3. Samples were electrophoresed under constant voltage for 30 min at 150 V. Gels were stained with a fixative solution containing toluidine blue (0.05% toluidine blue, 25% methanol, 5% glycerol) for 10 min, destained with the same fixative without toluidine blue, and then imaged in white light on a flatbed scanner.   54  Native Polyacrylamide Gel Electrophoresis (PAGE) Native PAGE was used to assess polyP-protein interactions (known as electromobility shift assay). Mini gels were handcast according to Laemmli?s gel system in the absence of detergent and reducing agent. 4x resolving and stacking gel buffer consisted of 1.5 M Tris-HCl (pH 8.8) and 1.0 M Tris-HCl (pH 6.8), respectively. Running buffer contained 25 mM Tris and 192 mM glycine (pH 8.3). 1-2 ?g of protein was incubated with 3 ?g polyP (or buffer as control) at ambient temperature for 10 min before adding sample buffer (50 mM Tris-HCl pH 6.8, 10% glycerol, 0.02% bromophenol blue). Proteins were electrophoresed at 100V constant voltage for 2 hours, stained with EZBlue Coomassie Brilliant Blue G-250 from Sigma-Aldrich and destained with several changes of water, and then imaged in white light on a flatbed scanner. Gel Filtration Proteins were used in the buffers as supplied by Complement Technology. The C5b,6 buffer consisted of 10 mM HEPES, 120 mM NaCl, pH 7.2 (HBS). The C7 buffer consisted of 10 mM Na3PO4, 145 mM NaCl, at pH 7.3 (PBS). Both proteins and phosphates (also in the appropriate buffers) were centrifuged at 21,000 g for 15 min to remove any precipitated material before use. HBS and PBS were prepared and filtered through a 0.22 ?m pore diameter Stericup and Steritop Express?PLUS filter from Millipore (Billerica, Massachusetts, USA), and the ?KTAmicro Chromatography System and the Superose 6 PC 3.2/30 column (both from G.E. Healthcare, Buckinghamshire, UK) were equilibrated with these buffers for 1 hour before using. The buffer used in the column was consistent with the buffer of the protein being analyzed. 100 ?L of sample   55  containing 20 ?g of protein with or without 10 mM monophosphate (P1) or polyP>1000 were injected into the filtration column and eluted at a rate of 5 ?L per minute. The retention time of the protein was monitored by measuring UV absorbance at 280 nm. Data for each curve were normalized to the point where the peak starts.     56  2.6. Supplemental Figures     Supplemental Figure 2.1 Standard curve for the total complement-mediated hemolytic assay. This was generated by incubating increasing concentrations of normal human serum with rabbit erythrocytes for 30 min at 37 oC, quenching the reaction with excess EDTA, and then measuring the A405 of the supernatant. Increasing absorbance corresponds to increasing hemogblonin release from lysed red blood cells. The linear region of the curve was determined to be between 20-80% lysis, corresponding to 2.5-4.5% serum. Since the effects of polyP were determined in pilot studies to be inhibitory, baseline lysis was fixed at ~80% (using 4.5% serum). The effects of polyP over a range of concentrations under these experimental conditions were subsequently evaluated (see manuscript ? Figure 2.1).     57        Supplemental Figure 2.2 Standard curve for the terminal pathway assay using serum as the source of complement. Serum concentration was fixed at 2%, and increasing concentrations of purified C5b,6 dose-dependently lysed chicken erythrocytes after 30 min at 37 oC. The linear region of the curve was achieved with 20-250 pM purified C5b,6. 250 pM of purified C5b,6 was therefore used to fix baseline lysis at ~80% to allow assessment of the inhibitory effects of polyP in serum.     58        Supplemental Figure 2.3 Standard curve for the terminal pathway assay using purified complement components. C7 (15 nM), C8 (10 nM), and C9 (25 nM) were in excess, and hemolysis of chicken erythrocytes after 30 min at 37 oC was determined as dependent on the concentration of purified C5b,6. The linear region of the curve was achieved with 2-20 pM purified C5b,6. 20 pM of purified C5b,6 was therefore used to fix baseline lysis at ~80% and this was used to assess the inhibitory effects of polyP.     59  3. General Discussion  From my experimental results, I conclude that polyP inhibits complement activity, specifically the terminal pathway, at least in part due to polyP interactions with C5b,6. Several critical questions remain: 1) Is this effect also seen with bacteria expressing cell-surface polyP as well as platelet-secreted polyP; 2) How may polyP act as an additional link between coagulation and complement; 3) How does the effect of polyP compare with other polyanions such as heparin; and 4) Are there other mechanisms by which polyP modulates complement activity? I will attempt to address these questions in the following general discussion.  3.1. Role of PolyP in Suppression of Complement by Bacteria  In this thesis, I have shown that polyP suppresses complement-mediated lytic activity. This is consistent with earlier reports. The Gram-negative bacteria Neisseria meningitidis and gonorrhoeae have polyP forming a capsule-like coating on the cell surface.153,249 The capsule plays an important role in protecting the organism, and mutants with defective capsule formation exhibit enhanced sensitivity to complement.250 Notably, the N. meningitidis mutant lacking the ppk gene for the synthesis of polyP show increased lysis in human serum.182 On the other hand, a mutant lacking the ppx gene for the degradation of polyP demonstrated resistance to serum-mediated lysis.181 Taken together, the accumulation of polyP by the bacteria, likely on the surface of the cell, is important for their protection against complement-mediated destruction.  Despite this, the mechanisms by which cell surface polyP protects Neisseria from complement have not been studied. Certainly, polyP may destabilize C5b,6 and prevent   60  its binding to the bacterial surface, as suggested my findings (Figures 2.6 and 2.7). However, other mechanisms may be at play as well. One notable mechanism by which N. meningitidis evades complement is through its ability to recruit host factor H (FH) through cell surface sialic acids.251 Through FH binding to cell surface polyanions, C3b inactivation and uncoupling of the convertase is enhanced, leading to reduced alternative pathway activation and ultimately complement-mediated lysis.76 It is possible that the polyanionic polyP may also assist in FH recruitment to the cell surface to diminish C3b activity. In support of this hypothesis, I have demonstrated through native gel electrophoresis that FH interacts with polyP (Figure 3.1). The anodal electrophoretic shift is consistent with that seen between FH and heparin.110 Immunoprecipitation experiments with immobilized polyP and serum samples also identified FH as a binding partner for polyP (Smith and Morrissey, personal communications).  I performed preliminary experiments to determine whether polyP enhances C3b inactivation to iC3b by FH and factor I (FI; Figure 3.2A). Purified C3b, FH, and FI were incubated together in the absence or presence of polyP, and cleavage of C3b was examined by reducing SDS-PAGE. Under the conditions used, fluid phase inactivation of C3b by FH and FI was unaffected by the presence of polyP>1000 (Figure 3.2B). However, it is possible that this reaction needs to take place on a surface in order for polyP to exert an effect since, in a physiologic environment, C3b is deposited on a cell surface and polyP is on the Neisseria cell capsule.153,249 A feasible method to assess this is to couple C3b, polyP, or both to zymosan (from the cell wall of Saccharomyces cerevisiae) and then assess FH/FI-mediated inactivation. In addition, FH also inhibits the alternative pathway by competing with factor B (FB) binding to C3b or accelerating    61   Figure 3.1 PolyP reduces the mobility of factor H in native gels. Representative figure of factor H mobility in native gels in the absence or presence of polyP. 1 ?g of protein were incubated with or without 6 ?g polyP45 or PolyP>1000 and resolved by native PAGE. The gel was stained with Coomassie blue for detection. Thrombin (IIa) and prothrombin (II) were used as the positive and negative controls, respectively. Both lengths of polyP causes a gel shift in factor H.     62   Figure 3.2 Fluid phase inactivation of C3b by factor H and factor I. (A) Schematic of C3b cleavage to iC3b by FH and FI and the fragments generated. (B) 1 ?M purified C3b (fluid phase) was incubated with 10 nM FH and 75 nM FI in the absence or presence of polyP>1000 for 30 min. Samples were quenched with sample buffer, and then resolved by SDS-PAGE under reducing conditions. Protein fragments were visualized by Coomassie staining. Lanes: (1) Ladder; (2) C3b alone; (3) 0 ?M polyP; (4) 1 ?M; (5) 2 ?M; (6) 5 ?M; (7) 10 ?M; (8) 20 ?M; (9) 50 ?M; (10) 100 ?M; (11) 200 ?M; (12) 500 ?M; (13) 1000 ?M.     63  the decay of the C3-convertase.72 The effects of polyP on these processes will need to be assessed as well. 3.2. Role of PolyP in Complement Suppression at Site of Injury PolyP is secreted from activated platelets at a site of injury, where it plays procoagulant and proinflammatory roles.15,16,158,195?198 Although this thesis only used synthetic polyP, one can presume that platelet-derived polyP can also inhibit the terminal pathway. This is supported by previous reports that both synthetic and platelet-derived polyP exhibit similar effects on coagulation.15,16,158,195 In this thesis, synthetically-derived polyP that was size-fractionated to be identical to those found in platelets also inhibit the terminal pathway (Figure 2.3). What could platelet-derived polyP be doing to complement in vivo? An important aspect to consider is the highly localized action of platelets. After injury, platelets are locally activated where they form an initial platelet plug and then incorporate into the mature fibrin clot. Following activation, they release their intragranular contents into the nearby surrounding environment to further promote coagulation. It is possible that the effects of polyP do not extend far beyond the clot, given the low estimated amount of polyP of 0.74 nmol/108 platelets.158 If all platelets are to be activated in blood (assuming 4x1011 platelets/L and 5 litres of blood), only approximately 3 ?M polyP will be released. Thus, its effects ? both on coagulation and complement ? may be localized to only the nearby cells and other platelets. A possible function for the anti-lytic effect of platelet polyP at the site of injury may be to protect local cells, such as endothelial cells and those in proximity to them, from further damage. Complement is activated by the exposure of the subendothelial   64  matrix252 as well as proteases from the now active coagulation system.200 Host cells in the local area, particularly those that are already damaged and more susceptible to additional damage, will need mechanisms to protect themselves from this burst of complement activity. The removal of injured cells needs to be tightly regulated to avoid the release of damage-associated molecular patterns, which would otherwise promote inflammation, and possibly necrosis from adjacent cells,253 and thus be a detriment to the healing process. PolyP may represent a mechanism to prevent host cell damage and promote the restoration of homeostasis.  In a similar manner, platelet polyP may protect other platelets from complement in order to further promote coagulation. It is well established that one of the mechanisms by which platelets are activated is by sublytic amounts of MAC deposition on its surface.131 However, too much MAC will lead to platelet damage, which may again cause unwanted inflammation as well as loss of normal platelet function (e.g. coagulation, complement). Platelet polyP may therefore be a way to protect the platelet in order to promote coagulation.  3.3. Comparing the Roles of PolyP and Other Polyanions on Complement Suppression Heparin and other polyanions are known to inhibit complement activity, including the terminal pathway.76,94,95,99?115 I therefore performed preliminary experiments to compare the effects of polyP>1000, unfractionated heparin (UFH) and various low-molecular-weight heparins (LMWHs; average molecular weights listed in Table 3.1), and double-stranded DNA on terminal pathway activity (Figure 3.3). Similar to the effects of polyP, the suppressive effect of the heparins on hemolytic activity is correlated    65          Table 3.1 Average molecular weights of different heparins. Preparation Mean Molecular Weight (Da)* Enoxaparin (Lovenox) 4,200 Nadroparin (Fraxiparine) 4,500 Dalteparin (Fragmin) 6,000 Tinzaparin 6,500 UFH (Sigma) 20,000 * Values obtained from Weitz 1997 N Engl J Med254 and Sigma Aldrich product sheet (Cat. no. H3393)     66   Figure 3.3 Comparison of the effects of polyP and other polyanions on terminal pathway activity. 1 ?g/mL of various low-molecular-weight heparins, unfractionated heparin (UFH), and polyP>1000 were added into the terminal pathway assay with serum, each performed in quadruplicate. Increasing heparin length correlated with increased suppression of terminal pathway activity. Double-stranded DNA from salmon testes was from Sigma-Aldrich (Cat No. D1626).   67  with the molecular weight of the molecule, with UFH exerting the same magnitude of suppression as polyP>1000. This suggests that the length of the chain, also proportional to the negative charge, is important for activity. This is also despite the fact that the negative charges on heparins are due to sulfates rather than phosphates. However, DNA, which is highly polyanionic due to the phosphate backbone, minimally affects terminal pathway activity. This indicates that neither charge nor the presence of phosphates alone will determine whether the molecule will suppress terminal pathway activity. Additional parameters, such as the charge density of the molecule, may influence the activity as well. Although the polyanions heparin and polyP have similar effects on the terminal pathway, it would not be necessarily reasonable to assume that polyP also affects complement activity where heparin also exerts an effect. A perfect example can be found in the polyanions? effects on coagulation: heparin is an anticoagulant while polyP is a procoagulant in vivo.15 The coagulation initiating effect of polyP is likely due to its ability to directly activate FXII and the contact pathway.15,16,195 While heparin has also been shown to initiate the contact pathway,255?257 the net effect of heparin in vivo is anticoagulation primarily due to its ability to potentiate antithrombin (AT) activity to inhibit thrombin and FXa.21 The ability of polyP to bind to AT has not been investigated, but it seems unlikely that it would potentiate AT inhibition of FXa because it lacks the pentasaccharide moiety required for AT conformational change and the effect on FXa.25 Because their chemical structures differ so greatly, it would be unreasonable to assume that polyP and other polyanions share identical activities. Indeed, polyP but not heparin increases endothelial barrier permeability,205 and preliminary experiments suggest that   68  heparin but not polyP is capable of inhibiting C4 activation by C1s (data not shown; performed by Dr. Jonathan H. Foley). 3.4. Role of PolyP in Modulation of Other Complement Activities Contrary to the trends of the growing body of literature on the mechanisms of coagulation-complement cross-talk, polyP suppresses complement-mediated lytic activity but promotes coagulation. Most mechanisms described so far suggest that the two systems work in parallel: a pro-coagulant molecule promotes complement activation, while an anti-coagulant molecule antagonizes complement activation.200 The few exceptions to this trend include plasmin (pro-fibrinolytic) cleavage of C3 and C5 to generate active cleavage products,117 and C4-binding protein (anti-complement) inhibition of protein S, which results in a more active coagulation cascade.258,259 However, with the data generated in this thesis so far, it is difficult to conclude that polyP only plays an anti-complement role in vivo. The main focus of this thesis was to investigate the effects of polyP on MAC lytic activity, which is an important but only one of many functions of complement. As it relates to the terminal pathway, polyP may be interfering with MAC formation on the cell surface but promoting the generation of the soluble and cytolytically inactive MAC (sMAC).260 Although sMAC cannot directly lyse target cells, it has been shown to participate in endothelial cell signalling, leading to increased vascular permeability, leukocyte migration, pro-inflammatory cytokine release, and even TF expression for the initiation of coagulation.137,261?263 T-cells can also be activated by the sMAC to proliferate and secrete pro-inflammatory cytokines.264 Thus, polyP   69  inhibition of complement-mediated cell lysis may still result in a pro-inflammatory response consistent with bradykinin generation through the contact pathway.15 The role(s) of polyP in complement-mediated phagocytosis and cell signalling have not been fully explored. It may be possible that polyP affects the generation of opsonic fragments on cell surfaces during the upstream pathways of complement, resulting in enhanced or suppressed phagocytosis by leukocytes. A potential candidate opsonin that may be affected by polyP is C3b, since a mechanism of C3b inactivation is through FH and I have demonstrated that polyP interacts with FH (Figure 3.1). PolyP may also affect anaphylatoxin generation or function. Using an immunoprecipitation approach, Smith et al. found that polyP also interacts with C3, C4a, and C5 (personal communications). Whether polyP affects the rate at which C3 or C5 are cleaved by their respective convertases to generate C3a and C5a was not investigated in this thesis but presents an interesting idea to examine. C4a is particularly interesting as its function as an anaphylatoxin in humans is unknown due to its low potency. Possibly, polyP may serve as a cofactor to bridge C4a (and other anaphylatoxins) with its native receptors to mediate its effects. In support of this idea, one study reported that polyP can interact with fibroblast growth factor-2 (FGF-2), physically stabilize it, and facilitate the binding of FGF-2 to its receptors on human fibroblasts.184        70  4. Conclusion  In this thesis, I have shown that polyP is not only involved in coagulation, but also in the complement system as a negative regulator of its lytic activity. I speculate that polyP secreted from activated platelets serves to protect local host cells from the heightened complement activity at a site of injury. My findings may also help to explain how infections, such as Neisseria meningitidis that express polyP on their surface, overcome host responses to allow them to proliferate and invade an organism.265 However, complement is a highly complex system, and there is much to explore than polyP effects on the terminal pathway.  Naturally, a next step will be to validate the physiologic relevance of polyP in animal models of human diseases, such as paroxysmal nocturnal hemoglobinuria (PNH) that is characterized by increased complement-mediated destruction of host red blood cells. It is hypothesized that polyP-treated mice will be protected from PNH-associated hemolytic anemia due to complement suppression. However, a major challenge in assessing polyP effects on complement in such in vivo models stems from the ability of polyP to induce thrombosis and possibly lethality of the model organism.15 Utilizing FXII-deficient organisms to prevent polyP-mediated activation of the contact pathway may be a feasible option to overcome this obstacle.  Furthermore, studies will need to be conducted to characterize other mechanisms by which polyP modulate complement, considering that I have found polyP interactions with upstream complement components such as factor H. This will be important in evaluating the potential of polyphosphate as a novel therapeutic agent or target.   71   This concludes my thesis, which also represents the first study to describe how polyphosphate may modulate the complement system. Hopefully, the findings presented here will inspire new insights into the importance of polyP in complement and physiology.    72  Bibliography 1. Davie, E. W. & Ratnoff, O. D. 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