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The PAI-1-vitronectin-vimentin ternary complex : mechanism of extracellular assembly and role in transplant.. Leong, Hon Sing 2008

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THE PAI-1-VITRONECTIN-VIMENTIN TERNARY COMPLEX: MECHANISM OFEXTRACELLULAR ASSEMBLY AND ROLE IN TRANSPLANT VASCULOPATHYbyHON SING LEONGB.Sc., The University of Alberta, 1999M.Sc., The University of British Columbia, 2002A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Pathology and Laboratory Medicine)THE UNIVERSITY OF BRITISH COLUMBIA(Vancouver)April 2008© Hon Sing Leong, 2008ABSTRACTThe active state of plasminogen activator inhibitor type-1 (PAI-1) is prolonged when itforms a complex with vitronectin (VN), a major serum protein. Active PAI-1 in the PAI-1:VNcomplex serves many functions related to fibrinolysis and cell migration but key to these effectsis its extracellular distribution. PAI-1:VN complexes can bind to exposed vimentin (VIM) onactivated platelet and platelet microparticles, resulting in the assembly of PAI-1:VN:VIM ternarycomplexes. However, the manner in which the vimentin cytoskeleton is presented extracellularlyis not well understood.I hypothesized that PAI-1:VN:VIM ternary complex assembly occurs on cell surfaceswhen microparticle release leads to exposure of vimentin cytoskeleton which can lead to eitherassembly of the ternary complex or become involved in an autoimmune response specific forvimentin.To follow the intracellular and extracellular fate of PAI-1, I constructed an expressionvector encoding PAI-1-dsRed, a fluorescent form of PAI-1, which would permit live celltracking of PAI-1 in megakaryocytes and endothelial cells. Secondly, to study how vimentin isexpressed on platelets and platelet microparticles, flow cytometry was used to isolate vimentinpositive platelets or PMP's and atomic force microscopy was performed to image platelets orPMP's at nanoscale resolution. From these studies, I propose a model of vimentin expression inwhich the junction of microparticle release results in the exposure of cytoskeletal vimentin onboth the cell and the microparticle. This exposed vimentin could potentially induce VNmultimerization on the same cell surface leading to incorporation of multiple PAI-1:VNcomplexes.iiFinally, I investigated how anti-vimentin antibodies can induce platelet:leukocyteconjugate formation. To achieve this, in vitro tests were performed to determine the binding siteof anti-vimentin antibodies (AVA's) and how they induce blood cell activation. Overall, myresults suggest that vimentin exposure in our model of microparticle release can lead to ternarycomplex assembly if suitable quantities of PAI-1 are released during platelet activation. In thesetting of transplant vasculopathy with high titres of AVA's, vimentin-positive granulocytes canbind these autoantibodies, which then leads to platelet activation and the formation ofplatelet:leukocyte conjugates.iiiTABLE OF CONTENTS ABSTRACT ^ iiTABLE OF CONTENTS ^ ivLIST OF TABLES .xiLIST OF FIGURES ^ ..xiiLIST OF ABBREVIATIONS ^ ..xivACKNOWLEDGEMENTS ^xviCO-AUTHORSHIP STATEMENT ^xviii^CHAPTER I: Introduction    11.1 Plasminogen activator inhibitor type-1 (PAI-1)     .11.1.1 Overview    11.1.2 Biochemical properties  ^ 11.1.3 Synthesis and cellular fates  ^31.1.4 PAI-1 in disease    ,41.2 Vitronectin (VN)   ^41.2.1 Overview    .41.2.2 Biochemical properties and interactions   ^41.2.3 Synthesis and cellular fates    .61.2.4 VN in disease    .71.3 Vimentin  ^81.3.1 Overview  ^81.3.2 Biochemical properties and interactions ^ 91.3.3 Vimentin as the cell surface receptor for PAI-1 ^ 10iv1.3.4^Vimentin in disease    121.4 Organ Transplant Vasculopathy     131.4.1^Introduction   141.4.2^Chronic organ rejection and MHC-mismatches   141.4.3^Antibody-mediated rejection and non-MHC autoantibodies ^ 151.5 Rationale, Hypothesis and Experimental Aims    161.5.1^Rationale   161.5.2^Hypothesis   171.5.3^Specific Aims   .171.5.4^Methodology Overview   181.5.5^Potential Relevance of Findings   181.6 References for Chapter I   20CHAPTER III: TARGETING OF RECOMBINANT PAI-1-dsRED AND VITRONECTIN TOSTORAGE GRANULES IN ENDOTHELIAL AND MEGAKARYOCYTE CELL LINES^3.1^Introduction^3.2^Materials and Methods   31333.2.1 PAI-1-dsRed plasmind construction ^ .333.2.2 Transfection of Eahy926 and MEG-01 cell cultures ^ ..333.2.3 Immunoblot analysis of conditioned media from transfectedEahy926 cells     ..343.2.4 Confocal microscopy of tPA-Pacific Blue:PAI-1-dsRedcomplexes in fibrin clot lysis ^ 343.2.5 Immunofluorescence staining of transfected MEG-01and Eahy926 cultured cells ^ .353.2.6 Immunofluorescence staining of TNF-a activated transfectedEahy926 cell cultures ^ 363.2.7 Videomicroscopic analysis of exocytosis of fibrinogen-Alexa488and PAI-1-dsRed from a-granules in MEG-01 cells ^37^3.3^Results ^ 383.3.1 PAI-1-dsRed synthesis and secretion by transfectedEahy926 cell culture ^ .383.3.2 PAI-1-dsRed forms a complex with addition of exogenoustPA^ 393.3.3 PAI-1-dsRed binds to immobilized vitronectin (VN) ^403.3.4 PAI-1-dsRed attenuates clot lysis in the presence ofexogenous tPA^  .413.3.5 PAI-1-dsRed localizes with P-selectin and vWFin Eahy926 cells ^ ...433.3.6 Activated endothelial cells express extracellular PAI-1-dsRedthat is associated with vimentin ^  .453.3.7 PAI-1-dsRed is targeted to a-granules in MEG-01 cellsfor rapid exocytosis ^463.3.8 Exocytosis of a-granules containing stores of PAI-1-dsRedin MEG-01 cells^ 473.4^Discussion ^ .49vi3.4.1 Overview^ 493.4.2 Thrombin induced MEG-01 cell a-granule exocytosis^513.4.3 PAI-1 colocalizes with vWF in Eahy926 cells ^ .513.5^References for Chapter III^ 53CHAPTER IV: DISTRIBUTION OF PAI-1:VITRONECTIN:VIMENTIN TERNARYCOMPLEXES ON ACTIVATED PLATELETS AND PLATELET MICROPARTICLES BYATOMIC FORCE MICROSCOPY4.14.2Introduction^Materials and Methods ^56..584.2.1 Ethics, blood preparation, and PAI-1 ELISA ^ ...584.2.2 Electrophoresis of High Molecular Weight Protein Complexesfrom Patient Plasma Samples ^ 584.2.3 FACS isolation of platelet microparticles and activatedplatelets expressing ternary complex ^ 594.2.4 Atomic force microscope (AFM) specifications ^ 604.2.5 Atomic force microscopy of platelet microparticles,VN-vimentin multimers and activated platelets ^ 614.2.6 Platelet-Rich Clot Formation and Staining for Vimentin,Vitronectin and PAI-1 ^ 624.4 Results ^ 634.4.1 High molecular weight complexes in post-AMI platelet-poorplasma contains elevated PAI-1, vitronectin, and vimentin ^ 63vii4.4.2 Platelet microparticles express vitronectin, vimentin and PAI-1on their surface as determined by FACS analysis ^654.4.3 AFM ultrastructural analysis of PMP's positive for ternarycomplex ^ .664.4.4 Microscopy of vitronectin-vimentin multimers on activatedplatelets ^ .674.4.5 Multimers of vitronectin-vimentin form a highly orderedultrastructure^ .70^4.5^Discussion^ .734.6^References for Chapter IV ^ 81CHAPTER V: VIMENTIN AUTO-ANTIBODIES INDUCE PLATELET ACTIVATION ANDFORMATION OF PLATELET-LEUKOCYTE CONJUGATES VIA PLATELET-ACTIVATING FACTOR5.1^Introduction^ 855.2^Materials and Methods^  .875.2.1 Blood collection and patient serum ^ ..875.2.2 Preparation of recombinant human vimentin ^ 875.2.3 Depletion of AVA's from patient sera 885.2.4 Flow cytometry and monoclonal antibodies (mAb) ^ 885.2.5 Complement dependent cytotoxic assay on AVA treatedpurified leukocytes ^ .895.3^Results ^ 90viii5.3.1 Effect of monoclonal and patient AVA's on whole blood ^905.3.2 Effect of other IgM antibodies on whole blood ^ 945.3.3 Effect of AVA IgM and anti-HLA A2 IgM on purifiedplatelets ^ .965.3.4 Effect of anti-vimentin antibodies (AVA) on purifiedNeutrophils^ .985.3.5 Supernatant of AVA-activated leukocytes induces plateletActivation^ 1005.3.6 AVA IgM-bound leukocytes release PAF ^ 103^5.6^Discussion^ 1055.7^References for Chapter V^ 109CHAPTER VI: CONCLUSIONS AND FUTURE DIRECTIONS6.1^Overall themes of dissertation ^ 1136.2^Strengths and limitations of thesis research ^ 1176.2.1 Chapter III ^ 1176.2.2 Chapter IV 1186.2.3 Chapter V ^ 1206.3^Evaluation of current knowledge and proposals and strategies for futuredirections ^ 1226.4^Three most significant contributions ^ 1256.4.1 Mechanism of microparticle release from activated bloodcells and endothelium. ^ 125ix6.4.2 The role of neutrophils in thrombus stabilization and structuralintegrity. ^  1266.4.3. Formation of platelet:leukocyte conjugates in transplantvasculopathy. ^  1266.5.^References for Chapter VI ..127APPENDIX I: List of Publications, abstracts, and presentations^ 129xLIST OF TABLESTable 4.1^Clinical data of patients with Acute Myocardial Infarction (AMI) ^ .63xiLIST OF FIGURES Figure 1.1^Summary of PAI-1's interactions with vitronectin (VN) and tPA ^ .2Figure 1.2^Summary of PAI-1-vitronectin-vimentin ternary complex formation. ^ 11Figure 3.1^Construction of pDsRed-PAI-1 vector and synthesis of chimericPAI-1-dsRed protein in transfected cells  ^.... ^39Figure 3.2^PAI-1-dsRed chimeric protein forms covalent complexes with exogenoustPA  ^40Figure 3.3^PAI-1-dsRed chimeric protein binds to immobilized vitronectin.   ^41Figure 3.4^PAI-1-dsRed attenuates t-PA mediated fibrin clot lysis. ^43Figure 3.5^Intracellular compartmentalization of PAI-1-dsRed in Eahy926 cells ^.44Figure 3.6^TNF-a treated Eah926 cells express PAI-1-dsRed:vitronectin:vimentincomplexes on their cell surface.   ^45Figure 3.7^Intracellular compartmentalization of PAI-1-dsRed in MEG-01 cells ^46Figure 3.8^Exocytosis of PAI-1-dsRed and Alexa 488-fibrinogen from MEG-01a-granules.   ^ 48Figure 4.1^Platelet poor plasma (PPP) from post-acute ischemic infarction(post-AMI) patients contain high molecular weight protein complexesconsisting of vimentin, vitronectin and PAI-1 ^ 64Figure 4.2^Flow Cytometric Analysis of PPP from post-AMI patients. ^65Figure 4.3^Atomic force microscopy on platelet microparticles isolated fromPPP collected from post-acute myocardial infarction patients. ^ .68Figure 4.4^Scan line analysis of filament-like structures present within membraneflaps of PMP's expressing CD41+vimentin ^ .69xiiFigure 4.5^Distribution of vitronectin-vimentin multimers on the cell surfaceof activated platelets ^ 71Figure 4.6^AFM analysis of the ultrastructure of vitronectin-vimentin multimers ^72Figure 4.7^Proposed model of vimentin exposure on activated platelets andplatelet microparticles and subsequent assembly of the PAI-1:VN:vimentin ternary complex ^ 74Figure 5.1^AVA monoclonals induce platelet:leukocyte conjugate formation andsurface expression of C3d and fibrinogen. ^92Figure 5.2^Patient sera with anti-vimentin autoantibodies (AVA's) induce plateletactivation and formation of platelet:leukocyte conjugates.  ^93Figure 5.3^AVA- and HLA- IgM's induce platelet:leukocyte conjugates which expresstissue factor and P-selectin.  ^95Figure 5.4^AVA-IgM does not directly induce platelet activation 97Figure 5.5^Localization of IgM to granulocytes and activated platelets and theircytotoxic effect on leukocytes.  ^99Figure 5.6^Effect of supernatant (SN) from IgM treated leukocytes on plateletactivation^ 102Figure 5.7^PAF inhibition attenuates platelet activation and blood cellagglutination.  ^ 104Figure 6.1.^Proposed mechanism of vimentin-induced vitronectin multimerization ^ 115Figure 6.2^Mechanism of platelet activation and platelet:leukocyte conjugateformation by anti-vimentin antibodies (AVA's) in whole blood^ 119LIST OF ABBREVIATIONSAFM^atomic force microscopyAMI^acute myocardial infarctionAVA's^anti-vimentin antibodiesDMEM^Dulbecco's modified eagle's mediadsRed^red fluorescent protein analogous to GFP (green fluorescent protein)Eahy926^name of endothelial hybridoma cell lineECM^extracellular matrixFACS^fluorescence activated cell sortingFBS^fetal bovine serumFITC^fluorescein isothiocyanateFS^forward scatterGFP^green fluorescent proteinHUVEC^human umbilical vein endothelial cellsIF^intermediate filamentskDa^kilo DaltonMEG-01^megakaryocyte cell linePAI-1^plasminogen activator inhibitor type-1PAI-1-dsRed plasminogen activator inhibitor type-1 fused with the dsRed fluorescent proteinPMP^platelet microparticlePPP^platelet-poor plasmaPRP^platelet-rich plasmaRPMI-1640 culture media named after its site of origin: Roswell Park Memorial InstitutexivRT-PCR^reverse transcriptase polymerase chain reactionSN^supernatantSS^side scatterTMAFM^tapping mode atomic force microscopyTNF-a^tumor necrosis factor- alphatPA^tissue-type plasminogen activatoruPA^urokinase-type plasminogen activatoruPAR^urokinase-type plasminogen activator receptorTVD^transplant vascular diseaseVN^vitronectinVIM^vimentinvWF^von Willebrand's FactorWPB^Weibel-Palade bodyxvACKNOWLEDGEMENTS "Education is an admirable thing, but it is well to remember from time to time that nothing thatis worth knowing can be taught." — Oscar Wilde (1854-1900)I don't really know what Oscar Wilde meant when he penned that, but I interpret the axiom likethis: the epiphany after a great trial of intellectual pursuit, a pursuit brought to a finale by ashackling realization. That there is more to come, with a darkness hugging the ocean horizon ofscientific marathon. This is how I feel after doing a PhD.I dedicate this thesis to my family: Phak Foo Leong (dad), Yut Yoong Leong (mom),Hon Fei Leong (brother), See Yuen Leong-Ng (sister), Johnny Ng (brother-in-law) and mybeautiful and patient wife, Eva. I did not get through a single day without thinking of all you as Iflailed in the trenches and did my time as a trainee. I hope that I make you all proud forever.I thank God for giving me the mental makeup to survive the past 8 years and in helpingme in some key moments that finally led to the completion of this PhD training.I acknowledge my supervisors, Dr. Thomas Podor and Professor Rose for the completionof this thesis. My first supervisor, Dr. Podor has suffered greatly medically for the past sevenyears, but his incredible spirit and drive have permitted me to understand and contribute to thefield in what we believe are important and salient issues regarding vimentin and vitronectininteractions. As indebted I am to Dr. Podor, I have learned a great deal about researchindependently. Dr. Podor has been much more than a supervisor; he has been a true mentor,scientist, and friend. I have gained a tremendous amount of experience and expertise, all becauseof his efforts to stay alongside my research with little regard for his own health. He is truly aninspiration to my future work and I hope to continue to be as dedicated to the pursuit of scienceas he has in the past five years, in spite of his medical conditions. I pray that his health returnsbecause the field of PAI-1 and vitronectin will surely require his energy and knowledge for manyyears to come.I also wish to acknowledge Professor Marlene Rose, who is a kindred spirit in the field ofvimentin alongside Dr. Podor. They both met in Vienna, Austria in 2004 in which a spiritedconversation about each other's interests in this intermediate filament resulted in a most fruitfulcollaboration that spanned the Atlantic Ocean. This international collaboration resulted in thedevelopment of the second half of my PhD studies, the role of vimentin in allograft rejection andits manner of expression in plasma that frequently leads to autoimmune responses in solid organtransplant patients. This collaboration proved to be the saving grace for my PhD project as theslight turn in focus became a bountiful exchange that builds on our knowledge of vimentin. Iwish to conclude by saying any successes found in this thesis is intimately linked to the foresightand tenacity of Dr. Podor and the graciousness and formidable insight provided by ProfessorxviRose. I only hope to be even mentioned in the same breath as my mentors, luminaries in theirown right.I want to acknowledge the great friends I have made during these past 8 years inVancouver (in random order). Bobby Yanagawa, who was the first person to make me realizehow good we have it in science and in Vancouver; the first person to suggest Toastmasters to me,a person whose example has showed me how to pursue science. Caroline Cheung for hercontinual presence and encouragement, for her shoulder as I cried and for her laughter as Isucceeded. Caroline is one person whose scientific demeanor and commentary will be wellpreserved in my mind and as I reflect on Vancouver and iCAPTURE. Melissa Westoby for herfriendship and her lightheartedness and strength in the laboratory and outside of it. HubertWalinski for demonstrating to me how senior graduate students can behave and how to properlynavigate the shallow waters of iCAPTURE and Vancouver's citizenry. To Alan Quan, who maypossibly be the greatest friend a poor pathetic graduate student can ever have, whose energy,thoughtfulness and attitude was always a refreshing break from all the dreary work and pain thatthis PhD has wrought upon me. Alan will always be in my plans as I move forward in scienceand industry. Ryon Bateman, a rock and the only big brother I have ever had. It's rare when youmeet someone that sees the potential in you and decides to help unlock it. Whether or not heregrets doing this, we shared some memorable moments in science and under his wing, I realizedsooner what it was like to be a real "imager" and a real scientist. He taught me some great one-liners and axioms as well as some great times in the lab and at the dinner table. Jerry Wong forbeing the science little brother that I mentored on an irregular basis. You made me realize thatsome good scientists DO go into medicine and that there is still some hope in this world forclinician scientists. Anna Meredith for her tireless pursuit of relevance and excellence inscientific endeavors and her caring ways in the lab. Beth Whalen for your time, talks, andtechnical help. Finally, Mahesh Balakrishnan, my brother from England/Bangalore, a manwhose standing will surely be realized in this world and in India (a world onto itself). Truly anamazing man with equally amazing principles and a fervour for excellence that is grounded byspirituality and legacy.Thank you all.xviiCO-AUTHORSHIP STATEMENTChapter II: I identified, designed, performed all experiments for this manuscript and Ianalyzed all data for this manuscript. I prepared 95% of the manuscript, with my supervisor Dr.Thomas Podor contributing the remaining 5%.Chapter III: I identified, designed, performed all experiments for this manuscript and Ianalyzed all data for this manuscript. I prepared 100% of the manuscript.Chapter IV: I identified, designed 60% of the experiments for this manuscript, Iperformed 95% of the experiments while Dr. Mahesh Balakrishnan contributed recombinantvimentin and the collection of human blood. I prepared 60% of the manuscript while ProfessorMarlene Rose contributed to the other 40%.xviiiCHAPTER I. INTRODUCTIONThe overall objective of this thesis is to understand the extracellular fates of a ternarycomplex composed of PAI-1, vitronectin and vimentin. These proteins both individually andtogether serve a broad scope of cellular functions related to cell motility and extracellular matrixmetabolism, but the primary benefit of this ternary complex is to localize PAI-1 activity to sitesof thrombus formation, specifically to sites of activated platelets and platelet microparticles.1.1. Plasminogen activator inhibitor tune - 1 (PAI- 1)1.1.1. Overview — As its name implies, PAI-1 is a major inhibitor of plasminogenactivators, such as tissue-type plasminogen activator (tPA) and urokinase plasminogen activatorinhibitor (uPA) [1]. Inhibition occurs when PAI-1 forms a covalent bond with the PA via itsreactive center loop, resulting in the formation of a PAI-1-PA complex, either tPA or uPA [1-3].The premier feature of PAI-1 is its reactive centre loop (RCL) which dictates its activity andforms covalent bonds with serine proteases such as PA's [1-3] and form an acyl-enzymecomplex in a positional conformation such that the catalytic sites on the PA's are blocked(Figure 1.1). Inhibition of t-PA prevents the conversion of plasminogen to its active form,plasmin, a protease with broad substrate specificity such as fibrin [4], fibronectin,thrombospondin, and von Willebrand Factor [5]. Inhibition oft-PA can prevent fibrinolysis aswell as reduce certain facets of extracellular matrix metabolism (ECMM) [6, 7]. Similarly,inhibition of u-PA also results in decreased cell adhesion and cell motility [8] and its effects arehighly relevant in angiogenesis and tissue remodeling [9].1.1.2. Biochemical properties — PAI-1 is a 50 kDa protein belonging to the serineprotease inhibitor (serpin) family that possesses a RCL that dictates its activity and inhibitory1Tissue typeplasminogenActivator (tPA)1. RCL of PAI-1forms covalentBond with tPAActive PAI-1open RCLN \PAI-1:VNcomplex 2. VN dissociatesfrom PAI-1Portion of RCL isincorporated intoPAI-1 and tPAis inhibitedFigure 1.1. Summary of PAI-1's interactions with vitronectin (VN) and tissue-type plasminogen activator (tPA).  PAI-1 contains a reactive centre loop (RCL) that when free, canbind to tPA via formation of a covalent bond (1). This interaction will cause dissociation of VNfrom PAI-1 (2) [10]. The inhibition of tPA occurs when the RCL is reinserted into PAI-1 moleculewhile the tPA is transferred to the other side of PAI-1, sterically hindering the active sites of tPA.ability. For instance, during tPA inhibition, the RCL and P1 site on PAI-1 will interact with tPAand form an intermediate Michaelis complex. This is followed by a cleavage in the Pl-Pl' bondon PAI-1 and the formation of a covalent acyl PAI-1-tPA complex [1-3]. This will also result inthe insertion of the once-free RCL back into the body of the PAI-1 molecule. Once the RCL iscompletely re-inserted, tPA becomes catalytically inactive in the final PAI-1-tPA complex.However, when the RCL slowly re-inserts into the PAI-1 molecule, PAI-1 can dissociate fromthe enzyme-inhibitor complex, leaving behind an inactive tPA molecule that may or may notcontain a part of the RCL [11].The position of the RCL dictates PAI-1 activity; active PAI-1 has a free and exposedRCL, but when the RCL is re-inserted into the PAI-1 molecule, it becomes the inactive or latentconformation of PAI-1 that is unable to bind and inhibit tPA. Because of the RCL's propensityto re-insert back into the PAI-1 molecule, the half-life of active PAI-1 is —1-2 hours [12]. LatentPAI-1 can be converted back to active PAI-1 by chemical denaturation and subsequent refoldingof purified latent PAI-1 [13, 14]. PAI-1 can also form complexes with vitronectin (VN) and al-2acid glycoprotein which stabilize the active conformation of PAI-1 [15]. In particular, thebinding interaction between PAI-1 and vitronectin (VN) allows it to follow similar localizationfates as vitronectin, thus broadening its participation and effects in various environments.1.1.3. Synthesis and cellular fates — PAI-1 is synthesized by a wide range of cell typessuch as megakaryocytes, endothelial cells, adipocytes, smooth muscle cells and fibroblasts — allof mesenchymal origin [16, 17] as well as some epithelial cell types [18]. Post-synthesis, theintracellular fate of PAI-1 has only been described in megakaryocytes, platelets, endothelial cellsand to a lesser extent, epithelial cells [19-21]. In terms of intracellular compartmentalization,PAI-1 is processed into storage granules such as a-granules in platelets and megakaryocytes.Stimulation by thrombin, collagen or calcium ionophore lead to immediate exocytosis of a-granules and release of PAI-1 [19]. A large percentage of PAI-1 stored within a-granules is ofthe latent and inactive conformation, perhaps indicative of the length of time spent in storage,which is beyond the half-life of active PAI-1 [22]. Endothelial cells are thought to synthesize themajority of circulating levels of active and latent PAI-1 in plasma [21] and variations in thesePAI-1 plasma levels have been correlated with a variety of diseases such as obesity,thromboembolic disease and atherosclerosis [23-26]. However, the manner in which PAI-1 isintracellularly compartmentalized and stored within endothelial cells is unclear andcontroversial; one report points to the Golgi as the main storage organelle [27], whereas anotherreport describes a loose cytoplasmic storage within endothelial cells while others reportcompartmentalization into storage granules [28], albeit in immortalized cell lines. Reports alsodemonstrate a mechanism of constitutive secretion into the lumen, however, it is unclear whetherthis secretion is mediated by exocytosis or passive release [29, 30].31.1.4. PAI-1 in disease — Because of the inhibitory nature of PAI-1 on plasminogenactivators (tPA and uPA), it is often implicated in the pathogenesis of a variety of diseasesranging from cancer metastasis to thrombosis as underscored by their pivotal roles in hemostasisand ECM metabolism, respectively. High plasma concentrations of PAI-1 which have beenobserved in individuals with insulin resistance and obesity can also lead to impaired fibrinolysisand an increased risk for cardiovascular disease [31]. Specifically, the long-term elevation ofPAI-1 in plasma or intramurally (within a thrombus or vessel) can induce fibrosis of vessels oratherosclerotic lesion development and may even induce thrombotic disorders [32-34]. It is alsobelieved that high plasma levels of PAI-lor an intracoronary thrombus with high PAI-1 contentmay counter the effects of thrombolytic therapy during its administration to patients undergoingan acute myocardial infarction [35, 36].1.2. Vitronectin (VN)1.2.1. Overview - Vitronectin is a major plasma glycoprotein predominantly synthesizedby the liver but is also synthesized at low levels by other tissues such as adipose, brain,heart, andskeletal muscle [37]. In plasma it exists at concentrations of 3-5 µM [38] and is also found inabundance within the extracellular matrix (ECM) [39, 40]. Apart from its ability to stabilizePAI-1 [41], VN also interacts with heparin [42, 43], collagen [39], urokinase plasminogenactivator receptor (uPAR) [44], components of the complement system [45] and thrombin/anti-thrombin III complexes [46]. Because of this wide range of biochemical interactions, VN playsan important mediating role in hemostasis, the innate immune system, angiogenesis and woundrepair by either acting as a ligand parter for other proteins and determining the distribution or4substrates for these VN-ligand complexes. Key to the majority of these interactions is thepresence of the somatomedin B domain and an Arg-Gly-Asp (RGD) sequence [47].1.2.2. Biochemical properties and interactions - Vitronectin exists in two majorconformations: monomeric and multimeric VN [48]. Monomeric VN is found in twoconformations, a 75 kDa form and a 65+10 kDa two-chain form. The 65+10 kDa form is formedby proteolytic cleavage of the 75 kDa form, but remains intact post-proteolytic cleavage [49].The majority of plasma VN exists in the monomeric form, whereas <2% of VN circulates asmultimeric VN by virtue of several disulfide bridge linkages. These high molecular weightmultimers of vitronectin [50] are highly active forms of VN [51] that are more readily availableto bind to ligand partners such as PAI-1, which maintains PAI-1 activity. Because of its multi-ligand partner (adhesive) properties, multimeric VN can potentiate the inhibition of fibrinolysisby associating with PAI-1 and mediating its binding to fibrin clots and vimentin [52, 53].By using sequence homology, the VN molecule can be defined by three major domains:the N-terminus which contains the somatomedian B domain; the central domain containingseveral hemopexin-like repeats; and the C-terminal domain. Within the N-terminus domain, thesomatomedin B domain (aa 1-44) harbors functional groups that are largely responsible for itsbinding interactions with PAI-1 [54]. Because of this interaction, VN participates in pericellularproteolysis of ECM through its localization and binding to PAI-1 and subsequent inhibition ofplasmin formation Immediately upstream of this is the RGD sequence (aa 45-47), a key elementin mediating cell motility and cell adhesion on ECM and adjacent cells via its bindinginteractions with various integrin receptors. Pericellular distribution and supply of VN can alsoregulate the extent of cell spreading and motility [55]. Adjacent to the RGD motif is the heparinbinding site, as well as a domain responsible for anti-thrombin III binding (aa 53-64) [56], and5two collagen binding sites [40] that mediate vitronectin's binding to matrix. Because of thisregion, VN may act as a pro-coagulant because of its ability to neutralize and clear heparin out ofthe circulation [40]. When this occurs, less heparin becomes available to anti-thrombin, resultingin higher thrombin levels and higher incidences of coagulation and fibrin formation [40].Beyond the N-terminus and within the central domain lie stretches of acidic and basicelements that, by ionic interaction, are likely involved in maintaining the 3-D structure of the VNmolecule. This vast stretch of VN (aa 132-459) contains 6 hemopexin-like repeating domainsthat contain several cysteines that participate and are [57] central to disulfide bridge formation,VN molecular structure and VN multimerization. Other than these structural contributions, abiological role for hemopexin-like repeats on VN remains unclear; although some reportsdescribe binding interactions with S. pyogenes to the extent where vitronectin "coats" themajority of the bacterial surface, hence suggesting an opsonization function for plasma VN[58].The C-terminus end of VN contains a binding site for plasminogen (aa 332-348), and asecond heparin binding site (aa 348-379) [59]. A second binding site for PAI-1 has also beendetermined at aa 348-370 [60], which enables VN to form both 1:1 or 1:2 stoichiometric ratiocomplexes with PAI-1[61].1.2.3. Synthesis and cellular fates — Initially classified as "serum spreading factor", VNis synthesized by a variety of cell types, but is predominantly synthesized within the liver andsecreted into the bloodstream to plasma concentrations of 200-400 ilg/mL (3-5p,M). VN isprimarily synthesized by hepatocytes [37] but extra-hepatic synthesis of VN is known to occur inbrain, fibroblasts, adipose tissue, heart and skeletal muscle albeit at a 25- to 100-fold loweramount compared to liver [37]. In vitro VN synthesis also occurs in macrophages, monocytesand human umbilical vascular endothelial cells (HUVEC). VN is a major constituent of ECM,6which exists predominantly in its multimeric form and bound to matrix and glycosaminoglycans(heparin) [62]. Tissue injury and wound healing is characteristically marked by VNaccumulation, within necrotic cells [63] or in the provisional matrix, subsequently promotingfibrosis via its interactions with PAI-1 [64-67].A vitronectin knockout mouse has been developed [68] for studies on the role ofvitronectin in hemodynamics and thrombus formation. It has also been used for studies onwound healing and myocardial infarction. When compared to wildtype models, the vitronectin -/- mice generate highly unstable thrombi that dissolve quickly [69] and demonstrate markedlyreduced wound healing as a result of decreased cell migration [70].1.2.4. VN in disease — VN is part of a group of proteins called acute phase responseproteins [71] whose plasma concentrations vary in response to the extent of inflammation. Anacute phase response that follows a typical inflammatory response can result in localvasodilation, platelet aggregation, neutrophil chemotaxis, and the release of lysosomal enzymes,histamines, kinins and oxygen radicals [71]. Systemic events can result in fever, hormonalchanges and even alterations in metabolism. For example, studies in rats and humansdemonstrate substantial changes in these levels during the acute phase response following tissueinjury [37, 72]. One such example of tissue injury is acute myocardial infarction (AMI), inwhich a thrombotic occlusion within a major coronary artery halts fresh blood perfusion ofmyocardium served downstream of the occlusion. This condition also triggers an acute phaseresponse [71] with a concomitant increase in VN plasma levels [73]. In the event of an AMI, itis conceivable that increased acute phase reactant proteins such as VN act as heparin-bindingproteins, serving to non-specifically bind heparin in circulation [74] which may decrease theanticoagulant effect of heparin because of its deposition into ECM and clearance from plasma —7a distribution which presents less heparin to anti-thrombin III. Abnormal plasma levels of VNare also present in patients with rheumatic disease [75]. In detail, increased VN present insynovial fluid and inflamed joints may promote cell motility and accelerate wound healing.Higher rates of VN synthesis in differentiating neuroblastic tumors have also been reported,implicating its roles in cell motility and adhesion on matrix [76].1.3. Vimentin1.3.1. Overview — Vimentin is a cytoskeletal protein that forms intermediate filaments(IF), a major component of a cell's cytoskeleton. Intermediate filaments composed of vimentinprovide a substantial amount of structural rigidity within a cell and structurally supports theorganelles and nucleus of a cell [77-79]. Moreover, intermediate filament networks arecortically distributed underneath the cell membrane to provide much needed mechanical supportduring cell-to-cell and cell-to-ECM interactions. IFs are so called because their diameter (8-12nm) is intermediate between thin actin microfilaments (7 nm) and thick microtubules (25 nm)[77-79].There are various classes of IFs with classification being based on sequence similarities.Prime examples of type I and II intermediate filament proteins are keratins; vimentin and desminare examples of type III IF proteins; and neuronal proteins are examples of type IV IF proteins.Vimentin IFs are found in cells of a mesenchymal lineage: endothelium, fibroblasts, smoothmuscle cells, erythrocytes, leukocytes, platelets, etc. [77-79]. Conversely, cardiac and skeletalmuscle synthesize desmin IF's in which the main function is to specifically stabilize andorganize sarcomeres by associating with the Z-disk of sarcomeres and connecting them with theZ-disks of other neighboring sarcomeres [80]. Vimentin IF networks resemble a radial8distribution alongside the shape of the cell, with IF's terminating at nuclear membranes anddesmosomes at cell membranes as well wrapping around organdies and the nucleus to providespatial distribution [77].1.3.2. Biochemical properties and interactions —Assembly of type III IF's canincorporate either vimentin or desmin, often occurring in immature myoblasts but not in fullydifferentiated cell types [81, 82]. Tetramers of vimentin are the basic subunit for IF assemblyand these will form long chains, two of which will associate with each other to form an a-helicaldimer chain, resulting in a complete vimentin IF with a diameter of 8-12 nm [78]. Intermediatefilament protein monomers have a common structural component, called the coiled-coil region(300-330 as long) and the diversity of IF's lies within the diversity of sequence and length of theN-terminus and C-terminus. The coiled-coil region is largely responsible as a longitudinalspacer and/or a lateral packing module, hence providing rigidity and structural integrity. Theamino-terminal has been shown to be essential for proper assembly of filaments [83-85] and isalso highly susceptible to proteolytic attack usually leading to posttranslational modification. N-terminal structural modifications of vimentin IF's may possibly dictate its intracellulardistribution and molecular organization in various physiological and pathological conditions[86]. Extracellularly, exposed vimentin IF's in activated or apoptosed cells may also undergocitrullination, a process that alters the biochemistry of vimentin in which arginine residues aredeiminized and converted into citrulline [87, 88]. This deiminization of vimentin has been foundto take place within the synovial fluid within joints, largely mediated by macrophages andmonocytes in disease contexts such as rheumatoid arthritis [87, 88].Apart from proteolytic modifications of vimentin, there are a number of importantproteins that associate with vimentin, one group being proteins such as kinesin and dynein that9bind to vimentin and traverse along microtubules to help vimentin IF's generate their radialnetwork throughout the cell [89, 90]. Another set of proteins also associate with vimentin butwith each interaction providing a different function: VN has been shown to multimerize whenmonomers first interact with exposed vimentin in the extracellular space [53]. Thismultimerization of vitronectin is not well understood but the binding site on vimentin responsiblefor this multimerization is found within the first 133 aa of the vimentin N-terminus and likelyupstream of the thrombin cleavage site (94 aa) on vimentin [53]. The observation of VNmultimerization as induced by exposed vimentin IF has many implications, particularly inexplaining how VN multimers are established within ECM and its origins.The Fc receptors of heavy chain immunoglobulins have also been described with theability to bind to vimentin IF's, an interaction that proposes immune-targeted clearance ofactivated and dead cells exposing vimentin to plasma [91, 92]. The first reports of this bindinginteraction were observed on endothelial cells and implicated complement-mediated lysis as theprimary means of clearing dead endothelial cells [91, 92], a proposed mechanism preceding thenow accepted pathways of apoptosis as a means of clearing cells.1.3.3. Vimentin as the cell surface receptor for PAI-1— PAI- 1:VN complexes accountfor 95% of active PAI-1 in plasma, at a normal concentration of 20 ng/mL [93]. The magnitudeand percentage of circulating active PAI-1 can vary significantly, particularly duringcoagulation, tissue injury and wound healing. PAI-1:VN complexes have been observed inabundance on fibrin fibils in clots and activated platelets with strong, high affinity bindinginteractions in which Ki >> le [52, 53]. Other reports have also demonstrated low affinitybinding interactions with PAI-1, ligands such as the Aa chain of fibrinogen (16210568), alphai-acid glycoprotein (16156651), but of a low affinity binding interaction (<<10 15). PAI-1 binding10interactions with vitronectin, more likely to occur given its plasma concentration, mediates thelocalization of PAI-1 to fibrin and activated platelets by interaction with yA/y'fibrinogenmonomers within fibrin fibrils [52] and exposed vimentin cytoskeleton on the surface ofactivated platelets and platelet microparticles [53]. More specifically, the N-terminus head ofvimentin interacts with vitronectin that results in multimerization of vitronectin on the vimentinfilament, with pre-existing PAI-1:VN complexes in plasma ready to be incorporated into themultimerized VN via VN homotypic interactions (Figure 1.2) [94].•Plasma vitronectin^PAI-1 :vitronectin • -■('A(vitronectinmultimerizationIncorporation ofPAI-1:VNcomplexesVimentin surfaceexposure byunknownmechanism••a-granuleexocytosisPLATELET ACTIVATIONFigure 1.2. Summary of PAI-1-vitronectin-vimentin ternary complex formation. Uponplatelet activation, platelet stores of VN and PAI-1 are released into plasma and is accompaniedby the exposure of vimentin IF's via an unknown mechanism. This exposed vimentin issusceptible to vitronectin multimerization which provides a form of vitronectin amenable tohomotypic interactions with pre-existing PAI-1:VN complexes in plasma. PAI-1:VN complexesbecome bound to this multimerized VN and the PAI-1-VN-VIM ternary complex is formed.Extracellular presentation of PAI-1 occurs when it forms a ternary complex withvitronectin and vimentin cytoskeleton that is exposed on the surface of an activated cell but thismechanism of cell surface expression occurs without the requirement for a transmembrane11protein receptor. However, it does provide a mechanism as to how active PAI-1 in complex withvitronectin can be bound to cell surfaces, i.e., platelets and platelet microparticles. Moreover,because VN has self-association properties to form VN multimers, PAI-1-VN complexes canbind to the growing VN aggregate, due to VN-VN homotypic interactions, thus incorporatingboth circulating plasmaVN, pre-exiting PAI-1:VN plasma complexes and PAI-1:VN complexesexocytosed during platelet activation [94]. Thus, physical exposure of platelet vimentincytoskeleton results in the PAI-1 localized on cell surfaces within thrombi, formation of ternarycomplexes that require multimerized VN.1.3.4. Vimentin in disease — Vimentin was initially used as a histological marker oftumor malignancy and the extent of cancer progression in breast carcinomas and melanomas.Morphologically, tumor growth and its cytoskeleton assembly can outpace cell surfaceadaptability, leading to the exposure of internal structure to the extracellular environment, thusevoking the description of cancer as "tumors: wounds that do not heal" [95]. Because IF proteincomposition is a defined characteristic for all cell types, IF type I-VI composition can reveal theextent of tumor cell differentiation and previous cell-type identities of the cancer cell mass [96].In terms of actual function, the abundance of vimentin IFs as observed in tumor cells both invitro and in vivo may yield augmented motility and invasiveness of tumor cells [97] with afibrillary pattern of distribution. Although not yet shown or determined, it is reasonable tospeculate that if vimentin is exposed on the surface of tumor cells, it may mediate binding ofmultimerized vitronectin, thus providing an added element of cell adhesion, particularly at sitesof cell damage or cell activation. Nonetheless, the hypothesis that over-expression of vimentinin some tumor cells confers a selective advantage remains to be elucidated.12Auto-antibodies specific for vimentin are a common diagnostic feature of diseases suchas systemic lupus erythematosus (SLE) [98], rheumatoid arthritis (RA) [99] and transplantvasculopathy [100]. The pathogenesis of SLE is not well defined but it is hypothesized thatimpaired clearance of apoptosed cells may be the source of vimentin that leads to anti-vimentinantibody (AVA) production [101]. It is suspected that IF-nuclear complexes from incompletelyapoptosed cells are responsible [98, 102]. To date, a pathogenic role for AVA has not beendescribed in SLE patients. Patients with rheumatoid arthritis also develop anti-vimentinantibodies, but these are specific for citrinullated vimentin, its deiminated form. Thiscitrinullated autoantigen may be generated from chronic destruction of synovial tissue, the softconnective tissue between joints. Inflammation of synovial tissue is often accompanied bymesenchymal fibroblast and chondrocyte proliferation, leading to vimentin release in theupwelling of synovial fluid within the cavity [103]. It is within this fluid-filled cavity thatmacrophages can release proteolytic enzymes that modify and citrullinate vimentin, which maypossibly be processed by the immune system to generate an AVA response [87]. Again, nopathological function of autoantibodies specific for citrullinated vimentin has been determined1.4. Organ Transplant Vasculopathv1.4.1. Introduction - Once commonplace, acute organ rejection has now been limited to<10% of all transplant recipients [104] due to the introduction of "immuno-suppresant" drugs.For example, CyclosporinTM inhibits the synthesis of proliferative cytokines in immune cells,such as IL-2, that would have resulted in the proliferation of more lymphocytes that wouldinduce a prolonged allo-immune response against the graft [105, 106]. However, despite a lowincidence of acute rejection and an immunosuppressant regiment, these highly vascularized13grafts often succumb to another form of rejection, known as chronic rejection, and this occurs at3-10 years post-transplantation [107]. Chronic rejection of heart and kidney grafts ischaracterized by obliterative arteriosclerosis as a result of chronic inflammation, medial necrosisand intimal thickening of all the vessels of the graft [108, 109].1.4.2. Chronic organ rejection and MHC-mismatches - Chronic rejection, also knownas transplant vasculopathy (TV) disease, is a multi-factorial disease that is distinct fromconventional lipid-induced atherosclerotic lesions [108, 109]. Conventional atherosclerosis ismorphologically distinct, as lesions are eccentric in their cross-sectional morphology and containlipid-rich, necrotic cores. Furthermore, these lesions are only found in some of the larger arteriesof the heart. On the contrary, TVD lesions are concentric in cross-section, and all vessels of theheart are uniformly narrowed, including the microvasculature and veins [108, 109]. Althoughthere is a noticeable lack of a lipid-rich core in the early to middle stages of the disease, lipid-lowering agents have demonstrated pleiotropic and anti-inflammatory effects, contributing toenhanced management of TVD [110-112]. However, considering that only the vasculature of thegraft is affected whereas other vessels in the body are not, the suppressed host immune system isimplicated in the chronic development of atheromatous lesions in all graft vasculature.Key to most immune responses are the MHC Class I (presents virally produced proteinsand self antigens) and II (presents phagocytosed/endocytosed antigens that undergo proteolyticprocessing) complexes which function to present foreign or self-antigen on the cell surface thatwill subsequently generate a cellular and humoral immune response [113, 114]. MHCcomplexes are synthesized and found on the surface of every nucleated cell, but not all MHCcomplexes are identical; in fact, there are two major classes of human MHC molecules in whichthere are up to 100 different MHC molecules. Each person contains an exclusive set of 6 MHC14Class I complexes and 6 MHC Class II complexes and in the event of an organ or tissuetransplant, efforts towards identifying the set of MHC molecules of a graft and finding a near-compatible host is critical in order to minimize the amount of MHC mismatch [113, 114].Although organs are allocated to patients as to minimize MHC mismatches between the host anddonor, there will usually be a minor mismatch and the host will develop an allo-immuneresponse against the graft [113]. Despite immunosuppression, a dampened immune responsewill nevertheless target the organ; in which two major immune outcomes will result: maturationof T-cells (cell-mediated immunity) and antibodies (humoral immunity) against the foreignMHC molecules [115-117] and in some cases, the damaged graft itself (non-MHC antibodies).1.4.3. Antibody-mediated rejection and non MHC autoantibodies - Chronic rejection isthe result of long term damage caused by low levels of activated graft-specific lymphocytes dueto immunosuppression and antibody-mediated targeting of the graft. These antibodies can begrouped into: MHC and non-MHC antibodies wherein MHC antibodies are specific for non-hostMHC molecules. Some prime examples of non-MHC antibodies are structural proteins such asmyosin, collagen and vimentin [100, 118, 119]. Both MHC and non-MHC antibodies exert theireffects on the graft by inducing complement-mediated lysis as seen by the abundance ofcomplement split products deposited within the graft vasculature [120]. This antibody-mediatedcomplement lysis of endothelium results in inflamed vasculature and dysfunction, thuspromoting atherogenic mechanisms and further immune cell deposition into the graft vasculature[121].Many studies have identified the intermediate filament vimentin as an importantautoantigen after allotransplantation [122] leading to the formation of non-MHC antibodiesspecific for vimentin. Significant levels of these non-MHC anti-vimentin antibody titres after15heart transplantation are associated with the development of graft vasculopathy [123], and thesepatients also have self-restricted vimentin-specific CD8+-T-cells [124]. Recipients of renalallografts suffering from chronic rejection also have significant titres of anti-vimentin antibodies[125]. Moreover, renal allografts placed in recipients with previously high titers of anti-vimentinantibodies have a higher predisposition to suffer from steroid-resistant acute rejection [125].The source of vimentin autoantigen is unclear in the context of transplant vasculopathy,but is suspected to originate from the cleavage of vimentin fragments off the surface ofapoptosing endothelial cells [126], leukocytes [127], and activated platelets [53], all of whichexist during the pathogenesis of TVD [126, 127]. As apoptotic cells and antigens released fromthese cells are processed by host dendritic cells and presented to self-MHC restricted recipient T-cells [126, 127], an antibody response may be mounted due to the high inflammatory state of thepatient and associated coactivated T-cell population [128]. Moreover, long ischemic timesincrease apoptosis in the graft, which is associated with increased levels of caspase-3 [129], thatcleave vimentin in the cytoskeleton early in apoptosis [130].The potentially pathogenic contribution of anti-vimentin antibodies cannot be ignored.Moderate to high levels of anti-vimentin antibody titres (AVA) in patients with cardiac graftsdemonstrate a strong correlation with the incidence of coronary artery disease [123]. However,the mechanisms of its pathogenic effects on the graft and hemostasis remain unknown.161.5. References for Chanter I1. Shore JD, Day DE, Francis-Chmura AM, Verhamme I, Kvassman J, Lawrence DA,Ginsburg D. A fluorescent probe study of plasminogen activator inhibitor-1. Evidence forreactive center loop insertion and its role in the inhibitory mechanism. J Biol Chem 1995;270:5395-8.2. 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J Leukoc Biol 2006; 79:489-98.27CHAPTER II: RATIONALE, HYPOTHESIS, AND EXPERIMENTAL AIMS2.1. RationaleThe extracellular distribution of PAI-1 has been determined to be governed by itsinteractions with VN and vimentin, and together they form a ternary complex that localizes ontothe surface of activated cells. What is not understood is how intracellular stores of PAI-1 fromplatelets become localized to the surface of activated platelets and platelet microparticles and thesource of vimentin for this surface distribution. What is also unknown is the conformation ofvitronectin required for the formation of the ternary complex: monomeric or multimeric VN.Although the extracellular presentation of vimentin cytoskeleton is prerequisite for ternarycomplex formation, the manner of vimentin presentation the mechanism of vimentin auto-antibody formation is unknown. Lastly, do these anti-vimentin antibodies have downstreameffects on cells that naturally express vimentin in the circulation?2.2. HypothesisTernary complex formation is dependent on the formation and release of microparticlesfrom the activated cell, morphologically termed microparticles. At the broken junction betweena platelet microparticle (PMP) and platelet that released the PMP, vimentin becomes brieflyexposed and vulnerable to, 1) vitronectin multimerization followed by, 2) PAI-1 and PAI-1:complex incorporation into the VN multimer aggregate. This exposed vimentin cytoskeletonbecomes the site of anti-vimentin antibody binding, leading to complement-mediated lysis.282.3. Specific Aims1. To develop a fluorescently red form of PAI-1 protein and to characterize this fusionprotein and verify its functionality as a PAI-1 analogue. This protein will be used totrack the intracellular and extracellular fate of endogenously synthesized PAI-1 uponstimulation of various transfected cell lines.2. To visualize the ultrastructure of purified ternary complex components and thesecomponents on activated platelets and platelet microparticles.3. To determine what cell type anti-vimentin antibodies bind to and what effects theseantibodies exert on those cells.2.4. Methodology OverviewIn the first project, cell culture, RT-PCR, nucleic acid ligation, subcloning, selectivebacterial culture and DNA sequencing to construct the fusion vector of PAI-1-dsRed wasperformed. Following this, cell transfection on cultured cells, confocal microscopy and live cellculture wide field fluorescence microvideography to track the PAI-1-dsRed proteinintracellularly and extracellularly was performed. To validate the protein chimera, westernimmunoblotting and ligand immunoblotting was performed to characterize the bindinginteractions of the fusion protein. In vitro fibrin clot formation was used to characterize PAI-1-dsRed binding and fibrinolysis. In the second project, flow cytometry and atomic forcemicroscopy to select platelet microparticles that expressed some if not all components of theternary complex was performed. In the third project, a variety of in vitro treatments on wholeblood were performed to compare the effects of anti-vimentin antibodies compared to positive-29control complement-fixing antibodies which were subsequently analyzed by flow cytometry.Cell cytotoxicity tests were used to assess cell type specificity of antibodies.2.5. Potential Relevance of Findings The cytoskeleton of platelets, neutrophils and endothelial cells is gaining more respect asa cause and effector for a variety of diseases and pathological processes. This thesis provides anovel look at how vimentin can interact with components of hemostatic mechanisms andimmune mechanisms by virtue of its biochemical properties. Proteins that interact and associatewith vimentin will gain more importance and provide insights in the treatment and managementof disease. Although many questions remain due to the conventional intracellularunderstandings of vimentin, this research combined with heightened understandings of it withinvarious cellular contexts such as cell activation or senescence [53, 131] will provide an impetusto focus on the cytoskeleton's role in natural processes and disease.30CHAPTER III: TARGETING OF RECOMBINANT PAI-1-dsRED AND VITRONECTINTO STORAGE GRANULES IN ENDOTHELIAL AND MEGAKARYOCYTE CELLLINES3.1. Introduction: Plasminogen activator inhibitor type-1 (PAI-1) is a serpin inhibitor family and aninhibitor of both urokinase-type and tissue-type plasminogen activator (uPA/tPA) [1, 2]. PAI-1plays a pivotal role in clot stabilization by inhibiting the fibrinolytic activities of fibrin-boundtPA. PAI-1 is expressed by a number of cell types, most notably endothelial cells [3-5],megakaryocytes [6, 7] and platelets [8, 9]. The active form of PAI-1 is stabilized when it formsa complex with vitronectin (VN), which prevents the re-insertion of the PAI-1 reactive centerloop back into the body of the molecule [10]. PAI-1-VN complexes are present in a-granules ofplatelets, and this complex assembly occurs when VN is endocytosed into a-granules thatcontain endogenously synthesized PAI-1 [7, 11].In platelets and megakaryocytes, PAI-1 is organized into a-granules, along with otherresident a-granule proteins such as VN, vWF and P-selectin [7, 9, 11]. Exocytosis of a-granulesand hence PAI-1, has also demonstrated to be secretogogue dependent [12]. In the context ofendothelial cells, studies to date do not report compartmentalization of PAI-1 to storage granulessuch as Weibel-Palade bodies (WPB's) [13]. However, both endothelial cells andmegakaryocytes are of mesenchymal lineage, and their storage granules - Weibel-Palade bodies'A version of this chapter will be submitted for publication. Leong, HS, Bateman RB, Walinski H, and Podor T.J.Targeting of Recombinant PAI-1-dsRed and Vitronectin to Storage Granules in Endothelial and Megakaryocyte CellLines.31and a-granules, contain similar resident proteins such as vWF and P-selectin. Although PAI-1 ispresent within a-granules, it has not been determined to reside in an endothelial storage vacuole,except for the Golgi. For example, Rosnoblet et al. have demonstrated that intracellular stores ofPAI-1 are confined to the Golgi, with a more recent report describing PAI-1 colocalization withGiantin, a Golgi protein marker in HUVEC cells [14].In this study, a fluorescent form of PAI-1 was developed to examine its intracellularcompartmentalization in endothelial and megakaryocytic cell lines without the use of epitopespecific antibodies. Intracellular PAI-1 may evade antibody detection because epitopes may bemasked due to complex formation with VN. We constructed a red fluorescent form of PAI-1which exhibits a spectral excitation/emission profile of 553/584 nm. This fusion protein consistsof a dsRed fluorescent protein fused to the C-terminus of the PAI-1 protein. The possibility ofprotein-tag interference and its potential impact on PAI-1 active-inactive states was addressed byassessment of the ability of PAI-1-dsRed to bind to tissue-type plasminogen activator (tPA) andvitronectin (VN), and its ability to attenuate fibrin clot lysis. Following this assessment, we thendetermined PAI-1 compartmentalization in an endothelial cell line (Eahy926) and subsequentlyits fate after TNF-a treatment. These properties were then compared in a transfectedmegakaryocyte cell line (MEG-01) in which PAI-1 organization into storage granules had beenpreviously described.323.2. Materials and Methods: 3.2.1. PAI-1-dsRed plasmid constructionRNA isolation was performed with Trizol reagent (Invitrogen Inc., Carlsbad, CA) onEahy926 cell culture grown to 80% confluency. Human plasminogen activator inhibitor type-1cDNA (PAI-1; GI accession number: 189541) was amplified via RT-PCR (Forward: 5' GGATCC GGG TTC CAT CAC TTG GCC CA 3' Reverse: 5'GAA TTC GTC TTT GGT GAA GGGTCT GC 3') using SuperScript II RNaseff RT and Platinum HiFi Taq polymerase (InvitrogenInc.). PCR product was ligated into a TOPO vector (Invitrogen Inc.) and upon transformationinto chemically competent cells, the PAI-1 cDNA was subcloned into the pDsRedNl plasmid(Clontech Inc., Mountain View, CA) in which PAI-1-dsRed transgene expression istranscriptionally controlled by a pCMV promoter. Sequencing was performed to verify theidentity and frame of the insert into the pDsRedNl plasmid.3.2.2. Transfection of Eahy926 and MEG-01 cell culturesHuman endothelial hybridoma Eahy926 were used because it is an endothelial cell linethat can be transfected (obtained from Dr. C Edgell, University of North Carolina) and werecultured on 30 mm six well plates grown to —70% confluency with DMEM supplemented with10% FBS. For transfections, —1 ug of PAI-1-dsRed plasmid and 3 ut of FuGENE6 (RocheDiagnostics Inc., Indianapolis, IN) was used per well, and was incubated without serum for 24hours. After 24 hours, the cells were washed and replenished with DMEM + 10% FBS. Atspecified timepoints post-transfection (0, 6, 12, 24, 48, 72 hours), 1 mL conditioned media wascollected for immunoblot analysis.333.2.3. Immunoblot analysis of conditioned media from transfected Eahy926 cellsSDS-PAGE of samples of conditioned media on 8% polyacrylamide gels was followedby transfer onto nitrocellulose membrane using a Bio-Rad mini-gel and transfer apparatus(BioRad Inc., Hercules, CA). Membranes blocked with 5% dry milk powder in TBS+0.05%Tween were subsequently probed with either: sheep a human PAI-1 IgG (1:1000 dilution;Affinity Biologicals Inc., Ancaster, ON), mouse a dsRed IgG (1:500 dilution; Clontech Inc.),sheep a human tPA IgG (1:1000 dilution — Affinity Biologicals Inc.), or sheep a human VN(1:1000 dilution — Affinity Biologicals Inc.). This was followed by secondary antibody detectionwith IgG-linked AP antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and the APdeveloping kit (BioRad Inc).VN ligand blots were performed by running lanes of 50 ng purified vitronectin (AffinityBiologicals Inc.) in 8% SDS-PAGE gels with the mini-gel apparatus and then transferred ontonitrocellulose membrane. Individual strips of membrane each representing a lane ofelectrophoresed VN were individually cut, incubated and shaken overnight with 1 mL ofconditioned medium at room temperature. After incubation, the strips were immunoblotted withantibodies specific for PAI-1 or dsRed. Positive and negative controls were strips that wereimmunoblotted with antibodies specific for VN and IgG-AP respectively.3.2.4. Confocal microscopy of tPA -Pacific Blue:PAI-1 -dsRed complexes in fibrin clot lysisTo assess PAI-1-dsRed functionality in terms of binding to tPA and its ability to attenuatefibrin clot lysis, tPA (Alteplase, Genentech, South San Francisco, CA) was labeled with PacificBlue-Maleimide agent as previously described (Molecular Probes Inc., Eugene, OR) [15].34Conditioned media containing PAI-1-dsRed (-2 mg/mL total protein) was incubated with 10 nMtPA-Pacific Blue for 5 minutes at 37°C and then added to a reaction mix consisting of 3 mMpurified fibrinogen, 0.1 mM fibrinogen-alexa488 (Molecular Probes Inc.) in Tyrode's buffer atpH 7.4. The clot was formed on a 24 x 50 mm coverslip by addition of 1.0 III, of 10 U/mLthrombin and then quickly overlaid with a 22 x 22 mm coverslip and incubated at 37°C in thedark for 30 minutes. The 22 x 22 mm coverslip was cleaved off the larger coverslip andconfocal microscopy used to visualize the distribution of PAI-1-dsRed and tPA-Pacific Blue onthe Alexa488-labeled fibrin lattice. To initiate clot lysis, 100 uL of platelet-poor plasma (PPP)warmed to 37°C was overlaid onto the clot and field of view. Immediately after addition of PPP,confocal scans of PAI-1-dsRed, tPA-Pacific Blue and fibrinogen-Alexa488 were acquiredsequentially every 30 seconds for 20 minutes. For confocal microscopy, a Leica invertedDMIRE2 microscope fitted with a TCS SP2 AOBS scanner was used. A Leica 63X/NA=1.20HCS PL APO objective for water immersion optics (cat. # - 506131) was used for all widefieldand confocal microscopy.3.2.5. Immunofluorescence staining of transfected MEG-Oland Eahy926 cultured cellsThe human megakaryoblastic cell line MEG-01 (ATCC, Manassas, VA) was culturedonto fibronectin coated glass coverslips with RPMI 1640 media supplemented with 10% FBSand 1 mM sodium pyruvate. Cells were pre-treated with 1.0 nM of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich Inc., Oakville, ON) prior to transfection to induce cell flatteningand maturation(8001904). The transfection protocol is as described above. Transfected cellswere fixed with 4% paraformaldehyde for 10 minutes and then incubated with PBS+ 0.01%Triton-X for 10 minutes. Immunofluorescence staining was performed with the previously35described primary antibodies at the same dilutions. Alexa488 conjugated secondary antibodieswere used according to the primary antibody used (Molecular Probes Inc.). Cells werecounterstained with Hoechst 33342 nuclear stain.Eahy926 cells were cultured onto coverslips in 30mm six well culture plates to a 75-80%confluency and transfected as described above. Transfected cells were fixed with 4%paraformaldehyde for 10 minutes and then incubated with PBS+ 0.01% Triton-X for 10 minutes.Transfected cells were stained with these antibodies: rabbit anti-human vWF (1:250 dilution,Dako, Mississauga, ON) with donkey anti-rabbit IgG-Alexa488 (Molecular Probes Inc.), or withgoat anti-human CD62P (1:500 dilution, Biovision, MountainView, CA) with donkey anti-goatIgG-Alexa633 (Molecular Probes Inc.).3.2.6. Immunofluorescence staining of INF-a activated transfected Eahy926 cell culturesEahy926 cells were cultured onto glass coverslips and cultured to 70% confluency withDMEM supplemented with 10% FBS. Three days following transfection with PAI-1-dsRedvector, cells were activated with 100 ng/mL of TNF-a for 12 hours prior to paraformaldehydefixation and staining. After blocking with sheep and donkey normal serum (5% in PBS), wellswere immunostained with sheep a-human VN IgG and donkey anti-sheep IgG-Alexa488 ormouse a-human vimentin (3B4) IgG with goat anti-mouse IgG-Alexa488. Sheep IgG or mouseIgG was used as isotype antibody controls. A Hoechst 33342 nuclear counterstain was used at1:1000 dilution post-antibody labeling.363.2.7. Videomicroscopic analysis of exocytosis of fibrinogen-Alexa488 and PAI-1-dsRed from a-granules in MEG-01 cellsTo examine whether stored PAI-1-dsRed could be rapidly released upon thrombinstimulation in megakaryocytes, stably transfected PMA-treated MEG-01 cells were cultured withAlexa488-labeled fibrinogen (50nM final) for 6 hours. Fibrinogen-Alexa488 is known to beendocytosed into a-granules via glycoprotein IIb-IIIa [16], and was used as a positive control fora-granule exocytosis. Endocytosis tracing of fluorescently labeled VN was impractical for thesestudies that required monomeric, native VN due to the multimerization of the protein duringconjugation, thus fibrinogen was used as a surrogate marker for an internalized a-granuleprotein. The cells were then counterstained with Hoechst 33342 for 10 minutes and then gentlywashed with RPMI 1640 media. A temperature and CO2 gas regulated stage specimen setup wasused to allow for time-lapse fluorescence imaging of exocytosis of PAI-1-dsRed and fibrinogen-Alexa488. Cells were stimulated by the addition of 100 IA, of thrombin (1.0 U/mL in PBS pH7.2). For wide-field fluorescence microscopy, a Leica DMIRE2 inverted microscope fitted witha 515-560/580/590 (nm) dichroic filter set for dsRed excitation/emission, and a 450-490/510/515(nm) dichroic filter set for Alexa488 excitation/emission was used. Wide field images weresequentially acquired every 5 seconds for 10 minutes with a Retiga EXi mono CCD camera(QImaging, Inc., Burnaby, BC) with an exposure time of 20 milliseconds per frame and thecamera set on automatic gain. Heat mirrors and a neutral density filter (3% transmittance)minimized sample overheating and fluorophore photobleaching.373.3. Results 3.3.1. PAI-1-dsRed synthesis and secretion by transfected Eahy926 cell cultureHuman PAI-1 cDNA was cloned by RT-PCR amplification using mRNA from culturedEahy926 cells to generate a —1.2 kb PCR product that was ligated into the pDsRedNl vector.Restriction enzyme digest analysis confirmed successful insertion (Figure 1A). DNA sequenceanalysis of this insert confirmed 100% identity with the published amino acid sequence of PAI-1(GI accession number: 189541). The resulting vector was named pDsRed-PAI-1 in which thePAI-1 cDNA sequence was fused with a 6 alanine residue sequence followed by the dsRedsequence (5' to 3' end).Transfection of Eahy926 cells with the pDsRed-PAI-1 vector resulted in the synthesisand secretion of two distinct forms of PAI-1, native PAI-1 (-45 kDa) and PAI-1-dsRed (-70kDa). As shown in Figure 3.1, conditioned media was collected at various time points post-transfection and upon western immunoblot analysis with a polyclonal antibody specific for PAI-L both forms of PAI-1, PAI-1-dsRed and native PAI-1 were detected. Since both forms of PAI-1 are under different transcriptional control, detection of PAI-1-dsRed protein expression andsecretion was not evident until 24 hours post-transfection. There was gradual accumulation ofnative PAI-1 over all time points as well as PAI-1-dsRed accumulation in later time points asevidenced by increasing intensity of bands over time at the 45kDa and 65kDa size level. Toassess potential bleedthrough for each fluorophore used, spectral control images were acquiredby collecting signal in wavelength ranges lower than the incident light used to excite thefluorophore or higher than the predicted emission range. For example, spectral controls fordsRed were: 530-560nm when using 563nm incident light; 600-650nm when using 488nmincident light for FITC/Alexa488.38A G64 <16^12.^ePDsRedvector4.7 kbPM-11.2 kb110 kDaBO kDa61 kDa44 kDa37 kDaPAI-l+dsRad (fusion)PM-1 (native).4■Figure 3.1. Construction of pDsRed-PAI-1 vector and synthesis of chimeric PAI-1-dsRed protein in transfected cells. Panel A) is a restriction digest of pDsRed-PAI-1vector to confirm insertion of human PAI-1 cDNA clone. Panel B) is an immunoblot ofconditioned media from cultured Eahy926 cells transfected with the pDsRed-PAI-1vector. A PAI-1 polyclonal antibody detected both native PAI-1 and PAI-dsRed inconditioned media.3.3.2. PAI-1-dsRed forms a complex with addition of exogenous tPAPurified tPA (200 ng) was added to 40 uL of conditioned media from the 48 hour timepoint, to determine if exogenous tPA could form complexes with both secreted PAI-1-dsRed andnative PAI-1. Upon immunoblotting with a tPA primary antibody, a doublet band was observedat the -110 and -135 kDa size range (Figure 3.2), thus representing tPA complex formation withnative PAI-1 (-110 kDa) and PAI-1-dsRed (-135 kDa). Moreover, immunoblotting with thedsRed primary antibody only recognized the PAI-1-dsRed fusion protein which was the upperband of the doublet. The matching intensities of both bands at -110 kDA and -130 kDa alsoindicates that exogenous tPA has similar binding affinities to both PAI-1 and PAI-1-dsRed.39"PAI-1-dsRed:tPAPAI-1 :tPA1130 kDa110 kDaAnti-dsRed^Anti-tPAFigure 3.2. PAI-1-dsRed chimeric protein forms covalent complexes withexogenous tPA. Lane 1 was immunoblotted with a dsRed monoclonal antibody andlane 2 was immunoblotted with a tPA polyclonal antibody. The dsRed antibodydetected one band representing PAI-1-dsRed-tPA complexes, whereas the tPAantibody detected both PAI-1-tPA and PAI-1-dsRed-tPA complexes.3.3.3. PAI-1-dsRed binds to immobilized vitronectin (VN)A VN ligand blot was performed to determine if the dsRed tag on the PAI-1-dsRedmolecule could hinder PAI-1-dsRed binding to immobilized VN [9]. Figure 3.3 demonstratesPAI-1-dsRed binding interaction with immobilized VN as confirmed by dsRed and PAI-1antibodies. Nitrocellulose lanes of electophoresed VN were incubated with conditioned mediaso that both PAI-1 and PAI-1-dsRed could bind to immobilized VN. The positive control in thefirst lane was not incubated with conditioned media and was stained with VN primary antibodyto demarcate the doublet banding pattern of VN. The doublet pattern present in the lane asstained by the polyclonal PAI-1 antibody suggests that both native and fusion forms of PM-1binded to the immobilized VN. The third lane was immunoblotted with dsRed antibody and thedoublet banding pattern confirmed the ability of PAI-1-dsRed to bind to both bands ofimmobilized VN. The negative control was a lane of immobilized VN incubated with mouseIgG followed by secondary antibody detection with goat anti-mouse IgG AP.401^2^375 kDa65 kDaAnti-VN^Anti-PAI-1 Anti-dsRed Mouse IgGnegativecontrolFigure 3.3. PAI-1-dsRed chimeric protein binds to immobilized vitronectin. Purified VN was electrophoresed and transfected to nitrocellulose. Each individual stripwas then incubated with conditioned media from transfected Eahy926 cells and thenimmunoblotted with antibodies specific for (lane 1) vitronectin, or PAI-1(lane 2), ordsRed (lane 3), or a mouse IgGi isotype control (lane 4). Lane 1 highlights the positionof immobilized two-chain vitronectin (75- and 65-kDa). Lane 2 demonstrates the bindingof both PAI-1 and PAI-1-dsRed present in the conditioned media that binded toimmobilized vitronectin. Lane 3 demonstrates the binding of only PAI-1-dsRed toimmobilized vitronectin from conditioned media.3.3.4. PALI -dsRed attenuates clot lysis in the presence of exogenous tPAFor production of PAI-1-dsRed alone, CHO cells were grown to —90% confluency andtransfected with the vector according to transfection protocols used for Eahy926 cells.Conditioned media was collected and used as a source of PAI-1-dsRed in these experiments.Fibrin clots were formed with purified human fibrinogen and trace amounts of fibrinogen-Alexa488, tPA-Pacific Blue, and in some clots to inhibit tPA-Pacific Blue, PAI-1-dsRed wasalso added. To initiate tPA-Pacific Blue mediated clot lysis, platelet poor plasma (PPP) was41overlaid onto these fibrin clots to initiate plasminogen conversion into plasmin, leading to clotlysis. Three channel fluorescence images of tPA-Pacific Blue, fibrinogen-Alexa488, and PAI-1-dsRed were acquired every 30 seconds to determine the effect of PAI-1-dsRed on tPA-mediatedclot lysis. Figure 3.4 illustrates the distribution of tPA-Pacific Blue, PAI-1-dsRed on anAlexa488-fibrin clot (Figure 3.4A-E) and the rate of clot lysis (Figure 3.4F) as measured by theamount of Alexa488-fibrinogen fluorescence intensity in the field of view remaining at each timepoint. Confocal microscopy revealed PAI-1-dsRed co-localization to areas of tPA-Pacific Bluesignal which were present intermittently throughout the fibrin clot as a punctate signal (Figure3.4C). Figures 3.4A-B depict the distribution of tPA-Pacific Blue and PAI-1-dsRed respectivelyand a composite of these channels in Figure 3.4C, demonstrates a high degree of colocalization.Figure 3.4E represents a composite of PAI-1-dsRed and fibrin-Alexa488 channels, and yellowsignal represents PAI-1-dsRed bound to Alexa488-labelled fibrils. In an experiment todetermine rates of clot lysis with or without PAI-1-dsRed (Figure 3.4F), clots supplemented withPAI-dsRed prior to PPP addition displayed a lower rate of clot lysis compared to the clotsupplemented with just tPA-Pacific Blue. At 10 minutes, fibrinolysis as mediated by tPA-PacificBlue resulted in —80% dissolution of the original clot (black plot line) compared to —10%dissolution of the clot supplemented with both tPA-Pacific Blue and PAI-1-dsRed (red plot line).The difference in initial RFI of Alexa488-fibrinogen/area is explained by the greater amount ofAlexa488-fibrin present within the field of view (black plot line vs. red plot line) prior toaddition of plasma.42Ftbrindgen-Alexa488•11 0•• 4PA-Pacific Blue100 232^300Time kseconds)FFigure 3.4. PAI-1-dsRed attenuates t-PA mediated fibrin clot lysis. Figures 4A - Bdepict the distribution of tPA-Pacific Blue and PAI-1-dsRed respectively in the fibrinclots formed with 0.1 U/mL thrombin, and trace amounts of fibrinogen-Alexa488 and 0.1mM tPA-Pacific Blue. Figure 3.4C depicts a composite of the pseudocolored Figures3.4A-B and reveals the colocalization of tPA-Pacific Blue and PAI-1-dsRed (depicted asmagenta signal). Figure 3.4D is a pseudocolor image of the Alexa488-fibrin clot, andagain depicted in green in Figure 3.4E which is a composite of Figure 3.4B & D withyellow signal indicating areas of colocalization of PAI-1-dsRed on fibrin fibrils. In Figure3.4F, (ii) is the rate of fibrinolysis in a fibrin clot treated with tPA-Pacific Blue (n=1), while(i) is the rate of fibrinolysis in a fibrin clot treated with t-PA and PAI-1—dsRed (n=1).Lysis was measured by the total relative fluorescence intensity of Alexa488-fibrin(ogen)in a randomly selected field of view.3.3.5. PAI-1-dsRed localizes with P-selectin and vWF in Eahy926 cellsFigure 3.5 depicts the intracellular localization of PAI-1-dsRed protein in relation to vWF(Figure 3.5A) and P-selectin (Figure 3.5B) in Triton X-100 permeabilized Eahy926 cells. PAI-1-dsRed was shown to significantly co-localize to granules containing P-selectin and vWF, bothresident Weibel-Palade body (WPB) proteins. Calculations to assess colocalization determinedthat PAI-1-dsRed demonstrated strong colocalization correlations for P-selectin (R=0.65) and43A BPAI-1 -tSc 1Iii"4!^.1*.•■• .Ex — 563 nmEm — 530-575 nm• Cp• "0 4• .Ex 7- 563 nm ••Em — 600-650 nmPAI-1-dsRedvWF (R=0.77) (Figures 3.5A-B). Colocalization correlation values were determined withImageJ software.Figure 3.5. Intracellular compartmentalization of PAI-1-dsRed in Eahy926 cells.Confocal microscopy of immunofluorescence stained Eahy926 cells transfected with thePAI-1-dsRed vector. Figure 3.5A depicts colocalization of PAI-1-dsRed (red) togranules containing vWF (green). The composite image reveals colocalization ofR=0.65 between vWF and PAI-1-dsRed. Figure 3.5B depicts colocalization of PAI-1-dsRed (red) to granules containing P-selectin (green) in another transfected Eahy926cell. The composite image reveals a colocalization R=0.77 between P-selectin and PAI-1-dsRed. Each scale bar represents 10.0 pm. The bottom images represent spectralcontrols for PAI-1-dsRed (two bottom right) and isotype labeling controls (Rabbit IgG forvWF and Goat IgG for P-selectin).44B3.3.6. Activated endothelial cells express extracellular PAI-1-dsRed associated with vimentinTransfected Eahy926 cells were activated with TNF-a to induce PAI-1 exocytosis and todetermine the nature of extracellular PAI-1 distribution on the activated endothelial cell surfaces[17-20]. In Figure 3.6A, PAI-ldsRed was found to co-localize with VN present on the surface ofnon-permeabilized activated Eahy926 cells, suggesting that PAI-1 is bound to the surface via aPAI-1:VN complex interaction. Figure 3.6B demonstrates the distribution of surface exposedvimentin on non-permeabilized Eahy926 cells which is exposed as a result of cell activation byTNF-oc treatment. PAI-1-dsRed was shown to co-localize with VN (Figure 3.6A) and wasassociated with exposed vimentin intermediate filaments (Figure 3.6B) suggesting that VN andvimentin surface expression is a requirement for PAI-1 binding on cell surfaces [9].Figure 3.6. TNF-a treated Eahy926 cells express PAI-1-dsRed:vitronectin:vimentin complexes on their cell surface. Eahy926 cells transfected with the PAI-1-dsRedvector were activated with 12 hours incubation of 100 ng/mL TNF-a. Non-permeabilizedcells were immunostained for either vitronectin or vimentin (green) and a Hoechst33342 counterstain. Figure 3.6A represents a TNF-a activated transfected Eahy926 cellrevealing colocalization of vitronectin (green) and PAI-1-dsRed (red) on the cell surface.Figure 3.6B represents the distribution of PAI-1-dsRed relative to exposed vimentin(green) on the surface of the activated Eahy926 cells. The arrow heads highlight sitesof PAI-1-dsRed binding to exposed vimentin cytoskeleton. Scale bar, 7.0 urn.45ABC3.3.7. PAI-1-dsRed is targeted to a-granules in MEG-01 cells for rapid exocytosisTransfected MEG-01 cells were also stained with a variety of antibodies to confirm PAI-1-dsRed co-localization to resident a-granule proteins such as VN (Figure 3.7). The PAI-1polyclonal antibody was able to detect the majority of PAI-1-dsRed as shown by the strongyellow colocalization signal in the channel composite image. The disparate signal in Figure3.7A is due to the pseudopodial extensions on that particular cell. Staining with VN and vWFantibodies demonstrated strong yellow colocalization signal in both channel composite images,confirming that PAI-dsRed is compartmentalized to a-granules. PAI-1-dsRed fluorescencesignal did not appear alone and was generally found to colocalize with PAI-1, VN and vWF.Figure 3.7. Intracellular compartmentalization of PAI-1-dsRed in MEG-01 cells.As represented in whole image projections, transfected MEG-01 cells were stained withantibodies specific for PAI-1 (A), vitronectin (B), and vWF (C) and each antigen waslocalized to a-granules. The far right image of each panel is the overlay composite inwhich yellow signal denotes colocalization of PAI-1-dsRed and the antibody stain.Scale bar, 10.0 urn.463.3.8. Exocytosis of a-granules containing stores of PAI-1-dsRed in MEG-01 cellsTo examine real-time exocytosis of PAI-1-dsRed from storage granules, transfectedMEG-01 cells were incubated with 0.1 mM Alexa488-fibrinogen overnight to use as a positivemarker for the dynamics of a a-granule protein. MEG-01 cells endocytosed Alexa488-fibrinogeninto a-granules and exhibited significant colocalization with PAI-1-dsRed stores (Figure 3.8A-B). Thrombin-induced (1.0 U/mL) exocytosis resulted in rapid (—I minute) release of largegranules containing both fibrinogen—Alexa488 and PAI-1-dsRed.In contrast, a second subpopulation of granules positive for both fluorophores within thecytosol demonstrated signal depletion over a longer time period (-5 minutes); suggestingmobilization and externalization of this second subpopulation of granules. Additionally, a thirdsubpopulation of granules positive for both fibrinogen-Alexa488 and PAI-1-dsRed remainedstationary throughout the acquisition time period. In Scan A&B, the peaks of the line scanrepresent the signal of granules containing both Alexa488-fibrinogen (in green) and PAI-1-dsRed (in red). Scan A represents granules that eventually become exocytosed and anothergranule that remains stationary throughout the acquisition period. This stationary granule mayrepresent immature granules that are not able to undergo exocytosis. Scan B represents twogranules that become exocytosed by depletion of signal and loss of a doublet peak.4751.014C601..110 sec00:00^00.45^05:45Al4^4. ^,..„,60^4.5^9.0^13.5^17.0^21.0PAI-1-dsRedAFibrinogen-Alexa 488Scan line length (microns)B^00:00^00:45^05:45^300Fibrinogen-Alexa 488PAI-1-dsRed 3.3^6.7^10.0^13.3^16.7Scan line length (microns)Figure 3.8. Exocvtosis of PAI-1-dsRed and Alexa 488-fibrinogen from MEG-01 a-granules. MEG-01 cells with stores of Alexa 488-fibrinogen and PAI-1-dsRed wereactivated with thrombin (10 U/mL) to observe exocytosis of granules containing PAI-1-dsRed. Figures 3.8A-B depict wide-field fluorescence images of fibrinogen-Alexa 488(upper panels in 3.8A-B) and PAI-1-dsRed (lower panels in 3.8A-B) in two differentMEG-01 cells at various time points (min:sec) after thrombin stimulation. Theaccompanying graphs (right side) for Figures 3.8A-B depict spectral line scans of PAI-1-dsRed signal (deep red curve: t=0 secs, red curve: t=45 secs, red dashed curve: t=405secs) and Alexa488-fibrinogen signal (deep green curve: t=0 secs, green curve: t=50secs, dashed green curve: t=410 secs). The yellow and white arrowheads in theimages represent the scan line used to analyze the image, and this data is presented inthe graphs to the right of the images.483.4. Discussion: 3.4.1. OverviewIn this report, we reveal that a novel PAI-1-dsRed fusion protein is targeted to granulescontaining vitronectin within endothelial and megakaryocyte cell lineages respectively. PAI-1-dsRed was able to form complexes with VN on nitrocellulose, formed SDS-stable complexeswith tPA in conditioned media, and on the fibrin fibril surface PAI-1-dsRed attenuated clot lysis.Moreover, PAI-1-dsRed was sorted into a-granules in megakaryocytic cell cultures, and wasexocytosed upon thrombin stimulation. Interestingly, in the context of the Eahy926 cells, PAI-1-dsRed was found to colocalize with vWF- and P-selectin- containing granules, which has notbeen previously described. Upon TNF-a activation, PAI-1 was found to translocate to surfacebound VN, and exposed vimentin intermediate filaments. These findings are consistent withprevious flow cytometry and immunogold studies on activated human and bovine endothelium invitro [19]. The vitronectin-dependent binding of PAI-1 to vimentin has also been demonstratedin activated platelets and platelet microparticles generated in vitro and in subjects with heartdisease undergoing acute exercise stress [9].PAI-1-dsRed is the first fluorescent protein form of PAI-1 for investigating protein-protein binding interactions, biogenesis and maturation of secretory granules, and theconstitutive and regulated pathways of exocytosis in megakaryocytic cells and endothelium. Ourfinding of PAI-1-dsRed colocalizing with vWF and P-selectin in WPB-like granules in Eahy926cells raises the question regarding the role of the fluorescent dsRed protein in regulating thesorting of the fusion protein. As a protein tag, dsRed was significantly large enough (-23 kDa)to cast concerns about its potential steric interference effect on PAI-1, a serpin highly dependenton the position of its reactive center loop (RCL) relative to its body. However, recognition of49both native and fused forms of PAI-1 by a polyclonal specific for PAI-1 now suggests that thedsRed tag does not interfere with the PAI-1 antibody binding epitopes. In terms of VN bindinginteractions with PAI-1-dsRed, positioning the dsRed tag at the C-terminus end of the PAI-1enabled the VN binding site (aa 115-167) [21] to remain unobstructed, thereby allowing active toinactive latent phases of the PAI-1 molecule to be primarily dependent on its interactions withVN. Similar strategies in fusing a GFP tag with vimentin and cathepsin B also preservedfunctionality [22, 23].Previous studies for tracking PAI-1 entailed the chemical conjugation of FITC or biotinto the PAI-1 molecule [24, 25]. In detail, specific conjugation of fluorophores such as FITC tothe P1' [26], P3 and P18 domain of PAI-1 [27] by insertion of a cysteine residue by site-directedmutagenesis permitted labeling of this sulfhydryl group with iodoacetamide derivatives offluorophores such as FITC without any observed inhibition of PAI-1:tPA interactions; in fact,these strategies further stabilized PAI-1 activity [24]. Biotinylation of PAI-1 also resulted inminimal loss of PAI-1 inhibitory activity as determined by VN ligand blotting and ELISA [7].In terms of protein tags, we have previously constructed a recombinant human PAI-1 fusionprotein containing the 6 residual peptide consensus sequences for heart muscle kinase (HMK) atthe amino terminus and shown it to be fully functional, while also allowing for radiolabellingwith 32P for quantitative binding studies [25]. However, our use of the PAI-1-dsRed fusion nowgives us the same advantages as all the other forms of labeled PAI-1 plus the advantage of its usein real-time secretory granule biogenesis tracking studies.503.4.2. Thrombin induced MEG-01 cell a-granule exocytosisTime-lapse video microscopy confirmed that thrombin stimulation of MEG-01 cellsinduced rapid exocytosis of a large majority of a-granules storing PAI-1, PAI-1-dsRed andfibrinogen within <45 seconds. A second population of a-granules containing PAI-1-dsRedremained static but demonstrated depletion of signal over a longer period of time. It appears thatthere are two classes of a-granules: larger more mature a-granules which possessed bothfibrinogen and PAI-1-dsRed stores primed for exocytosis, and smaller more peri-nuclear vesiclesthat only contained PAI-1-dsRed. It is conceivable that the lack of fibrinogen in these smallergranules may suggest an immature stage of these granules, perhaps containing freshlysynthesized PAI-1 shuttled from the Golgi apparatus. Therefore, it is a possibility that PAI-1-VN complexes are assembled in fully mature granules nearest the cell membrane because VNwould be expected to follow a similar endocytotic fate as fibrinogen [9, 28]. Similar observationsregarding differential fates of storage granules have been found in cultured HUVECs and theirWPB's when activated by thrombin. Vinogradova et al., observed two main fates of vWF-containing Weibel-Palade bodies, immediate exocytosis or centrosome-directed translocation tothe peri-nuclear space [29].3.4.3. PAI- 1 colocalizes with vWF in Eahy926 cellsAfter establishing the properties of PAI-1-dsRed as a native equivalent of PAI-1, wedetermined that it colocalizes with vWF and P-selectin in large, heterogeneous shaped granulesin Eahy926 cells. Given that vWF and P-selectin are well-described Weibel-Palade body (WPB)resident proteins, it is apparent that PAI-1-dsRed is internalized into these granules just as it is inmegakaryocytic cells and platelets [7]. Perhaps more relevant is that these results challenge51previous reports describing PAI-1 intracellular distribution [13, 14], in that our technique of PAI-1 intracellular colocalization does not require antibody detection of PAI-1. This is an advantagefor in vitro and in vivo tracing studies given the disadvantages of using an antibody that mayhave a poor binding signal because of steric hinderance from VN or other WPB proteins,masking binding epitopes on the PAI-1 molecule. Using the PAI-1-dsRed protein, we were ableto observe the punctate or ring-like distribution of exposed vimentin [32] and the distribution ofvimentin-vitronectin-PAI-1 ternary complexes at these sites on the surface of TNF-a activatedEahy926 cells. More specifically, PAI-1-dsRed was found to be primarily associated with VNand the exposed vimentin intermediate filament cytoskeleton, a finding only previously observedon activated platelets and platelet microparticles [9]. Overall, the punctate distribution of PAI-1-dsRed and VN (Figure 3.6A) indicates that cell activation results in the binding of PAI-1-dsRed:VN complexes from the conditioned media binding to this exposed vimentin cytoskeleton,forming the ternary complex as previously described [9]. To validate the ring-like, punctatedistribution of VN and PAI-1, previous reports have also described this observation of surfaceexposed vimentin in a ring-like morphology [32, 33]. Surface vimentin exposed to plasma, willact as a site for VN multimerization [9] and to an extent, PAI-1-dsRed:VN complexincorporation into this VN multimer via VN-VN homotypic interactions [34].The construction of the first endogenous red fluorescent form of functionally activehuman PAI-1 is a catalyst for studying the cell biology of PAI-1 and its role in vascularfibrinolysis. In addition to real-time fluorescence imaging in cultured cells, the PAI-1-dsRedfusion protein can be used for in vivo applications with transgenic mice, and as purified proteinfor ELISA assay design, and for studying interactions between PAI-1 and target ligands.523.5 References for Chapter III: 1. Gils A, Declerck PJ. Plasminogen activator inhibitor-1. Curr Med Chem 2004; 11:2323-34.2. Lindahl TL, Ohlsson PI, Wiman B. The mechanism of the reaction between humanplasminogen-activator inhibitor 1 and tissue plasminogen activator. Biochem J 1990;265:109-13.3. Schleef RR, Wagner NV, Loskutoff DJ. Detection of both type 1 and type 2 plasminogenactivator inhibitors in human cells. J Cell Physiol 1988; 134:269-74.4. MacGregor IR, Booth NA. 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Podor TJ, Singh D, Chindemi P, et al. Vimentin exposed on activated platelets andplatelet microparticles localizes vitronectin and plasminogen activator inhibitorcomplexes on their surface. J Biol Chem 2002; 277:7529-39.10. Zhou A, Huntington JA, Pannu NS, Carrell RW, Read RJ. How vitronectin binds PAI-1to modulate fibrinolysis and cell migration. Nat Struct Biol 2003; 10:541-4.11. Preissner KT, Holzhuter S, Justus C, Muller-Berghaus G. Identification of and partialcharacterization of platelet vitronectin: evidence for complex formation with platelet-derived plasminogen activator inhibitor-1. Blood 1989; 74:1989-96.12. Mousa SA, Bozarth J, Forsythe M, Tsao P, Pease L, Reilly TM. Role of plateletGpIIb/IIIa receptors in the modulation of platelet plasminogen activator inhibitors type-1(PAI-1) release. Life Sci 1994; 54:1155-62.5313. Rosnoblet C, Vischer UM, Gerard RD, Irminger JC, Halban PA, Kruithof EK. Storage oftissue-type plasminogen activator in Weibel-Palade bodies of human endothelial cells.Arterioscler Thromb Vasc Biol 1999; 19:1796-803.14. Ma GM, Halayko AJ, Stelmack GL, et al. Effects of oxidized and glycated low-densitylipoproteins on transcription and secretion of plasminogen activator inhibitor-1 invascular endothelial cells. Cardiovasc Pathol 2006; 15:3-10.15. Novokhatny VV, Ingham KC, Medved LV. Domain structure and domain-domaininteractions of recombinant tissue plasminogen activator. J Biol Chem 1991; 266:12994-3002.16. Handagama P, Scarborough RM, Shuman MA, Bainton DF. Endocytosis of fibrinogeninto megakaryocyte and platelet alpha-granules is mediated by alpha llb beta 3(glycoprotein IIb-IIIa). Blood 1993; 82:135-8.17. Schleef RR, Loskutoff DJ, Podor TJ. Immunoelectron microscopic localization of type 1plasminogen activator inhibitor on the surface of activated endothelial cells. J Cell Biol1991; 113:1413-23.18. Hill SA, Podor TJ. Serum-dependent modulation of the type 1 plasminogen activatorinhibitor binding to endothelial cell surfaces. Ann N Y Acad Sci 1992; 667:42-5.19. Podor TJ, Loskutoff DJ. Immunoelectron microscopic localization of type 1 plasminogenactivator inhibitor in the extracellular matrix of transforming growth factor-beta-activatedendothelial cells. Ann N Y Acad Sci 1992; 667:46-9.20. Podor TJ, Joshua P, Butcher M, Seiffert D, Loskutoff D, Gauldie J. Accumulation of type1 plasminogen activator inhibitor and vitronectin at sites of cellular necrosis andinflammation. Ann N Y Acad Sci 1992; 667:173-7.21. Padmanabhan J, Sane DC. Localization of a vitronectin binding region of plasminogenactivator inhibitor-1. Thromb Haemost 1995; 73:829-34.22. Linke M, Herzog V, Brix K. Trafficking of lysosomal cathepsin B-green fluorescentprotein to the surface of thyroid epithelial cells involves the endosomal/lysosomalcompartment. J Cell Sci 2002; 115:4877-89.23. Lochner JE, Kingma M, Kuhn S, Meliza CD, Cutler B, Scalettar BA. Real-time imagingof the axonal transport of granules containing a tissue plasminogen activator/greenfluorescent protein hybrid. Mol Biol Cell 1998; 9:2463-76.24. Shore JD, Day DE, Francis-Chmura AM, et al. A fluorescent probe study of plasminogenactivator inhibitor-1. Evidence for reactive center loop insertion and its role in theinhibitory mechanism. J Biol Chem 1995; 270:5395-8.5425. Podor TJ, Peterson CB, Lawrence DA, et al. Type 1 plasminogen activator inhibitorbinds to fibrin via vitronectin. J Biol Chem 2000; 275:19788-94.26. Shore JD, Vandenberg, E., Day, D., Olson, S.T., Sherman, R., Ginsburg, D., Kvassman,J. Fibrinolysis 1992; 6, Suppl. 2:Abstract 292.27. Strandberg L, Johansson, L. B.-A., Ny, T. Fibrinolysis 1992; 6 Suppl. 2.28. Wencel-Drake JD, Boudignon-Proudhon C, Dieter MG, Criss AB, Parise LV.Internalization of bound fibrinogen modulates platelet aggregation. Blood 1996; 87:602-12.29. Vinogradova TM, Roudnik VE, Bystrevskaya VB, Smirnov VN. Centrosome-directedtranslocation of Weibel-Palade bodies is rapidly induced by thrombin, calyculin A, orcytochalasin B in human aortic endothelial cells. Cell Motil Cytoskeleton 2000; 47:141-53.30. Volker W, Hess S, Vischer P, Preissner KT. Binding and processing of multimericvitronectin by vascular endothelial cells. J Histochem Cytochem 1993; 41:1823-32.31. de Boer HC, Preissner KT, Bouma BN, de Groot PG. Internalization of vitronectin-thrombin-antithrombin complex by endothelial cells leads to deposition of the complexinto the subendothelial matrix. J Biol Chem 1995; 270:30733-40.32. Hansson GK, Starkebaum GA, Benditt EP, Schwartz SM. Fc-mediated binding of IgG tovimentin-type intermediate filaments in vascular endothelial cells. Proc Natl Acad Sci US A 1984; 81:3103-7.33. Hansson GK, Lagerstedt E, Bengtsson A, Heideman M. IgG binding to cytoskeletalintermediate filaments activates the complement cascade. Exp Cell Res 1987; 170:338-50.34. Wu YP, Bloemendal HJ, Voest EE, et al. Fibrin-incorporated vitronectin is involved inplatelet adhesion and thrombus formation through homotypic interactions with platelet-associated vitronectin. Blood 2004; 104:1034-41.55CHAPTER IV: DISTRIBUTION OF PAI-1:VITRONECTIN:VIMENTIN TERNARYCOMPLEXES ON ACTIVATED PLATELETS AND PLATELET MICROPARTICLES BY ATOMIC FORCE MICROSCOPY. 4.1. Introduction: Elevated plasma levels of plasminogen activator inhibitor type-1 (PAI-1) have beenfound to be correlated with thrombotic events, a likely association due to its ability to inhibit thefibrinolytic branch of hemostasis [1-6]. PAI-1 can attenuate fibrinolytic breakdown of thrombiby inhibiting the key initiators of fibrinolysis, tissue-type and urokinase-type plasminogenactivators (tPA and uPA) [7], and the inhibitory activity of PAI-1 is further prolonged when itforms a complex with vitronectin (VN), an abundant plasma protein [8]. While VN can stabilizePAI-1 activity, VN can also direct PAI-1 localization in thrombi by mediating its binding to twomajor components: the surfaces of activated platelets and fibrin [9, 10].The assembly of the PAI-1:VN:vimentin ternary complex is largely responsible for the majorityof cell-surface bound PAI-1 on activated cells, namely activated platelets and plateletmicroparticles (PMP's). In detail, VN binds with high affinity to vimentin [9], a cytoskeletalintermediate filament protein expressed by mesenchymal cell lineages such as platelets [9, 11],endothelial cells [12, 13] and leukocytes [14]. We have previously determined that the headdomain of vimentin is exposed on the surface of activated platelets, acting as a site formultimerization of VN and incorporation of PAI-1:VN complexes [9]. In theory, PAI-1:VNcomplexes can also be incorporated into these vimentin-induced multimers due to homotypic'A version of this chapter will be submitted for publication. Leong, HS, Bateman RB, Walinslci H, van Eeden SVand Podor T.J. Distribution of PAI-1:Vitronectin:Vimentin Ternary Complexes on Activated Platelets and PlateletMicroparticles by Atomic Force Microscopy.56VN-VN interactions [15]. Although the exact proportion of each component is not fullyunderstood, the extent of vimentin-induced multimerization of VN is thought to dictate the extentof PAI-1 localization onto activated cell surfaces.Platelet microparticles (PMP's) are vesicular bodies that are released during plateletactivation and have been postulated to play a pro-thrombotic role in acute coronary syndromes[16 , 17]. It is thought that PMP's are shed from the tip or median of platelet pseudopodia [18,19], therefore we hypothesized that vimentin exposure occurs at the broken junction between thereleased PMP and the pseudopod of the activated platelet, which subsequently becomes availablefor PAI-1:VN:vimentin ternary complex formation. To understand this exposure of vimentin, weused atomic force microscopy to visualize PMP's that expressed some, if not all, components ofthe ternary complex. To do this, we isolated and imaged PMP's generated from expired plateletconcentrates or platelet-poor plasma (PPP) from post-myocardial infarction patients. PMP'swere isolated by FACS and their topography visualized by atomic force microscopy (AFM). Asimilar strategy was performed on isolated activated platelets in order to visualize VN-vimentinmultimers on the activated platelet surface. Finally, we used AFM to visualize VN-vimentinmultimers formed in vitro in a cell-free preparation to understand the ultrastructure of VN-vimentin multimers and to determine if these multimers have a distinct molecular organizationthat allows them to be recognized on the surface of activated platelets and PMP's.574.2. Materials and Methods: 4.2.1. Ethics, blood preparation and PAI-1 ELISAEthics approval was obtained from the University of British Columbia Clinical ResearchEthics Committee to collect whole blood from patients at 24, 48, and 72 hours post-myocardialinfarction. Whole blood was collected into 4.5 mL acid citrate dextrose vacutainers (BDBiosciences, Mississauga ON). Blood samples were then centrifuged at 1500xg for 15 minutesand the upper layer representing the platelet-poor plasma (PPP) fraction was removed and storedat -80°C. Expired platelet concentrates (10 days post-preparation) were provided by the NationalBlood Service (UK, Harefield Hospital). An ELISA kit to measure active PAI-1 in patient PPPwas used, in which 5 gL of PPP diluted in 20 gL of reaction buffer was assayed for in triplicate(Molecular Innovations, Ann Arbor, MI).4.2.2. Electrophoresis of High Molecular Weight Protein Complexes from Patient PlasmaSamplesDiluted PPP samples (4 gL PPP + 16 gL of PBS + 4 gL 6X sample buffer) andelectrophoresed on a native 8% acrylamide gel with a 5% acrylamide stacker gel. Native proteinseparation was visualized by staining with Coomassie blue, and acrylamide gel pieces withpositive staining in the stacker well interface were excised and then incubated in 400 gL ofelution buffer (0.25 mM Tris-HC1 buffer, pH=6.8; 0,1% (w/v) SDS) at 4°C overnight. Thispreparation was then ultrafiltrated with a Nanosep Centrifuge tube column (0.28 gm pore size;Pall Life Sciences, Ann Arbor, MI) by centrifugation at 8000xg for 20 minutes. After elution,the samples were boiled in sample buffer containing 5% 2-mercaptoethanol, and fractionated in10% SDS-PAGE gels. Separated proteins were transferred to nitrocellulose membrane, and after58blocking with blotting buffer (1X PBS, pH 7.4, containing 2% milk powder and 0.05% w/vTween-20), the membranes were incubated for 1 hour with antibodies directed against PAI-1,VN, and vimentin at a final concentration of 5 gg/mL (Affinity Biologicals Inc., Hamilton, ON).After washing, membranes were incubated with a 1:2000 dilution of alkaline phosphatase-conjugated rabbit anti-sheep IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 1 hourand then developed with AP Color Development Reagent Kit (BioRad Corporation Inc.,Hercules, CA).4.2.3. FACS isolation of platelet microparticles and activated platelets expressing ternarycomplexFACS analysis was used to assay PMP populations in PPP collected from patients at 48hours post-AMI. PPP (20 gL) was incubated for 15 minutes with a combination of: 1) mouse a-human CD41 a IgGi-RPE (BD Biosciences, Burlington, ON), sheep a-human vimentin IgG(Affinity Biologicals Inc, Hamilton, ON) with donkey a-sheep-FITC IgG (BD Biosciences), andrabbit a-human VN IgG (Affinity Biologicals Inc.) with goat a-rabbit Alexa633 IgG (MolecularProbes, Eugene, OR); or 2) mouse a-human CD41a IgGi-RPE (BD Biosciences), sheep a-humanvimentin (Affinity Biologicals Inc.) with donkey a-sheep FITC IgG (BD Biosciences) and mousea-human PAI-1 IgG (Molecular Innovations, Ann Arbor, MI), or mouse a-human CD62PIgGi (BD Biosciences) both labeled with goat a-mouse Alexa633 IgG (Molecular Probes Inc.)secondary antibody. The forward scatter (FS) PMT threshold was minimized to measure allparticles of any size with both significant FITC and Alexa633 fluorescence. Microspheres with a1.0 gm diameter (Molecular Probes Inc.) were analyzed to define a size standard in terms offorward scatter of microparticles. For PMP sample isolation, only particles that exhibited an FS59below that of the 1.0 gm microsphere forward scatter with significant dual FITC+Alexa633fluorescence was sorted onto mica for AFM imaging.Human blood was drawn into ACD tubes and PRP was prepared by centrifugation ofanti-coagulated blood at 200xg for 10 minutes and the supernatant transferred into another tube.This PRP fraction was centrifuged at 1000xg for 10 minutes, the resulting supernatant removedand the platelet-rich pellet was gently washed twice with calcium-free Tyrodes buffer andresuspended with modified Tyrodes buffer to a final cell concentration of 1x104 platelets/gL.One aliquot of diluted washed platelets was activated with 2 gL of 10 U/mL of thrombin andincubated for 30 minutes. Activated platelets were incubated for 15 minutes with a combinationof mouse a-human CD41 a IgG1 -RPE (BD Biosciences, Burlington, ON), sheep a-humanvimentin IgG (Affinity Biologicals Inc, Hamilton, ON) with donkey a-sheep-FITC IgG (BDBiosciences), and rabbit a-human VN IgG (Affinity Biologicals Inc.) with goat a-rabbitAlexa633 IgG (Molecular Probes). CD41+ve platelets with the highest FITC+Alexa633 dualsignal were gated and isolated by FACS.4.2.4. Atomic Force Microscope (AFM) SpecificationsThe instrument used for atomic force microscopy was a BioScope (Digital InstrumentsInc., Santa Barbara, CA), which is an AFM head piece mounted onto an inverted microscope(Axiovert 100, Zeiss Inc.). For sample preparation, proteins and cells were adsorbed onto apiece of freshly cleaved mica (2 cm2, Ted Pella Inc., Redding, CA). Tapping mode AFM wasused for all microscopy and was performed in ambient temperature and humidity using VeecoTapping Mode Etched Silicon cantilevers (k=40 N/m). A slow scan rate of 1-3 Hz was used tominimize sample disturbances giving a scan rate that was much slower (<25 000x) than the tap60rate. A maximum resolution of 512x512 pixels was used. The x- and y-scan directions werecalibrated with a 10x 10 pm2 grid. The z-direction was calibrated with 5 nm diameter goldparticles (Ted Pella Inc.) on a cleaved mica surface. The scans were tested for imaging artifact byvarying scan direction, scan size, and by rotating the sample.4.2.5. Atomic force microscopy of platelet microparticles, VN-vimentin multimers and activatedplateletsPMP's and activated platelets that presented significant expression of antibodies usedwere gated and sorted directly onto freshly cleaved mica sheets for AFM imaging (Ted Pella,Inc.). Approximately 200 particles were absorbed onto the mica sheet. AFM imaging ofprotein-protein interactions of VN and vimentin required pretreatment of mica sheets with adH2O wash, then adsorption of 10 lit of 5 mM MgC12 on the center of the mica sheet for 5minutes. Excess MgC12 was washed off with dH2O and then gently blow dried with dry nitrogengas. To form vitronectin-vimentin multimers, purified human VN (Molecular Innovations Inc.,Southfield, MI) and purified vimentin head domain protein [9] were incubated at varying molarratios and the multimerization reactions were carried out in PBS (pH 7.4) at 37 °C for 24 hours.To prepare protein samples for AFM imaging, 20 tiL of sample was plated on the center of thecoverslip and then left to adsorb onto the mica at room temperature for 10 minutes. Protein wasfixed by addition of 2 pL of EM grade 0.025% w/v paraformaldehyde for 5 minutes. The samplewas gently washed three times with 0.1 M ammonium acetate (Ultrasigma grade, Sigma-Aldrich,St. Louis, MO) and then gently dried with dry nitrogen gas. To quantify VN-VIM multimerstructure, images were processed using MATLAB and quantified by fractal analysis. The imageprocessing algorithm consisted of background signal subtraction, median filter, image61thresholding (Otsu's method [20]) and binarization. Fractal dimension was calculated using thebox dimension estimation method. The box dimension, Db, is defined as the exponent of N(d)1/dDb, where N(d) is the number of boxes of linear size (d) required to cover a set of pointsdistributed in a 2D plane.4.2.6. Platelet-Rich Clot Formation and Staining for Vimentin, Vitronectin and PAI- 1Human blood was drawn into ACD tubes and PRP was prepared by centrifugation ofanti-coagulated blood at 200xg for 10 minutes and the supernatant transferred into another tube.This supernatant was recalcified with 1 M CaC12 to a final concentration of 10 mM Ca2+ prior toclot formation. To generate a platelet-rich clot, 2 'IL of 10 U/mL human thrombin (Chronolog,Havertown, PA) was added to 20 ut of platelet-rich plasma and then quickly pipetted onto a 1mm thickness glass coverslip and left to incubate for 60 minutes at 37°C. The clot was incubatedfor 1 hour for both primary and secondary antibody incubations with these antibodies: sheepanti-human vimentin IgG antibody with donkey anti-sheep IgG-FITC secondary antibody, andmouse anti-human \TN IgG (clone - 1244) with goat anti-mouse IgG-Alexa594 secondaryantibody. After several PBS washes (1X, pH=7.4), the sample was mounted with a coverslip andanti-fade mounting media (Molecular Probes, Eugene, OR).624.4. Results: 4.4.1. High molecular weight complexes in post-AMI platelet-poor plasma contains elevatedPAI-1, vitronectin, and vimentinTwo PPP sets of patient samples (samples collected at t=24, 48, 72 hours post-AMI) wereelectophoresed on a non-reducing, native PAGE gel in order to separate the high molecularweight protein fraction from plasma, which also contains platelet microparticles. Coomassieblue stain revealed the accumulation of HMW protein complexes in the stacker gel (Figure4.1A). These HMW complexes were eluted from the native gel and further fractionated bySDS-PAGE and subjected to Western blot analysis. At all three time points, levels of PAI-1, VNand vimentin expression were greater or equal to levels found in normal human plasma. Themultiple bands (>47kDa) detected by the PAI-1 polyclonal antibody represents cleavagefragments of covalently-formed complexes that contain some of the PAI-1 molecule. Table 1presents the levels of active PAI-1 in the PPP in a group of patients 48 hours post-AMI. Patient#1 and #2 are part of this group. Levels of active PAI-1 in control PPP are <2.0 U/mL.Nurber of sullects (N) 6Male/Ferrele 6/0Aix (yrs) 571:3.3aigrette smoker 16.70%Complications pcst-analysis 33.30%Patient plasma Active PAI-1 (U/rri..) 5.2±1.1Ccntrol plasma Adive PAI -1 (U/rri) N=9 1.1±0.1Table 4.1. Clinical data of patients with Acute Myocardial Infarction (AMI). Active PA1-1 levels were determined in PPP collected from patients 48 hours post-AMIwith an ELISA assay specific for PAI-1.634- 61 kDa4- 24 kDa4- 18 kDaeluted protein frompatient #1eluted protein frompatient #2B24 hr^48 hr^72 hr24 hr^48 hr^72 hrControl4- 82 kDa-4- 47 kDa•PAI -1VitronectinVimentinPlatelet-Poor Plasmafrom patient #1Post-AMI24 hr^48 hr^72 hrAstacker wellseparatinggel interfaceFigure 4.1. Platelet poor plasma (PPP) from post-acute ischemic infarction (post-AMI) patients contain high molecular weight protein complexes consisting ofvimentin, vitronectin and PAI-1. A) illustrates the migration of PPP in a native PAGEgel and the entrapment of high molecular weight protein complexes in the stacker wellinterface. Following excision and elution of this high molecular weight fraction,immunoblot analysis in a 8% SDS-PAGE in B) demonstrates vimentin, vitronectin andPAI-1 protein levels to be higher in both post-AMI patients compared to control PPP.644.4.2. Platelet microparticles express vitronectin, vimentin and PAI-1 on their surface asdetermined by FACS AnalysisFigure 4.2 is an overview of the FACS analysis performed on a representative 48 hourpost-AMI PPP sample (Figure 4.2A) and a control PPP sample (Figure 4.2B). In the left panel,use of a CD41 antibody detected a population of CD41-positive particles with lower forward andside light scatter compared to beads with a diameter of —1.0 p,M, representing PMP's. With thisgating, a minimum of 10,000 counts were analyzed. The middle and right panels represent co-expression scatter plots of these CD41-positive PMP's that also co-express vimentin andvitronectin (middle histoplot); as well as vimentin and PAI-1 (right histoplot). These histoplotssuggest that at least two components of the ternary complex are present on a subpopulation ofCD41-positive PMP's in greater quantities (Figure 4.2A) compared to control PPP (Figure 4.2B).Figure 4.2. Flow Cytometric Analysis of PPP from post-AMI patients. Flowcytometry was performed to determine the amount of vitronectin, vimentin and PAI-1 onCD41-positive particles present in platelet-poor plasma. Each forward/side scatterhistogram represents a combination of PAI-1, vitronectin, vimentin vs. CD41 or vimentinco-expression. Each inset box in the FS vs. SS histrogram depicts the cell populationsorted for atomic force microscopy as determined by the upper limit of forward scatter ofUV microspheres with a diameter of 1.0 pm.654.4.3. AFM ultrastructural analysis of PMP's positive for ternary complexAs determined in Figure 4.2, PMP's positive for vimentin-vitronectin or vimentin-PAI-1were sorted onto mica coverslips for atomic force microscopy. Image analysis of these patientPMP's reveal a heterogeneous population of granular, round and vesicular particles with adiameter range of 350-900 nm. In Figure 4.3A, AFM images of PMP's expressingCD41+vimentin, as well as VN (top), or PAI-1 (second from top) or P-selectin (third from top)are presented in two images: a height dimension channel with a pseuodocolour LUT (nm) and aamplitude channel in grayscale that represents the topographical detail of the PMP. Asubstantial proportion of sorted patient PMP's exhibited extensive surface protein aggregationwhich may in part, reflect localization of the antigen-antibody complexes specific for multimericVN, vimentin and PAI-1. In these sample patient samples, a population of PMP's expressingCD41+vimentin+VN exhibited minor phospholipid membrane appendages extending from baseof the microparticle (Figure 4.3A second from top). These short membrane appendages or"skirts" had a low height approximating —5-7 nm and attached to the base of the microparticle.PMP's isolated from post-AMI patient PPP exhibited substantial surface topography asmanifest by the presence of many proteinaceous structures on the surface of the PMP's. Incontrast, PMP's generated in vitro by expired platelets did not present with such topography andpermitted ultrastructural analysis in assessing expression of ternary complex on the surface ofPMP's. In Figure 4.3B, PMP's co-expressing vimentin or vimentin-vitronectin also exhibitedextensive phospholipid appendage-like structures attached to the base of the microparticle. Itwas determined that 68% (17/25) of PMP's expressing VN+VIM had membrane flaps thatexhibited an average thickness of 6.3±1.3 nm. Moreover, 45% (5/11) of PMP's expressing66vimentin exhibited membrane flaps with a thickness of 5.8±0.2 nm. Figure 4.3C demonstratesthe manner in which a PMP expressing CD41+vimentin is analyzed in terms of (panel from leftto right) a height dimension, amplitude dimension, a scan line of the height dimension of thePMP, and a 3-dimensional perspective of the PMP depicting the height difference between thePMP and its membrane flaps. In this particular PMP, the scan line graph reveals that themembrane thickness to be —5.6 nm thick, which is within the 4-7 nm thickness range determinedby previous observations [21].In Figure 4.4, one particular PMP that expressed CD41 and vimentin was chosen forheight profile analysis because of filamentous structures associated with membrane flapsattached to the PMP (As seen in Figure 4.3B second from the top). In Figure 4.4A&B, thesefilamentous were analyzed via scan line analysis (green scan line in A and grey scan line in B).Figure 4.4C represents the height profile of this scan line and when compared to the membraneheight of 6.3nm, the two major peaks had thickness values of 11.3 and 9.1 nm, which is withinthe range of reported diameters of vimentin intermediate filaments (9 —11 nm)[22].4.4.4. Microscopy of vitronectin -vimentin multimers on activated plateletsAs previously described in [9], a subpopulation of activated platelets are known toexpress high amounts of vimentin and vitronectin on their surface, observations that lead to aproposed mechanism wherein vimentin expression is requisite for vitronectin multimerizationand consequent ternary complex formation. Extracellular distribution of the ternary complex onactivated platelets was imaged with confocal microscopy as shown in Figure 4.5A-D and withAFM in Figure 4.5E. Panels B and C illustrate the staining distribution of vimentin andvitronectin of an activated platelet and in panel D, yellow signal represents areas of co-67PAI-1+VIM 500nm^ 1.0VX-axis: 1.3 urnVN+VIM kekiS. 1.0 umCD62P+X-axis: 1.3 umLiposomeX-exis: 1.0 umVIMX axle: 2.0 pmPAI - 1+VIM X-axis: 1.3 urn550ni 1.5VX-axis: 1.3 urn Membrane "flaps"3D viewmembranethicsnessfi FII , 1A^ BScan line through PMPmembranethickness5 6or 4ioScan line (um)Figure 4.3. Atomic force microscopy on platelet microparticles isolated from platelet- poor plasma. A) depicts images of PMP's as isolated from post-AMI plasmathat express CD41+PAI-1+vimentin (VIM), CD41+VN+VIM, CD41+CD62P+VIM, (top tobottom). B) depicts images of PMP's isolated from expired platelet concentrates thatexpress CD41+VIM, CD41+VN+VIM, CD41+PAI-1+VIM (top to bottom). In B), themajority of PMP's expressing CD41+VIM presented membrane appendages at the baseof the PMP. Each PMP is presented as two types of images, the left column a heightimage channel as measured in nanometers (nm) and the right column an amplitudechannel as measured in volts (V). The height channel provides a quantitative Z-planeperspective while the amplitude channel provides a superficial topographicalperspective.1_1` 5,4 1S.1001.11,41168C 11.3 nm thickness15— 9.1 nm thickness10N50^50^100 150 200 250 300 350Figure 4.4 – Scan line analysis of filament-like structures present withinmembrane flaps of PMP's expressing CD41+vimentin.  A) depicts the amplitudechannel of PMP and B) depicts the height dimension of the PMP. The green and greyscan line in A) and B) respectively marks the scan line used to analyze the filamentousstructures. In C), the peaks represent the heights of the filaments when the membraneheight is considered. The two major peaks identified represent filament structures witha diameter of 11.3 and 9.1 nm (left to right). This is within the reported diameters ofintermediate filaments (9-1 1 nm in diameter).69localization of vimentin and VN throughout the activated platelet. In this same image, discreteVN signal was also observed as minor protrusions extending from the vimentin cytoskeleton.This latter observation is consistent with our previous findings demonstrating multimerized VNat sites of exposed vimentin intermediate filaments [9]. Panel A represents another activatedplatelet stained with isotype negative controls with their respective FITC- and RPE- IgGconjugates. Ultrastructural analysis using AFM was performed on thrombin-activated plateletscoexpressing vimentin and vitronectin as shown in Figure 4.5E. Height dimension analysisidentified the structures as being two platelets adhering to the mica surface, each having amaximal height of —720 nm (data not shown). Panel E is a merged image of the trace andretrace deflection channel. An organized field of globular proteins can be observed on thehighest plateau of each platelet, and this distribution of protein is distinct from rest of the flatsurface of the platelet. It is believed that the distribution of vimentin-VN complexes isrepresented on the cortical region of the activated platelet and similar to the distribution ofvimentin-VN complexes in Panel D. In general, activated platelets with strong VN and vimentinsurface immunoreactivity exhibited a disc-like morphology with a central depression.Furthermore, the perimeter of these activated platelets was elevated and supported a distributionof carpet-like vimentin and VN multimers disproportionate to one side of the platelet.4.4.5. Multimers of vitronectin-vimentin form a highly ordered ultrastructureCell-free experiments with purified proteins were performed to simulate the formation ofVN multimers bound to the platelet vimentin cytoskeleton that become exposed during plateletactivation. The AFM was used to visualize the ultrastructure of the polymeric VN-vimentin70Figure 4.5. Distribution of vitronectin-vimentin multimers on the cell surface ofactivated platelets. A-D) depicts images of an activated platelet as acquired byconfocal microscopy. A) is an isotype antibody stained activated platelet, red signal B)represents vimentin and green signal C) represents vitronectin. The formation ofvitronectin-vimentin multimers is highlighted by yellow colocalization in D). E) is amerged AFM image of the trace and retrace deflection channel of an aggregate ofactivated platelets. Washed platelets were activated with thrombin and then stainedwith antibodies for vimentin and vitronectin. Only dual-labelled platelets were isolatedby flow cytometry sorting for AFM imaging. The distribution of vimentin-vitronectincomplexes on the cortical regions of the activated platelets as highlighted by the insetboxes is analogous to the distribution of the vimentin-vitronectin complexes in D).complex at a nanometer resolution. The reaction was primarily carried out in PBS (pH 7.4),while similar results were achieved with distilled water. Moreover, varying the incubation timesfrom 15 minutes to 24 hours did not affect the overall ultrastructure of the VN-vimentinmultimers (data not shown). Figure 4.6A demonstrates an AFM image of monomeric VN platedat a concentration of 5 iig/mL (1.3 1AM). VN monomers on average are -3 nm in height as seenin the corresponding scan line height analysis graph (right). Figure 4.6B represents vimentinhead domain protein (VIM133) which is approximately 13 kDa, at a height dimension of -0.8nm. Polymeric VN-vimentin aggregates as imaged by AFM illustrated a highly organized lattice710.6 0.802^•T:  1  ^0^0.2^0.4^0.6^0.8' 41® ]200 400 1000Figure 4.6. AFM analysis of the ultrastructure of vitronectin-vimentin multimers.The head domain of vimentin (VIM133) and purified vitronectin was used to formvitronectin-vimentin multimers. The above images represent height dimensions. A)represents monomeric vitronectin (0.8 nM) and B) represents monomeric VIM133 (0.6nM). Fractal dimension analysis (Db) of A) and B) are 1.32 and 1.11 respectively. C)Represents ultrastructure of vitronectin-vimentin multimers at a 3:2 molar ratio with afractal dimension of 1.74. D) represents vitronectin-vimentin multimers formed at amolar ratio of 6:1 with a fractal dimension analysis of 1.20.72ultrastructure in which no free protein was found unbound to the lattice (Figure 4.6C). In thisimage, no structures had a height that exceeded —3nm, suggesting that vimentin induces aconformational change such that VN height becomes <1.6 nm. This would indicate thatvimentin induces a structural change in VN that destabilizes its intramolecular structure.Fractal dimension analysis calculations were performed to determine the order andorganization of imaged structures by AFM. Fractal dimension analysis of monomeric VN andVIM133 protein generates Db's of 1.32±0.30, 1.11±0.30 indicating that the images have nosignificant structural organization. The image of polymeric VN-VIM aggregates (Figure4.6C&D) has a fractal Db value of 1.74±0.21, 1.63±0.70 exceeding the minimum Db value of1.5 required for significant order and degree of organization and thus suggesting the polymericstructures contain a high degree of structural organization. Figures 4.6E&F also had Db valueslower than 1.5, indicating that there was no major structural organization or pattern ofmultimerization.4.5. DiscussionThis study is a continuation of previous work describing the manner in which vitronectinmediates the binding of PAI-1 to the surface of activated platelets and PMP's by binding toexposed vimentin on the surface of activated platelets and PMP's [9]; moreover, the expressionof vimentin and vitronectin on PMP's has been verified by proteomic analysis [23]. We providefurther support for this mechanism with observations regarding the ultrastructure of PMP's andthe nature of vimentin exposure. The source of PMP's for these experiments was platelet-poorplasma from expired platelet concentrates [24, 25] and platelet-poor plasma from post-acutemyocardial infarction patients [26]. In summary, vimentin may become exposed when a PMP is73activated plateletwith pseudopodB)A)I1^'microparticle' —I tip of pseudopodvitronectin multimerizationon vimentinexposed vimentinpseudopod onI activated platelePAI-1:VN:VIM Icomplex^Iassembly^IPAI-1:VNMicroparticle release from pseudopod ^............. .................................. .. .............. .............. ...............Figure 4.7. Proposed model of vimentin exposure on activated platelets andplatelet microparticles and subsequent assembly of the PAI-1:VN:vimentin ternary complex. Microparticles can be released at the tip of pseudopods in activatedplatelets, A). We describe vimentin exposure at the interface of the broken junctionbetween the pseudopod and PMP. This exposure of vimentin will cause plasma-derived VN to undergo multimerization, which will be followed by incorporation of eitherfree PAI-1 or PAI-1:VN complexes to this multimerized VN. This assembly of ternarycomplex can occur on both the microparticle and activated platelet.74released from the pseudopod of an activated platelet, resulting in a broken junction between thetwo vesicular bodies. This broken junction can then lead to a very brief disruption of membraneat which there is exposure of vimentin cytoskeleton to the lumen. However, any membranedisruption will immediately re-seal due to the hydrophobic nature of the phospholipid tail inbilayer membranes, but some vimentin may become partially exposed as illustrated in a proposedmodel in Figure 4.7. Exposed vimentin on both the PMP and activated platelet becomes theassembly site of vitronectin multimerization and subsequent PAI-1 incorporation, hence theformation of the PAI-1:VN:vimentin ternary complexes. We have also visualized theultrastructure of vimentin-vitronectin multimers in a cell-free environment to further understandthe organization of the ternary complex when assembled on activated platelets and PMP's.Despite the wealth of literature supporting PMP counts as a potential diagnostic marker,the ultrastructure of PMP's and their formation from platelets is somewhat unclear due to opticallimitations of real-time imaging of microparticle release (diameter <1.0gm) from cells as smallas platelets (diameter of approximately 3 gm). However, various reports utilizing scanningelectron microscopy and confocal microscopy have described PMP formation occurring at the tipand/or median of platelet pseudopodia [18, 19, 27], as well as at the edge of the platelet body[28, 29]. Currently, flow cytometry is the primary methodology for high-throughput analysis ofPMP's but this methodology is beset by a major caveat wherein the incident light (488 nm) andforward scatter detector in commercially available flow cytometry instruments do not accuratelyanalyze and size particles with diameters smaller than the incident wavelength [30-33]. Theangular distribution of light scatter from sub-micron particles analyzed by the 488nm incidentlight does not accurately yield consistent size readouts [31, 33]. However, impedance flowcytometry instruments can overcome this physical limitation to permit accurate quantification of75PMP size dimensions [34] and is based on a technology that is free of optical limitations.Despite the physical limitations associated with conventional flow cytometry, we performed aFACS isolation of CD41(+)ve particles with a diameter < 1.0 pm that expressed vimentin,vitronectin and PAI-1 which were then imaged by AFM to provide accurate morphologicalanalysis.Vimentin intermediate filaments are not normally present on the surface of viable cells.Vimentin intermediate filaments are intracellular with a cortical distribution underneath the cellmembrane with intimate associations with microfilament and microtubule networks [35-37].During platelet activation, cytoskeleton reorganization results in the formation of plateletpseudopods, as evidenced by filamentous rings at the greatest circumference of the pseudopod[38-40] and treatment with cytoskeleton inhibitors such as cytochalasin D can prevent pseudopodformation and microparticle formation [19, 41]. In this report, we isolated vimentin-positivePMP's from expired platelet concentrates and observed minor phospholipid membraneappendages attached to the base of the PMP as visualized by AFM. Topographically, thesurfaces of these vesicles were smooth and continuous, with little suggestion of filament-likestructures on their surface. We infer that these vesicles are the tips of pseudopods and the originof vimentin lies at the base of the PMP between the phospholipid membrane flaps at the base ofthe PMP as visualized by AFM. In Figure 4.5, intermediate filament-like structures wereobserved within these membrane flaps, providing insight as to how vimentin may be exposedunderneath the base of the PMP.The membrane flaps observed via AFM are of artifactual nature, because the amphipathicnature of phospholipid molecules in aqueous solutions strongly dictates an arrangement thataccommodates the non-polar hydrophobic tails so that they are oriented towards each other while76the polar hydrophilic heads are oriented outside. In the event of membrane disruption, gaps willquickly re-seal as hydrophobic tails rearrange themselves and membrane is reorganized tomaintain surface continuity and minimize phospholipid tail interactions with water.One key requirement for membrane sealing is the presence of free Ca2+, in which Ca2+-dependent exocytosis mechanisms within a cell recruit internal stores of phospholipid in the formof endomembranes to the cell surface, providing more phospholipid for re-sealing [42, 43].However, PMP's formed in expired platelet concentrates contain citrate (-9 mM), whichneutralizes the ability of Ca2+ to recruit and mobilize stores of endomembrane, if exists withinthe PMP to its surface [44]. Most importantly, PMP's formed in vivo will not express membraneappendages at their base because physiological concentrations of Ca 2+ will contribute toresealing of membrane over or around vimentin. The membrane appendages visualized at thebase of PMP's isolated in expired platelet concentrates (Figure 4.3) are artifactual due to: 1) thespreading of PMP membrane adjacent which is adjacent to exposed vimentin spreading on themica coverslip and to a lesser extent, 2) storage conditions such as high citrate plasmaconcentrations that retard recruitment of phospholipid to the surface of PMP's. However, theseartifacts have also provided some insight regarding a potential mechanism describing vimentinexpression on PMP's and activated platelets.The ultrastructure of in vitro formed VN-vimentin multimers revealed a tightly knitlattice structure that rapidly incorporated all free VN and vimentin molecules. The AFM is anexcellent means to understand protein-protein interactions, particularly the manner in whichvimentin can induce structural changes in VN, a normally globular protein stabilized by its di-sulfide bridges into a tertiary structure —3 rim in height. It is concievable that the ionicinteractions between the highly charged VIM133 peptide and VN resulted in the unravelling of77vitronectin into a more linear structure, thus resulting in a VN monomer height of <1.6nm.Furthermore, the unravelling of VN from a globular to a linear structure could reveal crypticbinding sites for other ligands. The AFM has the potential to explore these possibilities of howproteins can induce tertiary structure change without the use of epitope-specific antibodiescombined with ELISA or surface plasmon resonance analysis. Considering that no structure inthe VN-vimentin multimers produced a height >3nm, it is clear that vimentin is a key activator or"unraveling" agent for vitronectin and warrants further experimentation onto itself.The VN-vimentin multimer is a tightly knit and highly organized lattice and resembledother similar patterns of VN and vimentin distribution as imaged by confocal and AFM onactivated platelets expressing high amounts of VN and vimentin (Figure 4.6). In particular, theuniform fields of protein-like structures distributed over the surface of the vitronectin-richplatelet in Figure 4.5E are analogous to this tightly knit lattice. However, no such lattice-typestructures are observed on VN-positive PMP's as imaged by AFM (Figure 4.3B and 4.4),indicating that activated platelets are more likely to present with VN-vimentin multimers due to agreater availability of vimentin on a cell much larger than a PMP. Although the lattice is highlyorganized, an obvious structural signature representing VN-vimentin multimers was notdelineated. However, it can be concluded that PMP's may be limited in the amount of PAI-1expressed on their surface because of the low proportion of vimentin that may be extracellularlypresented, while activated platelets have both a higher capacity of vimentin extracellularpresentation and a larger surface for which VN-vimentin multimers can spread out onto.Vimentin exposure may be relevant when considered in the context of thrombusformation, in which PAI-1 is bound to the surface of activated platelets and PMP's by way ofmultimerized VN. Presumably, the extent of vimentin exposure dictates the amount of PAI-178presentation within thrombi which will determine the extent of thrombus stabilization againstplasminogen activators. It also dictates the amount of multimerized VN within the thrombus,possibly contributing to thrombus structural integrity by homotypic interactions with other cellsor fibrin with multimerized VN on its surface [15]. Recent murine studies of intravital imagingmodels of photochemically-induced thrombosis have highlighted the requirement of VN for clotstabilization. Photochemically induced clots require more time to reach vessel occlusion in VN-/- mice and these clots are unstable and have earlier clot dissolution times compared to wild typemodels [45]. However, mice with both a VN-/- and PAI-1 -/- genotype generate comparableclot formation and clot lysis times when compared to VN-/- mice [46] but the basis for thissimilarity is still unclear. It was previously hypothesized that VN had dual contributory roles ininhibiting fibrinolysis as well as stabilizing thrombi by binding to integrins allb -33 and a503 . Thelack of variation between PAI-1-/- & VN-/- mice and their single gene knockout counterpartsstrongly suggests that VN plays a primary, if not central role in fibrinolysis and that perhapsabsence of VN leads to inactive PAI-1 and subsequent lack of clot stabilization. These intravitalstudies also support the requirement of VN multimerization for the deposition of active PAI-1onto the surface of activated platelets as a means to contribute to the stability of a growingthrombus during blood shear and plasminogen activator secretion by endothelium.Due to the high affinity of PAI-1-VN complexes to vimentin intermediate filaments,thromboembolic fragments with high PAI-1 content can resist significant clot lysis despitetreatment with thrombolytic therapy [47-50]. Moreover, PAI-1 present on platelet microparticles(PMP's) may act as a secondary pool of active PAI-1 and potentiate other downstreamthrombogenic events due to their sub-micron size and clot stabilizing properties [9]. However,the extent of these clot stabilitory properties on activated platelets and platelet microparticles will79be dependent on the extent of VN-vimentin multimerization on the activated cell surface and theamount of PAI-1-VN incorporation into this ternary complex. Hence, the expression andproportion of ternary complex will differ between PMP's and activated platelet populations dueto cell surface availability and vimentin exposure.804.6. References for Chanter IV:1. Pandolfi A, Cetrullo D, Polishuck R, Alberta MM, Calafiore A, Pellegrini G, VitacolonnaE, Capani F, Consoli A. Plasminogen activator inhibitor type 1 is increased in the arterialwall of type II diabetic subjects. Arterioscler Thromb Vasc Biol 2001; 21:1378-82.2. Vaughan DE. PAI-1 and atherothrombosis. J Thromb Haemost 2005; 3:1879-83.3. Muller JE, Tofler GH, Stone PH. Circadian variation and triggers of onset of acutecardiovascular disease. Circulation 1989; 79:733-43.4. Kohler HP, Grant PJ. 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A direct interaction between actin and vimentinfilaments mediated by the tail domain of vimentin. J Biol Chem 2006; 281:30393-9.37. Kuroda M, Porter KR. Cytoskeleton in vitro: preparation of isolated cytoskeletons withthree-dimensional architecture. J Biochem (Tokyo) 1987; 101:1413-27.38. Schliwa M. Action of cytochalasin D on cytoskeletal networks. J Cell Biol 1982; 92:79-91.8339. Debus E, Weber K, Osborn M. The cytoskeleton of blood platelets viewed byimmunofluorescence microscopy. Eur J Cell Biol 1981; 24:45-52.40. Taylor RG, Lewis JC. Microfilament reorganization in normal and cytochalasin B treatedadherent thrombocytes. J Supramol Struct Cell Biochem 1981; 16:209-20.41. Carroll RC, Butler RG, Morris PA, Gerrard JM. Separable assembly of plateletpseudopodal and contractile cytoskeletons. Cell 1982; 30:385-93.42. Terasaki M, Miyake K, McNeil PL. Large plasma membrane disruptions are rapidlyresealed by Ca2+-dependent vesicle-vesicle fusion events. J Cell Biol 1997; 139:63-74.43. Steinhardt RA, Bi G, Alderton JM. Cell membrane resealing by a vesicular mechanismsimilar to neurotransmitter release. Science 1994; 263:390-3.44. Hirayama J, Azuma H, Fujihara M, Homma C, Yamamoto S, Ikeda H. Storage ofplatelets in a novel additive solution (M-sol), which is prepared by mixing solutionsapproved for clinical use that are not especially for platelet storage. Transfusion 2007;47:960-5.45. Reheman A, Gross P, Yang H, Chen P, Allen D, Leytin V, Freedman J, Ni H. Vitronectinstabilizes thrombi and vessel occlusion but plays a dual role in platelet aggregation. JThromb Haemost 2005; 3:875-83.46. Koschnick S, Konstantinides S, Schafer K, Crain K, Loskutoff DJ. Thromboticphenotype of mice with a combined deficiency in plasminogen activator inhibitor 1 andvitronectin. J Thromb Haemost 2005; 3:2290-5.47. Zunker P, Schick A, Padro T, Kienast J, Phillips A, Ringelstein EB. Tissue plasminogenactivator and plasminogen activator inhibitor in patients with acute ischemic stroke:relation to stroke etiology. Neurol Res 1999; 21:727-32.48. Nicholls SC, Hoffer EK, Chandler WL. Failure of peripheral arterial thrombolysis due toelevated plasminogen activator inhibitor type 1. Blood Coagul Fibrinolysis 2003; 14:729-33.49. Huber K. Plasminogen activator inhibitor type-1 (part two): role for failure ofthrombolytic therapy. PAI-1 resistance as a potential benefit for new fibrinolytic agents. JThromb Thrombolysis 2001; 11:195-202.50. Potter van Loon BJ, Rijken DC, Brommer EJ, van der Maas AP. The amount ofplasminogen, tissue-type plasminogen activator and plasminogen activator inhibitor type1 in human thrombi and the relation to ex-vivo lysibility. Thromb Haemost 1992; 67:101-5.84CHAPTER V: VIMENTIN AUTO-ANTIBODIES INDUCE PLATELET ACTIVATIONAND FORMATION OF PLATELET-LEUKOCYTE CONJUGATES VIA PLATELET-ACTIVATING FACTOR5.1. IntroductionAuto-antibodies to the intermediate filament vimentin are associated with rheumatoidarthritis (1), systemic lupus erythematosus (SLE) (2-4) and rejection of solid organ transplants(5-9). Vimentin is a cytoskeleton intermediate filament protein present in cells of mesenchymalorigin; these include leukocytes, endothelial cells and smooth muscle cells. More recently,vimentin has been observed on the cell surface of apoptotic cells (10-12) and thrombin-activatedplatelets (13). The production of anti-vimentin antibodies (AVA) in certain diseases is probablycaused by excessive exposure to vimentin on apoptotic cells, since it is known that caspasedependent cleavage of vimentin, with exposure of vimentin on the cell surface, are necessaryrequisites for apoptosis (14). The functions of AVA are unknown, and in particular whether theyhave an active role in disease pathogenesis.Prevalence of coronary artery disease in SLE patients is high, with heightened states ofimmune activation and pro-thrombotic activity thought to be key contributory factors to diseasepathogenesis in these patients (15). Lupus autoantibodies have been implicated as observed byimmune deposits present in kidney and heart (16). In the context of transplantation, AVA havebeen associated with a different type of atherosclerosis, namely graft vascular disease (GVD) (9).GVD is the most common complication following heart or renal transplantation; it is'A version of this chapter has been published/accepted for publication. Leong HS et al. Vimentin Auto-AntibodiesInduce Platelet Activation and Formation of Platelet-Leukocyte Conjugates via Platelet-Activating Factor. J LeukoBiol, Feb 1, 2008; 83(2): 263-71.85characterized by intimal occlusion and fibrosis of donor arteries and veins (17;18).A pro-coagulant microvasculature is associated with pathogenesis of GVD. The presenceof fibrin deposition (19) and depletion of t-PA (20) in the microvessels of the heart, is predictiveof heart transplant recipients who will develop GVD. Similarly, deposition of the complementcomponent C4d within allografts is characteristic of GVD (21). These studies suggest thatsynergy between thrombotic events and complement fixing antibodies contribute to GVD andpossibly to atherosclerosis in patients with autoimmune diseases. We hypothesize thatantibodies to the autoantigen vimentin, particularly of the IgM subclass, may interact withvimentin expressing platelets with possible pathogenic consequences. This hypothesis wastested in vitro, by adding AVA to normal whole blood, platelet-rich plasma and leukocyte-richplasma, and investigating its effect on formation of platelet/leukocyte conjugates and plateletmicroparticl es.865.2. Materials and Methods: 5.2.1. Blood collection and patient serumMHC (major histocompatibility complex) Class I HLA A2+ve A3-ve subtype individualsconsented to provide normal whole blood which was collected into 4.5mL CTAD tubes and thendiluted 1/10X in Tyrodes buffer. Platelet-rich plasma (PRP) was obtained by collecting thesupernatant fraction of blood centrifuged at 200g for 10mins. Post-transplant serum collectedfrom cardiac transplant recipients for diagnostic reasons was used with permission from RoyalBrompton and Harefield Hospital ethics committee and University of British Columbia ethicsboard. Patient serum had been screened for presence of anti-vimentin and HLA antibodies aspreviously described (9;22).5.2.2. Preparation of recombinant human vimentinThe cDNA for human vimentin was isolated from a human umbilical vein endothelialcell cDNA expression library by PCR, introducing a 5' NdeI restriction site and a BamHI site atthe 3' end of the cDNA (Primers: forward 5' ATA GAG CAT ATG TCC ACC AGG TCC GTGTCC; reverse 5' GCG CTC GGA TCC TCT TAT TCA AGG TCA TCG TG). The PCR productwas sub-cloned into NdeI / BamHI of pET15b (Novagen; Merck Biosciences, Nottingham, UK),a bacterial expression vector, and transformed into BL21 E.coli (BL21/vimentin; Novagen).Crude preparations of recombinant human vimentin were prepared and extracted according to thepET System Manual (Novagen) and purified on a His-Bind resin column under denaturingconditions, using a His-Bind Purification Kit (Novagen). Purification was confirmed by SDSPAGE (a single band at 58kD was observed) and mass spectrometry (not shown).875.2.3. Depletion of AVA's from patient seraRecombinant human vimentin protein (1 mg/mL in 6 M urea, preparation) wasconjugated to agarose beads as described in kit instructions (AminoLink Plus ImmobilizationKit). Vimentin-conjugated agarose beads (50gL) were incubated with 50 1AL of patient serum at25°C overnight and then centrifuged at 1000g for 10mins and the supernatant fraction and wascollected, termed depleted patient serum. ELISA assays [10] demonstrated that this treatmentreduced AVA titres to —15% of original titres (Mean non-depleted 1061±52.9; mean depleted160±29.3).5.2.4. Flow cytometry and monoclonal antibodies (mAb)Equal amounts (25 lig/mL final concentration) of mAb mouse IgM antibody (to A2, A3,from One Lambda), anti-vimentin antibodies (AVA) 13.2-IgM, V9-IgG (both mouse anti-human- Sigma) or sheep anti-vimentin (Affinity Biologicals) were added to 201AL of diluted (1/10X)whole blood and incubated at 37°C for 30 or 45mins. 201AL of patient sera was added to 201AL ofdiluted blood for 30 or 45mins. CD41-RPE, C3d-FITC, or fibrinogen-FITC antibodies (mouseanti-human, rabbit anti-human, rabbit anti-human respectively - BD Biosciences) at a finalconcentration of 251.1g/m1 were used to label each sample to detect platelets and expression ofC3d or fibrin. CD62P-APC (mouse anti-human - BD Biosciences) and tissue factor-FITCantibodies (rabbit anti-human - American Diagnostica) were also added to blood at similarconcentrations. Isotype controls, FITC rabbit IgG (Beckman Coulter), APC-mouse IgGl(Beckman Coulter) and RPE-mouse IgG1 (BD Biosciences), were added at the sameconcentrations. To label leukocytes, Hoechst 33342 (Molecular Probes) diluted in PBS (1 pg/L88final concentration) was subsequently added for another 30mins. Labelled leukocytes wereidentified as 'Hoechst-positive cells' during flow cytometric analysis. To track binding of mAbIgM antibodies in whole blood, FITC-donkey-anti-mouse IgM (Sigma) was added at a finalconcentration of 25 gg/mL. In order to determine numbers of vimentin-positive cells in wholeblood, 13.2 IgM was added to washed buffy coat leukocytes. Washing buffy coat leukocyteswere resuspended in 100IAL of PBS, to which 13.2 IgM was added at a final concentration of 25gg/mL. After 30 mins incubation at 4°C, cells were washed again and FITC-goat anti-mouseIgM was added (final concentration 10 gg/mL) for another 30 mins at 4°C. Cells were analyzedusing a BD FACS-Aria instrument with 405, 488 and 635 nm single line lasers was used and>30,000 events were analyzed for every sample. Measurement of platelet microparticles wasperformed using 1.0 1.1M beads (Invitrogen cat#F13080). The PAF inhibitor, CV-6209, wasobtained from Calbiochem (Nottingham, UK).5.2.5. Complement dependent cytotoxic assay on AVA treated purified leukocytesTo prevent contamination by platelets, a modified method of purifying leukocytes wasperformed. Normal blood was collected into acid citrate dextrose tubes (BD Biosciences) andleukocyte-rich preparation (LRP) was prepared by centrifugation of 10 mL of whole blood for 10minutes at 200g and the platelet rich plasma (PRP) supernatant removed. An additional 1.0 mLof Tyrodes buffer was added to the remaining whole blood, mixed by inversion and thencentrifuged again under the same conditions. The supernatant was again removed and remainingblood was lightly layered onto Lympholyte H solution (Cedarlane Labs, Hornby, ON) in a 15mL Falcon tube. This was centrifuged at 200g for 20 minutes. Upon centrifugation, four layerswere present and a Pasteur pipette was used to suction out the leukocyte layer (typically 2 mL89from a 10 mL whole blood preparation). This layer was centrifuged at 200g for 5 minutes toremove residual platelets which was confirmed by flow cytometry. The supernatant containingplatelets was discarded and the pellet was resuspended in modified Tyrodes buffer. Flowcytometry demonstrated that these leukocytes were —60% neutrophils; the remainder weremonocytes and lymphocytes and used for two tests, the cytotoxic assay and preparation ofsupernatants from AVA activated neutrophils.Leukocytes were diluted with modified Tyrodes buffer to 5x105 cells/AL and 1ALaliquots were placed in single wells of a Terasaki plate. To these were added 1 AL of HLA-A2IgM, HLA-A3 IgM or 13.2-IgM antibody (0.5 pig/mL) in each well in duplicate. After 30mins,5AL of rabbit complement (Cedarlane Labs) was added and incubated for another 30mins. Cellviability was assessed by adding 2p,L of FluoroQuench (One Lambda) to each well. A Zeissinverted microscope fitted with a Qlmaging Retiga EXi colour cooled camera and QCapture Prosoftware (Qlmaging Inc.) was used to acquire images. A live/dead filter set (ChromaTechnology Corp) was used to visualize the ethidium bromide and acridine orange viabilitystains in which viable cells demonstrated a fluorescent green nuclear signal and dead cellsdemonstrated a fluorescent orange nuclear signal.5.3. Results:5.3.1. Effect of monoclonal and patient AVA 's on whole bloodTreatment of whole blood with AVA 13.2-IgM resulted in a depletion of platelet counts(determined by numbers of CD41-positive cells) as well as an increase in %platelet:leukocyteconjugates (calculated as a percentage of CD41+Hoechst cells/ Hoechst-positive cells) compared90to whole blood treated with AVA V9-IgG antibody or not (Figs 5.1A vs. 5.1B&C). The effect ofAVA 13.2 IgM was inhibited in the presence of recombinant human vimentin (Fig 5.1D).Fibrinogen and C3d deposition was evaluated on leukocytes and platelets before and aftertreatment with AVA 13.2 IgM. Post AVA13.2-IgM treatment, fibrin(-ogen) expression onleukocytes increased from 9.6±1.3% to 33.0±8.6% and fibrin(-ogen)expression on plateletsincreased from 7.8±1.2% to 89.3±2.2% (Fig 5.1E). When assessing C3d expression, increasesfrom 12.9±2.1% to 59.2±6.3% on leukocytes and increases from 4.2±1.2% to 41.1±13.6% wereobserved after AVA 13.2-IgM treatment (Fig 5.1E). Control experiments showed that AVA 13.2IgM treated blood cells do not non-specifically bind FITC rabbit IgG (Fig 5.1F&G). Theseresults demonstrate that whole blood treatment with AVA induces platelet:leukocyte (P:L)conjugate formation, and deposition of fibrin and C3d on platelets and leukocytes. For negativecontrol, A3 IgM did not induce deposition of fibrin and C3d on platelets and leukocytes (data notshown).To compare the activity of the AVA monoclonal with AVA from transplant patient sera,normal blood was then treated with patient sera containing high titre IgM AVA (mean titre1061±52.9) or the same sera depleted of AVA (mean titre 160 ±29.3), using vimentin coatedagarose beads. These sera did not contain alloantibodies of AVA of the IgG subclass. Patientsera containing high AVA titre lead to a decrease in the ratio of free platelets to leukocytescompared to whole blood treated with control serum or sera depleted of AVA IgM (Fig 5.2A).In Fig 5.2B, an increase in platelet/leukocyte conjugates was observed when whole blood wastreated with patient sera with high AVA IgM compared to normal blood incubated with controlserum (from a transplant patient negative for AVA) or sera depleted of AVA IgM. To controlfor the possibility that vimentin coated beads may remove immunoglobulin non-specifically, we91P:L=4.6%AVA V9 I gGrn"mr r 1 nil-IDSSC-AIo'AVA IgM treatmentE)^10080•a.)60 •a)40.0ID: L=1 .3%vimentin protein& AVA 13.2 IgM0F)Untreated blood+Rabbit IgG-FITC1.4%20.1Leukocytes^Platelets^LeukocytesAVA IgM treated blood +Rabbit IgG-FITCPlatelets2.3%rrrC-ANormalF!TC-AFigure 5.1. AVA monoclonals induce platelet:leukocyte conjugate formation and surface expression of C3d and fibrinogen. Figures 5.1A-D are forward/side scatterplots of blood under different treatments incubated for 30 mins. The green populationrepresents platelets; the red represents nucleated leukocytes. Incubation with 13.2 IgM(A) resulted in platelet depletion and P:L conjugate formation, whereas incubation withV9 IgG (B) gave similar results to untreated (C) blood. P:L conjugate formation inducedby 13.2 IgM was inhibited by recombinant vimentin protein+13.2 IgM (D). Figure 5.1Esummarizes data on the percentage of platelets and leukocytes expressing fibrinogen(white bars) and C3d (black bars), before and after AVA IgM treatment. Representativeof 5 experiments. *denotes (compared to no treatment, p<0.05; t-test, 2-tailed)compared to untreated.92A)^ B) 4 03 53 02• • 200is 15UnControl^Patient 1 Patient 2 Patient 3 Patient 4 Patient 5^ Control^Patient 1 Patient 2 Patient 3 Patient 4 Patient 5Patient PatientFigure 5.2. Patient sera with anti-vimentin autoantibodies (AVA's) induce plateletactivation and formation of platelet:leukocyte conjugates.  Normal blood wastreated with patient sera containing high titre AVA (filled bars), control serum (labelledcontrol patient), or the same patient sera depleted of AVA using agarose coated beads(unfilled bars) for 30 mins. In A), the extent of platelet activation was characterized bythe ratio of CD41-positive cells to Hoechst-positive cells. In B), formation ofplatelet:Ieukocyte conjugates was characterized by the percentage of cells co-expressing Hoechst+CD41 over the total number of Hoechst-positive cells.93treated patient serum known to contain high titres of antibodies to HLA (human leukocyteantigens) with vimentin coated agarose beads, this had no effect on the HLA antibody titre (notshown). These results demonstrate that the AVA's in patients' sera can also induce formation ofP:L conjugates.5.3.2. Effect of other IgM antibodies on whole bloodIn view of the fact that the AVA V9 IgG had no effect on platelets and that the majorityof transplant patients produce IgM and not IgG AVA (9), the remainder of the study focused onIgM antibodies. We next determined whether the effect of AVA was comparable to the effectsseen with IgM antibodies to another cell surface ligand such as the Major HistocompatibilityComplex (MHC) antigen subtypes, HLA-A2 and HLA-A3. All leukocytes express MHCantigens. Hence, AVA 13.2-IgM was tested against two other IgM mAbs specific for HLA-A2and HLA-A3 and blood used for these experiments were from individuals who were HLA-A2positive and HLA-A3 negative. Treatment of whole blood with AVA 13.2-IgM and HAL-A2IgM resulted in significant P:L conjugate formation (Fig 5.3A,B), compared to untreated blood(Fig 5.3D). The HLA-A3 antibody had no effect on blood from the HLA-A2 positive individual(Fig 5.3C). Upon flow cytometric analysis (Fig 5.3E), the percentage of P:L conjugates werehigher in whole blood treated with AVA13.2-IgM (17.2±2.6%) and HLA-A2 IgM (64.3±2.7%)compared to treatment with HLA-A3 IgM (4.8±0.5%) and endogenous levels in untreated wholeblood (5.9±0.5%). In order to investigate the phenotype of platelets attached to leukocytes,94E)100-F)CL50w 80 * CD (c)a) a 40T6 0----c060 I-I-F—o 0)30—I 40 O 2020* (j),10MOM0 0No treatment^HLA- HLA- AVA IgM No treatment^HLA- HLA-A2 IgM A3 1gM A2 IgM A3 1gM*AVA IgMHLA-A2 IgM treated blood+ Mouse IgG-APC1.8%G) H)HLA-A2 IgM treated blood+ Rabbit IgG-FITC0.9%Figure 5.3. AVA- and HLA- IBM's induce platelet:leukocvte conjugates whichexpress tissue factor and P-selectin. Whole blood was either treated with AVA 13.2(A) A2 IgM (B) A3 IgM (C) or untreated (D). The effects of IgM treatment on plateletsand leukocytes were expressed as forward/side scatter plots in Panels A-D (the bluearea indicates P:L conjugates, the red population indicates nucleated cells, the greenpopulation indicates platelets, and the black population represents activated plateletsthat did not bind the CD41 antibody). Panel E summarizes the extent ofplatelet:leukocyte conjugate formation in all four groups. Panel F summarizes theextent of tissue factor(+)ve (white bars) and P-selectin(+)ve (black bars)platelet:leukocyte conjugates in all four groups. Both E and F summarize experimentsperformed four times. *denotes (compared to no treatment, p<0.05; t-test) compared tountreated.95expression of P-selectin and tissue factor was examined on platelets within P;L conjugates. AfterHLA-A2 IgM treatment, 27.2±5.1% and 35.6±2.9% of platelets within P:L conjugates weretissue factor and P-selectin positive (Fig 5.3F). Following AVA 13.2 IgM treatment, 10.8±1.3%and 10.1±1.2% of platelets within P;L conjugates were also tissue factor and P-selectin positiverespectively (Fig 5.3F). In contrast, untreated whole blood and HLA-A3 IgM treated wholeblood, which contained few P:L conjugates had very few platelets that were also positive fortissue factor or P-selectin (<5%). Addition of HLA-A2 or 13.2 IgM antibody to whole blooddid not cause non-specific binding of FITC rabbit IgG or APC-mouse IgG respectively (Fig5.3G&H for HLA-A2 IgM), data not shown for 13.2 IgM AVA.5.3.3. Effect of AVA IgM and anti-HLA -A2 IgM on purified plateletsIn order to compare the effects of IgM antibodies on platelets in the absence ofleukocytes, platelet rich plasma (PRP) was used. In Fig 5.4, panels A-D show forward/sidescatter plots from a single experiment to illustrate formation of platelet microparticles afteraddition of AVA and anti-HLA IgM antibodies to PRP. Platelet microparticles (PMP) arereleased by activated platelets and are characterized as exhibiting significant CD41-PE signalwith a forward scatter less than a 1.0 gm bead particle. Platelets were unaffected by AVA 13.2-and HLA-A3 IgM treatments but addition of HLA-A2 IgM induced platelet microparticleformation. In Fig 5.4E, the results of 4 experiments are summarized. Analysis of normalplatelet-rich plasma yielded 11.0±1.1% PMP, indicating that —11% of CD41-PE positiveparticles in the sample were of microparticle size and morphology. When this plasma wastreated with AVA 13.2 IgM, the %PMP at 4.8±0.8% was lower than the %PMP observed inPRP. Treatment with HLA-A2 IgM resulted in 40.0±7.5% PMP, presumably a result of96AVA 13.2^HLA-IgM^A3 1gMNormal HLA-A2 IgME)Fiqure 5.4. AVA-IqM does not directly induce platelet activation.  Panels A-D areforward/side scatter plots in a density contour format. Results of platelet activation areexpressed as "% PMP"; in which # of CD41-positive particles with a diameter below1.0pm, over the total number of CD41-positive events. Results of %PMP aftertreatment of PRP with 13.2 IgM(B), A2 (C) and A3 IgM(C) compared to untreated (A) for30 mins are summarized in (E). Representative of 4 experiments. * denotes (p<0.05; t-test) compared to normal treatment.97complement-mediated lysis by the HLA-A2 IgM (23). Treatment of PRP with the A3-IgMresulted in 7.9±1.3%, a level similar to untreated PRP. These observations strongly suggest thatunlike A2-IgM, the 13.2 IgM has a negligible effect on resting platelets which are negative forcell surface vimentin, but constitutively express MHC class I antigens. Overall, the resultssuggest that the platelet depletion observed in the whole blood and not in PRP by AVA-IgMwere caused by an initial interaction of AVA-IgM with leukocytes with no platelets binding tothem; subsequently leading to platelet activation and vimentin surface expression on platelets.5.3.4. Effect of anti-vimentin antibodies (AVA) on purified neutrophilsTo determine which leukocytes in whole blood bind the various mAb IgM antibodies,anti-mouse IgM-FITC secondary antibody was used to track localization of HLA-A2 IgM, HLAA3 IgM and AVA 13.2 IgM antibodies which had been added to whole blood (Fig 5.5A-D).HLA-A2 IgM was found to bind to all leukocytes (Fig 5.5A). In contrast, the HLA-A3 IgM wasnot found to specifically bind to any cell population (Fig 5.5B). The 13.2-IgM antibody wasshown to specifically bind to monocytes, neutrophils, and activated platelets (Fig 5.5C), asdetermined by the light scattering properties of these cells. Minimal binding of the secondaryantibody alone was observed in untreated blood (Fig 5.5D). These results were confirmed byaddition of AVA 13.2 IgM to washed bully coat leukocytes, followed by FITC anti-mouse IgM(Fig 5.5E), in which it can be seen that approximately 15% of leukocytes binded to AVA 13.2IgM. These vimentin-positive cells were predominantly of neutrophil identity as determined9810^leSSC-A SSC-Ale^leSSC-AII? leleFigure 5.5. Localization of IgM to granulocytes and activated platelets and theircytotoxic effect on leukocytes. FITC goat anti-mouse IgM was added to whole bloodto track A2 IgM (A), A3 IgM (B) and 13.2 IgM AVA (C) or untreated blood (D). The whitepopulation denotes cells with positive IgM binding. Representative of 3 experiments.Panel E presents flow cytometry of whole blood to which has the polyclonal sheep anti-vimentin antibody has been added to normal whole blood. A2 IgM, A3 IgM and 13.2IgM were added to purified leukocytes and incubated with excess complement to induceantibody-mediated cell death (F). Viability was assessed by ethidium bromide/acridineorange labelling of treated cells. Representative of 3 experiments.99by their light scattering properties. Hence, a subpopulation of leukocytes expressing vimentinare the potential binding site for AVA-IgM antibodies.In order to determine whether AVA were cytotoxic and could mediate complementdependent cell lysis, purified leukocytes from an HLA-A2 positive individual were incubatedwith AVA 13.2-IgM, HLA-A2 IgM or HLA-A3 IgM in the presence of exogenous rabbitcomplement (Fig 5.5F); not surprisingly, incubation with HLA-A2 IgM, resulted in >75% celldeath and incubation with HLA-A3 IgM resulted in minimal cell death. Interestingly, the AVA13.2-IgM induced substantial cell death (-50-75%) upon addition of excess exogenouscomplement. Patient sera containing high AVA titres (but negative for antibodies to HLAantigens) were also tested for complement dependent cytotoxicity; 18/21 sera were cytotoxic forleukocytes (killing 20-100% of cells), in contrast 12/54 sera without AVA showedleukocytotoxic activity (data not shown).5.3.5. Supernatant of AVA -activated leukocytes induces platelet activationTo determine if AVA-bound granulocytes release mediators that subsequently activateplatelets, leukocytes were purified and treated with AVA13.2-IgM, HLA-A2 IgM or HLA A3IgM for 30 mins and the supernatant was transferred to purified platelets (PRP). Plateletactivation was assessed by generation of platelet microparticles (PMP) at 30 mins. Supernatantfrom HLA-A3 IgM did not result in significant PMP formation (Fig 5.6B) compared to untreatedcontrol (Fig 5.6A). Supernatant from leukocytes treated with AVA 13.2-IgM resulted insubstantial platelet microparticle formation (Fig 5.6C). Quantitative data from these experimentsare presented in Fig 5.6E. The large amount of PMPs generated by transfer of supernatant fromHLA-A2 IgM treated leukocytes is probably partially due to carry over of HLA-A2 IgM100antibody, which causes direct activation of HLA-A2 positive resting platelets (Fig 5.4C).Although the supernatant from AVA 13.2 IgM treated neutrophils contains both releasedinflammatory mediators and the AVA IgM, we demonstrated previously (Fig 5.4B,E) that theAVA IgM alone does not have an activating effect on platelets. Hence, the activation observedin Fig 5.6C indicates activation is due to the released inflammatory mediators and not the AVA13.2 IgM.101SN from HLA-A3 IgMtreated LRPSN from AVA 13.2 IgMtreated. LRPSN from HLA-A2 1gMtreated LRPI gMQuiescent platelets SN from HL -A2 SN from HLA-A3 SN from 132 AVAIgtvl^IgME)350Fiqure 5.6. Effect of supernatant (SN) from IqM treated leukocytes on plateletactivation. Results are expressed as an analysis of platelet particles according to sizeand only CD41 + particles are presented above. Data points in black are CD41-positive,and data points in white are CD41-positive cells with a forward scatter lower than a 1.0pm diameter fluorescent counting bead, which are considered to be plateletmicroparticles. Panel A shows untreated platelets. In B), SN from A3-IgM treatment didnot result in platelet microparticle formation greater than control (A). Treatment of PRPwith SN from AVA-IgM resulted in platelet aggregation and PMP formation (C), while SNfrom A2-IgM resulted in the highest generation of platelet microparticles compared to alltreatments. Quantitative data shown in E. Representative of 3 experiments. * denotes(p<0.05; t-test) compared to no treatment.1025.3.6. AVA IgM-bound leukocytes release PAFThe previous experiments demonstrated that AVA binding to neutrophils results inrelease of factor(s) that cause platelet activation and formation of PMP's, one of which may bePlatelet-Activating Factor (PAF). To test this hypothesis, blood was briefly pretreated with CV-6209, an inhibitor of the PAF receptor (PAFR) at a final concentration of 11.1M before beingincubated with AVA13.2-IgM. Preliminary experiments had shown that luM CV-6209 issufficient to produce 100% inhibition of thrombin mediated platelets activation (not shown).After 45 minutes of IgM incubation, normal blood treated with the AVA13.2-IgM demonstratedplatelet depletion (Fig 7B), but when CV-6209 pre-treated blood was incubated with AVA 13.2-IgM, platelet counts were not depleted and were similar to levels found in untreated blood (Fig7C,D). Interestingly, although PAF-inhibition prevented disappearance of whole platelets, it didnot prevent them becoming covered in C3d (Fig 7E).1030002500020000150001000050000le`SSC-A-71-]10'E)90*DCo0 80'C) 70'60'50rn0040302010IPSSC-AD)SSC ANo treatment AVA 13.21gMAVA 13.21gM+ CV-6209No treatment AVA 13.2 AVA 13.21gM1gM^+CV-6209Figure 5.7. PAF inhibition attenuates platelet activation and blood cellagglutination. Whole blood was untreated (A), or incubated for 45 minutes with 13.2IgM AVA in the absence (B) or presence (C) of the PAF receptor inhibitor CV-6209.Flow cytometry of treated blood was used to determine platelet depletion (summarizedin D) and C3d expression (summarized in E, where white bars represent leukocytes andblack bars represent platelets). The white coloured data points in scatterplots A-Crepresents platelets with C3d-positive signal, and the position of platelets is marked asa bracket in (A). Pretreatment of whole blood with CV-6209 attenuated plateletdepletion, but an increase in C3d deposition on platelets was still observed (A vs. C).Platelet counts were determined by analyzing 5000 leukocyte events for each sample.Representative of 4 experiments. * denotes (p<0.05; t-test) compared to no treatment.1045.6. Discussion: Anti-vimentin antibodies have been described in a number of diverse conditionsincluding autoimmunity (1;2;24), chronic infections (25), and clinical rejection of solid organallografts (5-9). It has been recently demonstrated that vimentin immunised mice undergoaccelerated rejection of their cardiac allografts (26) but the mechanisms were not elucidated.This is the first study to demonstrate interactions between IgM anti-vimentin antibodies andleukocytes and suggest a mechanism for their pathogenicity. The study was initiated by theobservation that activated platelets express cell surface vimentin (13) leading us to hypothesizethat AVA would have an effect on platelet activation or thrombosis. Treatment of normal wholeblood with the AVA13.2-IgM monoclonal resulted in platelet:leukocyte conjugate formation anddepletion of platelet counts. This was accompanied by induction of P-selectin on plateletsattached to leukocytes (Fig3F), and deposition of fibrinogen, C3d and tissue factor on plateletsand leukocytes. P-selectin is known to be a crucial molecule in P:L conjugate formation (27).That these effects can be produce by patient's antibodies was demonstrated by use of serum fromcardiac transplant recipients, which were selected on the basis of their high IgM AVA titre; whenthese serum samples were depleted of AVA's, the formation of platelet:leukocyte conjugates wasprevented and platelet counts were unaffected.To compare the effects of the AVA IgM to other antibodies known to bind to platelets,IgM molecules specific for HLA antigens were chosen. Antibodies to A2, but not A3 had aneffect on blood from an A2 individual, confirming the importance of the antigen binding part ofthe IgM molecule and discounting the possibility that IgM antibodies could non-specificallyactivate platelets to form P:L conjugates. Experiments using purified platelets as opposed towhole blood described an important difference between A2-IgM and AVA-IgM, namely that105only anti-HLA antibodies bind to purified platelets and AVA IgM did not. This is not surprising,since it is known that only activated and not quiescence platelets express surface vimentin (13).Podor et al. also described vimentin expression on platelet microparticles (13); this wouldexplain the decrease in %PMP observed when AVA 13.2 IgM was added to purified platelets(Figs 4A,B). We suggest that AVA IgM binds to the vimentin-positive PMPs in normal platelet-rich plasma, resulting in complement-mediated lysis and further degradation of plateletmicroparticles.Using whole blood and a secondary antibody detecting 13.2-IgM, it was determined thatAVA's bind to approximately 12 % of circulating neutrophils; this has been verified by Moisanet al who described a similar number of vimentin positive neutrophils in normal blood anddemonstrated them to be spontaneously apoptosing neutrophils (28). We have not formallyproven that the neutrophils that bind 13.2 IgM are undergoing apoptosis, but in view of theliterature describing the presence of vimentin on apotosing cells of various types (10;12;29), thisseems a probable explanation for the association between AVA and neutrophils. The possibilitythat AVA's bind to neutrophils that subsequently release factors to activate resting platelets wasconfirmed by using supernatant from AVA treated leukocytes and adding it to platelets andobserving PMP formation. Thus, platelet activating factor (PAF) was implicated as a primaryfactor leading to platelet activation because it can be rapidly synthesized and released byactivated neutrophils. In our studies leukocytes expressed tissue factor following AVA treatmentof whole blood (Fig 3F); this is also likely to have originated from activation of neutrophils byAVA (30;31); however, unlike PAF which has direct platelet agonist effects Tissue Factor doesnot directly activate platelets (32). PAF binds to Platelet-Activating Factor Receptor (PARF)present on platelets and leukocytes (33). Upon PAF binding, calcium channels are opened,106initiating activation of the platelet. In our experiments, although the platelet count was normalwith the inhibitor, platelets were still expressing C3d; this is probably because the PAF inhibitordid not affect release of activating factors from the neutrophils, but inhibited opening of thecalcium channels necessary for cell lysis. It is unlikely that PAF is the only mediator released byAVA-activated granulocytes. In summary, we hypothesize that AVA induce platelet activationand PMP formation via four stages; 1) activation of neutrophils and release of platelet activatingfactors, expression of tissue factor 2) induction of P-selectin, vimentin and tissue factor onplatelets 3) binding of fibrinogen to activated platelets (via GPIIbIII a) and formation of P:Lconjugates and 4) binding of AVA to activated platelets and generation of PMP's. The latterprocess is likely to be mediated by complement as demonstrated by the presence of C3d onplatelets and ability of AVA to fix complement and cause leukocyte lysis in vitro (Fig 5C).Previous studies have shown sensitivity of platelets to complement mediated lysis (23;34). Wehave not yet formally demonstrated the complement dependence of this process usingcomplement inhibitors.The formation of platelet:leukocyte conjugates by AVA is an observation that sheds lighton possible hemostatic mechanisms leading to GVD development in allografts. It may also beimportant for atherosclerotic disease progression in autoimmune diseases such as SLE, which arecharacterised by AVA. When platelet:leukocyte conjugates are formed, the effectiveness ofthese leukocytes to roll and tether to activated endothelium is substantially increased (35;36).The release of tissue factor or its expression on activated granulocytes may also potentiate T-cellactivation (37) alongside its pro-coagulation effects.This study has described effects of IgM AVA on leukocytes which are antigen dependent.In 1984, Hansson et al. demonstrated Fc dependent binding of non-specific IgG to vimentin107exposed on the surface of damaged endothelial cells (38); in the current study the failure of V9 tobind to senescent neutrophils. The fact that A3-IgM and A2-IgM behave in a different way to13.2-IgM and the fact that vimentin coated agarose beads deplete only AVA from patients serum(and not IgG HLA antibodies) argues against vimentin acting as a general Fc receptor forcirculating immunoglobulin. Whether IgG AVA interact with leukocytes in a similar mannerwarrants further investigation.It is interesting to speculate that the effects of AVA's described here, in vitro, may bepartly responsible for the neutropenia and thrombocytopenia typically present in lupus patients.Indeed the effects of AVA on vimentin positive senescent neutrophils, may be analogous to theeffect of anti-neutrophil cytoplasmic autoantibodies which are associated with specific forms ofsystemic vasculitis (39). In the latter case, it is known that cytokine treatment exposes theseautoantigens proteinase-3 and myeloperoxidase on the neutrophil surface, although the in vivostimulus of such exposure is not known. In conclusion, the mechanism we describe here mayreflect a novel mechanism of how autoantibodies lead to thrombosis and atherosclerosis.1085.7^References for Chapter V1. Vossenaar, E. R., Despres, N., Lapointe, E., van der, H. A., Lora, M., Senshu, T., vanVenrooij, W. J., Menard, H. A. 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(2002) Vimentin exposed on activated platelets and plateletmicroparticles localizes vitronectin and plasminogen activator inhibitor complexes on theirsurface. J.Biol. Chem. 277, 7529-7539.14. Morishima, N. (1999) Changes in nuclear morphology during apoptosis correlate withvimentin cleavage by different caspases located either upstream or downstream of Bcl-2action. Genes Cells 4, 401-414.15. Frostegard, J. (2005) SLE, atherosclerosis and cardiovascular disease. J Intern.Med. 257,485-495.16. D'Andrea, D. M., Coupaye-Gerard, B., Kleyman, T. R., Foster, M. H., Madaio, M. P.(1996) Lupus autoantibodies interact directly with distinct glomerular and vascular cellsurface antigens. Kidney Mt. 49, 1214-1221.17. Chapman, J. R., O'Connell, P. J., Nankivell, B. J. (2005) Chronic renal allograftdysfunction. J Am.Soc.NephroL 16, 3015-3026.18. Ramzy, D., Rao, V., Brahm, J., Miriuka, S., Delgado, D., Ross, H. J. (2005) Cardiacallograft vasculopathy: a review. Can.J Surg. 48, 319-327.19. Labarrere, C. A., Nelson, D. R., Faulk, W. P. (1998) Myocardial fibrin deposits in the firstmonth after transplantation predict subsequent coronary artery disease and graft failure incardiac allograft recipients. Am.J.Med. 105, 207-213.20. Labarrere, C. A., Pitts, D., Nelson, D. R., Faulk, W. P. (1995) Vascular tissue plasminogenactivator and the development of coronary artery disease in heart-transplant recipients.N.Engl.J.Med. 333, 1111-1116.21. Minami, K., Murata, K., Lee, C. Y., Fox-Talbot, K., Wasowska, B. A., Pescovitz, M. D.,Baldwin, W. M., III (2006) C4d deposition and clearance in cardiac transplants correlateswith alloantibody levels and rejection in rats. Am.J Transplant. 6, 923-932.22. Newell, H., Smith, J. D., Rogers, P., Birks, E., Danskine, A. J., Fawson, R. E., Rose, M. L.(2006) Sensitization following LVAD implantation using leucodepleted blood is not due toHLA antibodies. Am.J Transplant. 6, 1712-1717.23. Sims, P. J., Faioni, E. M., Wiedmer, T., Shattil, S. J. (1988) Complement proteins C5b-9cause release of membrane vesicles from the platelet surface that are enriched in themembrane receptor for coagulation factor Va and express prothrombinase activity. JBiol.Chem. 263, 18205-18212.11024. Sato, Y., Matsumori, A., Sasayama, S. (1994) Autoantibodies against vimentin in a murinemodel of myocarditis. Autoimmunity 18, 145-148.25. Yang, Y., Fujita, J., Bandoh, S., Ohtsuki, Y., Yamadori, I., Yoshinouchi, T., Ishida, T.(2002) Detection of antivimentin antibody in sera of patients with idiopathic pulmonaryfibrosis and non-specific interstitial pneumonia. Clin.Exp.Immunol 128, 169-174.26. Mahesh, B., Leong, H. S., McCormack, A., Sarathchandra, P., Holder, A., Rose, M. L.(2007) Autoantibodies to vimentin cause accelerated rejection of cardiac allografts. Am.JPathol. 170, 1415-1427.27. Larsen, E., Celi, A., Gilbert, G. E., Furie, B. C., Erban, J. K., Bonfanti, R., Wagner, D. D.,Furie, B. (1989) PADGEM protein: a receptor that mediates the interaction of activatedplatelets with neutrophils and monocytes. Cell 59, 305-312.28. Moisan, E., Girard, D. (2006) Cell surface expression of intermediate filament proteinsvimentin and lamin B1 in human neutrophil spontaneous apoptosis. J Leukoc.BioL 79, 489-498.29. Boilard, E., Bourgoin, S. G., Bernatchez, C., Surette, M. E. (2003) Identification of anautoantigen on the surface of apoptotic human T cells as a new protein interacting withinflammatory group IIA phospholipase A2. Blood 102, 2901-2909.30. Maugeri, N., Brambilla, M., Camera, M., Carbone, A., Tremoli, E., Donati, M. B., de, G.G., Cerletti, C. (2006) Human polymorphonuclear leukocytes produce and expressfunctional tissue factor upon stimulation. J Thromb.Haemost. 4, 1323-1330.31. Nakamura, S., Imamura, T., Okamoto, K. (2004) Tissue factor in neutrophils: yes. JThromb.Haemost. 2, 214-217.32. Orfeo, T., Butenas, S., Brummel-Ziedins, K. E., Mann, K. G. (2005) The tissue factorrequirement in blood coagulation. JBiol.Chem. 280, 42887-42896.33. Centemeri, C., Colli, S., Tosarello, D., Ciceri, P., Nicosia, S. (1999) Heterogeneousplatelet-activating factor (PAF) receptors and calcium increase in platelets andmacrophages. Biochem.Pharmacol. 57, 263-271.34. Sims, P. J., Rollins, S. A., Wiedmer, T. (1989) Regulatory control of complement on bloodplatelets. Modulation of platelet procoagulant responses by a membrane inhibitor of theC5b-9 complex. JBiol.Chem. 264, 19228-19235.35. Ludwig, R. J., Schultz, J. E., Boehncke, W. H., Podda, M., Tandi, C., Krombach, F., Baatz,H., Kaufmann, R., von Andrian, U. H., Zollner, T. M. (2004) Activated, not resting,platelets increase leukocyte rolling in murine skin utilizing a distinct set of adhesionmolecules. J Invest Dermatol. 122, 830-836.11136. Esposito, C. J., Popescu, W. M., Rinder, H. M., Schwartz, J. J., Smith, B. R., Rinder, C. S.(2003) Increased leukocyte-platelet adhesion in patients with graft occlusion afterperipheral vascular surgery. Thromb.Haemost. 90, 1128-1134.37. Shrivastava, S., McVey, J. H., Dorling, A. (2007) The interface between coagulation andimmunity. Am.J Transplant. 7, 499-506.38. Harmon, G. K., Starkebaum, G. A., Benditt, E. P., Schwartz, S. M. (1984) Fc-mediatedbinding of IgG to vimentin-type intermediate filaments in vascular endothelial cells.Proc.NatLAcad.Sci.U.S.A 81, 3103-3107.39. Pankhurst, T., Savage, C. 0. (2006) Pathogenic role of anti-neutrophil cytoplasmicantibodies in vasculitis. Curr.Opin.PharmacoL 6, 190-196.112CHAPTER VI: CONCLUSIONS AND FUTURE DIRECTIONS 6.1 Overall themes of dissertationIn this dissertation, I present data on the dissection of the intracellular and extracellularfates of a ternary complex composed of vimentin, vitronectin and PAI-1. First, I constructed afluorescent form of PAI-1 to track the intracellular localization of the anti-fibrinolytic componentof the ternary complex within cells such as megakaryocytes (MEG-01 cell line) and endothelialcells (Eahy926 cell line). I later determined that the majority of synthesized PAI-1 did not bindto the surface of megakaryocytes post-thrombin activation but did bind to the surface of activatedendothelial cells. Previous to these observations, we demonstrated that PAI-1 synthesized byendothelial cells was organized into storage granules that also contained vWF and P-selectin.This was a novel observation, as previous work had shown that PAI-1 was notcompartmentalized to a storage granule, but was instead loosely dispersed within the cytoplasmof transfected endothelial cells and megakaryocytes. It was previously determined that PAI-1was bound to the surface of activated cells such as platelets and platelet microparticles by firstbinding to vitronectin (VN) in PAI-1:VN complexes, the VN mediating its localization toexposed vimentin on the cell surface. Hence, it became important to understand the mechanismof vimentin exposure on activated endothelial cells and platelets.A morphological analysis ensued whereby a novel technique was devised to visualizesurfaces of platelets and platelet microparticles at a high resolution (nanometer level) to identifyvimentin and multimers of vitronectin as sites of ternary complex assembly. Using acombination of FACS and atomic force microscopy, only PMP's expressing CD41 a and acombination of PAI-1, VN and vimentin were isolated and individually imaged. AFMmorphological analysis revealed platelet microparticles with a diameter range from 350-800 nm,113and surface morphology differed between PMP's isolated from expired platelet concentrates orfrom AMI-patient plasma. PMP's from expired platelet due to Platelet Storage Lesion (PSL)concentrates demonstrated continuous, smooth surfaces with sparse protein distribution whereasPMP's from patient-AMI plasma demonstrated a highly irregular and decorated surfacecharacterized by protein distribution. The surface morphology of PMP's generated by PSLreflects their in vitro origin and instrinsic mechanism of activation whereas PMP's in patient-AMI plasma are generated in vivo and thus more susceptible to association with other plasmaproteins such as acute phase proteins, coagulation proteins such as fibrin and Factor VII, as wellas interactions with apoptotic or necrosed cells and their releaseates. In contrast, PMP's formedin vitro by platelet storage lesion present with far less surface irregularities, likely due to the lackof possible binding partners released in an activated in vivo setting. Another key observationwas membrane flap-like appendages that extended from the base of vimentin positive PMP's ofwhich some contained intermediate filament structures as verified by AFM. The heightdimension of these membrane flaps corresponded to previous AFM measurements ofphospholipid bilayer membrane of —5 nm.We have also uncovered a mechanism of how vimentin can induce vitronectin activationas determined from the cell-free experiments with purified VN and VIM133 peptide and AFM.The purified VN imaged by AFM demonstrated a height of 3 nm, presumably the native andinactive conform of VN. However, addition of VIM133 peptide which contains extensiveregions of basic amino acid residues, caused the formation of VN-vimentin multimers with amaximum observed height of 1.6 nm, a noticeable lack of 3 nm VN monomer structures. Theseobservations also hold true for conditions in which molar ration of VN:vimentin was 3:2 or 6:1.In Figure 6.1, we propose a mechanism of how VN activation may occur via the unraveling of114NfS4.1^1^^II+444 ++++1 AMMO.the VN molecule when the basic resides of VIM 133 ionically interact with the 340-380 aa regionof VN that contains acidic residues, thus revealing the basic resides contained in 50-100 aa onVN. This unravelling and subsequent exposure of basic resides can induce furtherdestabilization of intramolecular ionic bonds in another VN molecule, inducing co-operativebinding between VN to induce VN multimerization.Vimentin N-terminus (133aa)340-380 aaSomatomedinDomain50-100 aaInactive vitronectin (globular form)Figure 6.1. Proposed mechanism of vimentin-induced vitronectin multimerization.Vitronectin contains a region of basic amino acids (50-100 aa) and a region of acidicamino acids (340-380 aa) that interacts in an ionic binding interaction that contributes tofolding and its globular tertiary structure. When the region of basic amino acids onvimentin interacts with the acidic region on VN, it may induce a major conformationalchange such that the VN molecule "unravels" and in doing so, reveals the endogenousbasic amino acid region to other inactive VN, subsequently inducing further "unravelling"on another inactive VN, thus propagating VN-VN interactions to the extent of VNmultimerization.2.115From the observations made in Chapters III and IV, I propose a model that supports ourinitial hypothesis of how the ternary complex is assembled on activated platelets and plateletmicroparticles in Figure 6.2. Essentially, the model describes how the ternary complex is formedat the sites of microparticle release whereby vimentin cytoskeleton of platelet pseudopods thatrelease PMP's become externalized at the pseudopod-PMP breakage junction. At thesepsuedopod-PMP junctions, this exposed vimentin may be readily susceptible to a range ofprotein interactions such as VN multimerization and incorporation PAI-1-VN complexes, whichmay originate from pre-existing plasma sources or from exocytosis during platelet activation.Vimentin is typically not exposed to plasma because of its properties as a cytoskeletonprotein; my proposed mechanism of vimentin exposure provides a hypothesized manner ofvimentin presentation that may be relevant in discussions involving vimentin autoantibodygeneration during organ transplant vasculopathy. Moreover, the effects of these anti-vimentinantibodies (AVA's) were not understood, and whether or not they exert any pathogenic effect inthe context of organ transplant vasculopathy. We were able to determine that AVA's in normalwhole blood specifically bind to a subpopulation of senescent granulocytes expressing vimentinon their surface. As shown in Figure 6.2, when AVA's bind to this subpopulation of senescentgranulocytes, they induce complement-mediated cell lysis leading to cell death and during thisprocess, the release of platelet agonists such as tissue factor and platelet-activating factor (PAF)may occur. The release of these platelet agonists lead to platelet activation, platelet:leukocyteconjugate formation and PMP formation. Lastly, these observations are in agreement with otherpublished reports describing elevated levels of markers of platelet activation in the literature andthe contribution of activated platelets to atherogenesis in transplant vasculopathy [1, 2].1166.2. Strengths and limitations of thesis research6.2.1. Chapter IIIThe primary aim of this dissertation was to understand the role of vimentin in hemostasisand antibody-mediated complement fixation and how vimentin is exposed on the surface of cells.The means in understanding how vimentin is exposed on the surface of cells, did not yield to thetypical conventions or techniques of biomedical research. One of the main findings of thisdissertation is that the majority of synthesized PAI-1 is not bound to the surface of the activatedmegakaryocyte, a cell model analogous to its much smaller associate, the platelet. Theseobservations were made by constructing a chimeric form of PAI-1, fusing a fluorescent redprotein, dsRed, to the C-terminus of the PAI-1 cDNA sequence. Although not an extraordinaryprocess, extensive validation was performed to ensure its suitability as an intracellular marker ofPAI-1. This process of validation is comparable to work performed on other GFP/dsRed fusionchimeras such as tPA-GFP [1, 2], granzyme B-GFP [3], keratins K8-GFP K1 1 -GFP [3],vimentin-GFP [4], thrombopoietin receptor Mpl-dsRed [5]. This is considered a strength of thisdissertation, a validation that enabled investigation of PAI-1 trafficking andcompartmentalization in megakaryocytes and endothelial cells, observations that are animportant contribution to vascular biology, hemostasis and ECM metabolism.One of the key limitations in chapter III of this dissertation was the lack of PAI-1-dsRedquantification on the surface of activated megakaryocytes, which proved to be difficult duringflow cytometric analysis. Both thrombin- and calcium-ionophore- activated megakaryocytesproved to be unamenable to flow cytometry due to a subpopulation of large cellular aggregatesobstructing fluidics; aggregation possibly due to thrombin and cell adhesion. Our inability toperform flow cytometric analysis did not allow for PAI-1-dsRed signal comparison at pre- and117post-activation states of megakaryocytic and microparticle fractions. However, judging by thegeneral lack of PAI-1-dsRed on the surface of thrombin-activated megakaryocytes as visualizedby videomicroscopy (Figure 3.8.), the proportion of surface-bound PAI-1-dsRed is minimal.However, more work needs to be done to determine why ternary complex is expressed onactivated endothelium as determined in Figure 3.6. Major determinants in ternary complexformation may depend on two major criteria: 1) cell surface area available for microparticlerelease and; 2) cortical vimentin cytoskeleton network underneath the cell membrane.Endothelial cells may hold a greater capacity in ternary complex assembly because of their largersurface area and the density of underlying vimentin cytoskeleton network compared to plateletsand megakaryocytes [6-8].6.2.2. Chapter IVAnother key strength of this dissertation is the use of AFM to visualize individual plateletmicroparticles (PMP's). Prior to this work, imaging of PMP's was limited to transmissionelectron microscopy but with no ability to specifically image PMP's expressing just ternarycomplex. These images provided insight as to how vimentin may be exposed on the surface ofactivated cells, which can lead to extracellular ternary complex assembly. The combination ofFACS-mediated isolation of ternary complex-positive PMP's for AFM imaging is a noveltechnique that can be extended to visualizing protein distribution on the surface of larger cellssuch as endothelial cells, cardiomyocytes, and leukocytes.My proposed model (Figure 4.6) describing the mechanism of vimentin cell surfaceexpression also provides insights relating to the origin of vimentin autoantibody production. Ibelieve allograft implantation induces immune responses that immediately target graft1180 minPlatelet-Activating factor1r■ p roir^#•/^*.Ata di ^ae*ilmentln..--P-selector9#• %45 min iiC.CAIVOO.CS< ECD(79.3 3• O,as 2• 2—cp0• cucn3 5'24'<syC)0Anti-vimenUn 1gMantibodyPlatelet Leukocyteconjugate formationr---->Complement fixation^ via AVA IgM41. Quiescent platelet4111111111, Activated plateletgel Platelet microparticleNeutrophilendothelium, inducing the production of endothelial microparticles [9, 10]. The formation ofthese endothelial microparticles may result in vimentin expression on both the microparticle andendothelial cell and upon processing by antigen processing cells, potentially leading to an auto-immune response against vimentin. We have also determined the binding site of these vimentinautoantibodies in normal whole blood and determined how they can induce blood cell activation(Figure 6.1). The model of vimentin surface expression by microparticle release requires furtherinvestigation, for example, imaging of vimentin-positive platelets with cryo-electron microscopy.However, I propose a model in Chapter IV in which AFM images of platelet microparticles(PMP) provide insight as to how vimentin is formed, albeit interpretations based on artifactualmembrane appendages attached to the base of the PMP.There is another important limitation to be considered in Chapter IV regarding atomicforce microscopy of activated platelets. Although only CD41+ve platelets with high vitronectinsignal were sorted for AFM imaging, it was difficult to translate surface topography with our invitro observations on purified vitronectin and vimentin multimers. Furthermore, the fields of thesupposed vitronectin-vimentin multimers also contain antibodies specific for vitronectin andvimentin, causing interpretation to be even more speculative. Despite these potential artefacts, itis possible that the areas of high topographic activity are fields of multimerized proteincontaining vitronectin and vimentin. Overall, this chapter showcases the potential of atomicforce microscopy in providing insights with the ultrastructure of protein-protein complexes, andsubmicron ultrastructure of biological samples such as microparticles.6.2.3. Chapter VAnother strength of this dissertation is the comprehensive evaluation of anti-vimentinantibodies (AVA's) and their pathogenic effects on whole blood, a role previously unknown and120thought to be benign in effect. By using an in vitro effect but using both commercially availableand patient-derived AVA's, I was able to determine the mechanism of AVA-induced blood cellactivation. Similar reports describing the cytotoxic effects of other autoantibodies such as anti-neutrophil cytotoxic antibodies (ANCA) have established pathological effects of auto-antibodieswhile others such as anti-dsDNA antibodies and their effects remain unclear. Although theirpresence remains associative in relationship to the actual pathogenesis of vasculitis in kidneys;they are postulated to activate and induce neutrophils to release their proteolytic contents, thuscausing inflammation of the vessel wall [11, 12]. Chapter V described a mechanism of howAVA can induce blood cell activation by determining the binding site of AVA in normal wholeblood — neutrophils.The limitations associated with this chapter are primarily application-related and stemfrom the fact that this work was performed in vitro. Some of the key experiments we designedhad utilized mouse whole blood of a vimentin -/- genotype. We initially hypothesized that AVAwould not have any effect on vimentin-/- blood when compared to AVA on normal blood.Surprisingly, AVA did not exert any effect in terms of activation on both normal and vimentin-/-whole blood (data not shown). Similar comparisons with isolated leukocytes and AVAcytotoxicity tests also generated inconclusive results, bringing into doubt a cross-speciesrelevance of AVA and pathogenesis. Definitive studies based on AVA specificity for vimentinon activated murine platelets and leukocytes must be performed, both from washed and plasmasources. As well, it is conceivable that other murine plasma proteins bind to exposed vimentin,sterically hindering AVA binding to surface exposed vimentin on platelets and leukocytes.However, it has been determined by our laboratory that immunization of mouse models withhuman vimentin does not lead to murine AVA production and brings to light a cross-species121difference in vimentin homologues, a 446/453 amino acid residue similarity between Musmusculus and Homo sapiens. Hence, all future in vitro tests should use enough murine specificAVA in order to adequately compare the observed in vitro effects in normal human blood andmurine blood.6.3. Evaluation of current knowledge and proposals for future directionsPAI-1 exerts its pleiotrophic effects in both plasma and on extracellular membranesurfaces, but the mechanism of its localization to cell surfaces is still unclear despite continuedprogress. One major mechanism of PAI-1 extracellular presentation is related to its interactionswith a classical receptor, uPAR (urokinase-type plasminogen activator receptor). In detail, PAI-1 binds to uPA (urokinase-type plasminogen activator) that is already associated with the cellsurface receptor, uPAR, which coordinates both intracellular signals and extracellular proteolysisfor cellular motility. In this classical receptor-based mechanism, PAI-1 binds directly to uPA,thus inhibiting uPA and preventing the formation of plasmin[13]. At this site, VN can also bindto uPAR via its somatomedin B domain, although it is not definitively understood whether VNcan mediate PAI-1 binding to the VN-uPAR complex. It is also unclear whether or not a uPAvacancy is required for VN complex formation with the uPAR receptor [14, 15] in order for VNto recruit PAI-1 localization to the uPAR receptor.There is also a non-classical receptor mechanism of PAI-1 localization in which Podor etal. revealed the ability of VN to mediate PAI-1 localization to activated cell surfaces by bindingto exposed vimentin cytoskeleton on activated platelets and platelet microparticles[16]. Thisnon-classical receptor based mechanism partially explains PAI-1 expression on activated cellssuch as platelets, thus implicating PAI-1's anti-thrombolytic effects within thrombi. However,122the acceptance of this mechanism became dependent on the understanding of how vimentin isextracellularly presented. The proposed model in Figure 6.1 provides a plausible model for 1)vimentin surface expression leading to 2) PAI-1:vitronectin:vimentin ternary complex assemblyon cells such as platelets and endothelial cells. Future studies must determine the proportion ofPAI-1 bound to either the uPAR or vimentin receptor; studies that can utilize the PAI-1-dsRedintracellular probe. This is the ultimate objective of the PAI-1-dsRed probe, to permit theunderstanding of the extracellular fate of PAI-1 and the mechanism of its localization: whetherby the exposure of vimentin or by a receptor-based mechanism such as uPAR. I believe that theextracellular fate of PAI-1 will differ between cell types offering another dimension of thepleiotrophic effects PAI-1 exerts in the areas of cell motility, cell adhesion and fibrinolysis.The PAI-1-dsRed probe also catalyzed the understanding of PAI-1 trafficking withinendothelial cells, which will bring forth new ventures in controlling this factor via modulation ofPAI-1 secretion, hence regulating pathways such as hemostasis, fibrosis and vascular patency.This intracellular marker will also rejuvenate studies once centered on intracellular trafficking ofPAI-1 in platelets and endothelial cells as well as extracellular imaging studies on hemostasis viaintravital imaging by photochemical induced thrombosis animal models. This probe will permitreal-time evaluation of pro-thrombotic factors and their spatial distribution during thrombusformation in these intravital imaging experiments. In disease models of pulmonary fibrosis, thegeneration of lentiviral vectors or murine models with endothelium expressing only PAI-1-dsRedcan provide further understanding of the endothelium's role in disease progression in thisdisease.Atomic force microscopy of PMP's has proved to be an exciting contribution in the fieldof microparticle biology, shedding light on the morphology of PMP's. Once referred to as123"platelet dust" [17] and primarily assessed by flow cytometry, we have provided the firstdefinitive images of PMP's providing major insight to their origin, structure and potential as"miniature envoys", expressing other important proteins, e.g. tissue factor [18]. Moreover, theirsmall size (<1.0gm in diameter) renders their level of interactions to an almost soluble phase andit would be of interest to evaluate the functionality of CD41 integrin to determine its capability tobind to fibrin/fibrinogen. The ability of PMP's to bind to fibrin/fibrinogen will determine theirability to be incorporated into thrombi and whether they can contribute to clot strength in amanner analogous to activated platelets in a thrombus. If not, then their ability to express PAI-1may only be realized at a soluble level and not within a cell surface/cell adhesion/cell motilityenvironment.Investigations regarding the requirement of vimentin for ternary complex formation onthe surface of platelets and endothelial cells can be readily determined by pre-treating platelets orcultured endothelial cells with inhibitors such as cytochalasin D [19]. These inhibitors cause thevimentin intermediate filament cytoskeleton to collapse and reorganize tightly around thenucleus, dissolving the original cortical distribution of the intermediate cytoskeleton. Whenthese pre-treated platelets or endothelial cells are treated with agonists such as thrombin or TNF-a, the surface expression of ternary complex (PAI-1:VN:VIM) can be evaluated asmicroparticles are released from pseudopods formed via activation. This is one example inwhich pharmacological vimentin inhibition may provide an avenue of downregulated surfaceexpression of PAI-1.This vimentin inhibition or general cytoskeleton inhibition via cytochalasin D or otherinhibitors may provide avenues of insight as we continue to build on the knowledge gained fromvimentin exposure from activated endothelium and endothelial microparticles. A collapse of124vimentin cortical cytoskeleton and actin cytoskeleton by these inhibitors in graft endotheliumwill decrease microparticle formation [20] and may prove beneficial by preventing the release ofgraft endothelial microparticles available for antigenic processing. However, grafts wouldrequire this treatment prior to implantation to prevent cytoskeleton disruption in the host bloodcells. Aims toward reducing the processing of graft microparticles may result in the decreasedformation of non-MHC antibodies, or autoantibodies. Although there are no current strategies toprevent the formation of antibodies targeting the MFIC molecules of organ grafts, cytoskeletalreorganization by pharmacological inhibitors may be a means to limit the formation and impactof non-MHC antibodies, which are thought to arise from the destruction of graft cells caused bythe host immune system. This experimental methodology would also prove to be beneficial inpreventing the assembly of ternary complex on the surface of endothelium, and decreasing theamount of PAI-1 bound to the activated cell surface. Whether the origin of PAI-1 is platelet orendothelial derived, the absence of a vimentin anchor for the ternary complex would ultimatelydecrease the incorporation of PAI-1 into atherosclerotic lesions, thus decreasing futurethrombotic complications [21-23].6.4. Three most significant contributions6.4.1 Mechanism of microparticle release from activated blood cells and endotheliumMicroparticles are sub-micron sized (< 1.01.1m) vesicles that are released from the surfaceof activated cells such as platelets, leukocytes and endothelial cells. I set forth a mechanism ofmicroparticle release and how PAI-1 may be bound to the surface of these activated cells and onthe surface of microparticles. This mechanism is important because it explains in part how a125variety of proteins such as PAI-1 can be bound to the surface of activated cells without aclassical receptor, ie. integrins.6.4.2. The role of neutrophils in thrombus stabilization and structural integrity.I have performed microscopic and biophysical assessments on thrombi collected viaaspiration of intracoronary culprit lesions from infarct patients. I have determined thatneutrophils are specifically bound to thrombus fibrin via CD1 1 b on their surface. This bindinginteraction contributes to —15% of clot strength and can be inhibited via inhibitory antibodiesagainst the CD1 lb integrin. I have also shown that neutrophils may be partially responsible forthrombolytic resistance because azurophilic granule release can result in the biochemicalmodification of fibrin, rendering it unrecognizable by other proteins or fibrin-specific antibodies.6.4.3. Formation of platelet:leukocyte conjugates in transplant vasculopathy.Anti-vimentin antibodies (AVA) are a prognostic indicator of cardiovascular disease incardiac transplant recipients but their mechanism of action in the pathogenesis in chronic organrejection was unclear. I determined that anti-vimentin antibodies specifically bind to apopulation of senescent leukocytes, primarily of neutrophil and monocyte cell type. WhenAVA's bind to these leukocytes, platelet-activating factor was secreted which in turn, activatedplatelets. This activation of platelets subsequently led to the formation of platelet:leukocyteconjugates. When these platelet:leukocyte conjugates are present in the circulation of thesepatients, it is postulated that there may be increased infiltration of leukocytes and platelets intothe graft vasculature. This increased infiltration may exacerbate graft function, resulting incardiovascular disease and validating use of AVA titres as a prognostic indicator of graft life.1266.5. References for Chapter VI: 1. Jovin IS, Taborski U, Szalay Z, Friedel A, Segieth I, Jovin A, Heidinger K, Schreiner K,Frass 0, Klovekorn WP, Muller-Berghaus G. Trapidil decreases the aggregation ofplatelets from heart transplant recipients ex vivo. Transplant Proc 2006; 38:1523-5.2. Fateh-Moghadam S, Bocksch W, Ruf A, Dickfeld T, Schartl M, Pogatsa-Murray G,Hetzer R, Fleck E, Gawaz M. Changes in surface expression of platelet membraneglycoproteins and progression of heart transplant vasculopathy. Circulation 2000;102:890-7.3. Yoon KH, Yoon M, Moir RD, Khuon S, Flitney FW, Goldman RD. Insights into thedynamic properties of keratin intermediate filaments in living epithelial cells. J Cell Biol2001; 153:503-16.4. Yoon M, Moir RD, Prahlad V, Goldman RD. Motile properties of vimentin intermediatefilament networks in living cells. J Cell Biol 1998; 143:147-57.5. Zhang YP, Tang YS, Chen XS, Xu P. Regulation of cell differentiation by hNUDC via aMpl-dependent mechanism in NIH 3T3 cells. Exp Cell Res 2007; 313:3210-21.6. Dellagi K, Vainchenker W, Vinci G, Paulin D, Brouet JC. Alteration of vimentinintermediate filament expression during differentiation of human hemopoietic cells.Embo J1983; 2:1509-14.7. Dellagi K, Tabilio A, Portier MM, Vainchenker W, Castaigne S, Guichard J, Breton-Gorius J, Brouet JC. Expression of vimentin intermediate filament cytoskeleton in acutenonlymphoblastic leukemias. Blood 1985; 65:1444-52.8. Tablin F, Taube D. Platelet intermediate filaments: detection of a vimentinlike protein inhuman and bovine platelets. Cell Motil Cytoskeleton 1987; 8:61-7.9. Garcia S, Chirinos J, Jimenez J, Del Carpio Munoz F, Canoniero M, Jy W, Horstman L,Ahn Y. Phenotypic assessment of endothelial microparticles in patients with heart failureand after heart transplantation: switch from cell activation to apoptosis. J Heart LungTransplant 2005; 24:2184-9.10. Meehan SM, Limsrichamrern S, Manaligod JR, Junsanto T, Josephson MA,Thistlethwaite JR, Haas M. Platelets and capillary injury in acute humoral rejection ofrenal allografts. Hum Pathol 2003; 34:533-40.11.^Falk RJ, Terrell RS, Charles LA, Jennette JC. Anti-neutrophil cytoplasmic autoantibodiesinduce neutrophils to degranulate and produce oxygen radicals in vitro. Proc Natl AcadSci USA 1990; 87:4115-9.12712. Reumaux D, Duthilleul P, Roos D. Pathogenesis of diseases associated withantineutrophil cytoplasm autoantibodies. Hum Immunol 2004; 65:1-12.13. Chazaud B, Bonavaud S, Plonquet A, Pouchelet M, Gherardi RK, Barlovatz-Meimon G.Involvement of the [uPAR:uPA:PAI-1:LRP] complex in human myogenic cell motility.Exp Cell Res 2000; 258:237-44.14. Madsen CD, Ferraris GM, Andolfo A, Cunningham 0, Sidenius N. uPAR-induced celladhesion and migration: vitronectin provides the key. J Cell Biol 2007; 177:927-39.15. Waltz DA, Natkin LR, Fujita RM, Wei Y, Chapman HA. Plasmin and plasminogenactivator inhibitor type 1 promote cellular motility by regulating the interaction betweenthe urokinase receptor and vitronectin. J Clin Invest 1997; 100:58-67.16. Podor TJ, Singh D, Chindemi P, Foulon DM, McKelvie R, Weitz JI, Austin R, BoudreauG, Davies R. Vimentin exposed on activated platelets and platelet microparticleslocalizes vitronectin and plasminogen activator inhibitor complexes on their surface. JBiol Chem 2002; 277:7529-39.17. Wolf P. The nature and significance of platelet products in human plasma. Br J Haematol1967; 13:269-88.18. Ahn YS. Cell-derived microparticles: 'Miniature envoys with many faces'. J ThrombHaemost 2005; 3:884-7.19. Pryzwansky KB, Merricks EP. Chemotactic peptide-induced changes of intermediatefilament organization in neutrophils during granule secretion: role of cyclic guanosinemonophosphate. Mol Biol Cell 1998; 9:2933-47.20. Yano Y, Kambayashi J, Shiba E, Sakon M, Oiki E, Fukuda K, Kawasaki T, Mori T. Therole of protein phosphorylation and cytoskeletal reorganization in microparticleformation from the platelet plasma membrane. Biochem J 1994; 299 ( Pt 1):303-8.21. Landmesser U, Hornig B, Drexler H. Endothelial function: a critical determinant inatherosclerosis? Circulation 2004; 109:1127-33.22. Schafer K, Muller K, Hecke A, Mounier E, Goebel J, Loskutoff DJ, Konstantinides S.Enhanced thrombosis in atherosclerosis-prone mice is associated with increased arterialexpression of plasminogen activator inhibitor-1. Arterioscler Thromb Vasc Biol 2003;23:2097-103.23. Sobel BE, Taatjes DJ, Schneider DJ. Intramural plasminogen activator inhibitor type-1and coronary atherosclerosis. Arterioscler Thromb Vasc Biol 2003; 23:1979-89.128APPENDIX I: List of Publications, abstracts, and presentationsA. Peer-Reviewed PublicationsLeong HS, Mahesh BM, Day JR, Smith JD, McCormack AD, Podor TJ, Rose ML..VimentinAuto-Antibodies Induce Platelet Activation and Formation of Platelet-Leukocyte Conjugates viaPlatelet-Activating Factor. J Leuk Biol Nov 1 2007 (Epub)Mahesh B, Leong HS, McCormack A, Sarathchandra P, Holder A, Rose ML.. Autoantibodies tovimentin cause accelerated rejection of cardiac allografts. Am J Pathol. 2007 Apr; 170(4): 1415-27.Walinski HP, Gyorffy SF, Leong HS, Slaughter GR, Dawood F, Pate GE, Liu PP, Parker TG,Podor TJ.. Exercise increases tissue-type plasminogen activator expression in ratcardiomyocytes. Thromb Haemost. 2006 Dec;96(6):859-61.Cheung C, Luo H, Yanagawa B, Leong HS, Samarasekera D, Lai JC, Suarez A, Zhang J,McManus BM.. Matrix metalloproteinases and tissue inhibitors of metalloproteinases incoxsackievirus-induced myocarditis. Cardiovasc Pathol. 2006 Mar-Apr;15(2):63-74.B. Reviews and ChaptersHeine, H., Leong, H.S., Rossi, F., McManus, B.M., Podor, T.J.. Conditional Gene Expression inMyocardium: an Overview. Molecular Cardiology: Methods and Protocols.C. Manuscripts Submitted and In PreparationLeong HS, Bateman RM, Walinski HP, Podor TJ. Targeting of recombinant PAI-1-dsRed andvitronectin to storage granules in endothelial and megakaryocyte cell lines. J Thromb Hemo(submitted)Leong HS, Bateman RM, Whalen B, Meredith A, VanEeden S, Walinski HP, Podor TJ..Distribution of PAI-1:vitronectin:vimentin ternary complexes on activated platelets and plateletmicroparticles by atomic force microscopy. Blood (in preparation)Spiro J, Leong HS, Ghimire G, Dalby M, Kharbanda R, Mitchell A.. An Unusual Cause of ST-Segment Elevation Myocardial Infarction; a case report. Nat Clin Pract Cardiovasc Med(submitted)D. Oral PresentationsLeong HS, Mahesh BM, Day JR, Smith JD, McCormack AD, Podor TJ, Rose ML.. VimentinAntibodies Induce Formation of Platelet-Leukocyte Conjugates and Blood Agglutination via129Platelet-Activating Factor. Oral presentation. San Francisco, USA. April 25-29, 2007.International Society for Heart and Lung Transplantation.Leong HS, Mahesh BM, Day JR, Smith JD, McCormack AD, Podor TJ, Rose ML. VimentinAntibodies Induce Formation of Platelet-Leukocyte Conjugates and Blood Agglutination viaPlatelet-Activating Factor. Oral presentation. Manchester, UK. March 28-30, 2007. BritishTransplant Society Annual Congress.E. AbstractsLeong HS, Ghimire G, Spiro JR, Kharbanda R, Mitchell AG, Mason MJ, Ilsley C,Podor TJ, Rose ML, Dalby MC.. Characterization of human in vivo intracoronary thrombi fromacute myocardial infarction suggests a role for CD1 1 b. Orlando, USA. November 9-14, 2007.American Heart Association Basic Science Sessions.Ghimire G, Leong HS, Spiro JR, Kharbanda R, Rose ML, Dalby MC.. Increased numbers ofactivated T-cells from coronary artery aspirate in patients with acute myocardial infarctionundergoing primary angioplasty. Orlando, USA. November 9-14, 2007. American HeartAssociation Basic Science Sessions.Mahesh B, Leong HS, McCormack A, Holder A, Sarathchandra P, Smith J, Rose ML..Antivimentin Antibodies Cause Acute and Chronic Damage In MHC-Matched Allografts. SanFrancisco, USA. April 25-29, 2007. International Society for Heart and Lung Transplantation.Mahesh B, Leong HS, McCormack A, Holder A, Sarathchandra P, Smith J, Rose ML..Antivimentin Antibodies Cause Acute and Chronic Damage In MHC-Matched Allografts.Manchester, UK. March 28-30, 2007. British Transplant Society Annual Congress.Leong HS, McManus BM, Podor TJ.. Increases in Activated Platelet Microparticles ExpressingSurface PAI-1 Vitronectin- and Vimentin-Ternary Complexes in Mice Allograft Recipients.Madrid, Spain. April 3-9, 2006. International Society for Heart and Lung Transplantation.Leong, HS, Bateman, RM, van Eeden, S, Jiao, YK, Walinski, H, Podor, TJImaging of Highly Organized Vitronectin-Vimentin Polymeric Complexes that Express on theSurface of Activated Platelets and Platelet Microparticles using Atomic Force Microscopy.Sydney, NSW, Australia, August 6-12, 2005. International Society of Thrombosis andHaemostasis.Leong, HS, Bateman, RM, Walinski, H, Podor, TJChimeric PAI-1-dsRed Fusion Protein Characterization in MEG-01 Megakaryocytic Cells:Model for PAI-1 Trafficking from a-Granules to Cell Surfaces. Sydney, NSW, Australia, August6-12, 2005. International Society of Thrombosis and Haemostasis.Bateman, RM, Leong, H, Walley KR, Podor TJThe Effect of Thrombin Concentration on Fibrin Clot Structure Imaged by130Multiphoton Microscopy and Quantified by Fractal Analysis. Sydney, NSW, Australia, August6-12, 2005. International Society of Thrombosis and Haemostasis.Bateman, R.M., Leong, H., Podor, T., Hodgson, K.C., Kareco, T., Walley, K.R.. The Effect ofThrombin Concentration on Fibrin Clot Structure Imaged by Multiphoton Microscopy andQuantified by Fractal Analysis. Honolulu, Hawaii, July 31-Aug 4, Microscopy andMicroanalysis, 2005.Bateman, R.M., Hodgson, K.C., Leong, H.S., Podor, T., Walley K.R.. Intravital and Ex vivoMicrovascular Imaging using Dual Fluorescence Multiphoton Microscopy. Jena, Germany, Mar18-23, Focus on Microscopy, 2005.Walinski, H., Lowe, R., Bohunek, L., Pate, G., Leong, H., Hamburger, J., McManus, B.M.,Podor, T.. Vitronectin deficient mice exhibit reduced cardiac remodeling and wound healingfollowing acute myocardial infarction. San Diego, CA. April 2-6, FASEB 2005.Leong, H.S., Walinski, H., Westoby, M.A., Podor, T.J.. Characterization of PAI-1-dsRedChimeric Protein Expression by Cultured Megakaryocyte and Endothelial Cells. Toronto, ON,June 1-5, International Vascular Biology Meeting 2004.Leong, H.S., Walinski, H., Westoby, M.A., Podor, T.J.. Characterization of PAI-1-dsRedChimeric Protein Expression by Cultured Megakaryocyte and Endothelial Cells. Winnepeg,MB, May 6-9. CIHR Young Investigators Forum 2004.F. AwardsPersonnel funding: Canadian Blood Services PhD traineeship (2006-2008)Michael Smith Foundation PhD traineeship (2006-2008)Royal Brompton & Harefield Hospital Honorary Fellowship (2007)British Heart Foundation Travel Fellowship (2006)Heart and Stroke Foundation Focus on Stroke PhD Traineeship (not accepted)CIHR/CBS travel awards (2005&2006)CIHR/Dept of Surgery PhD Traineeship for Transplantation (2005-2006)CIHR/CBS PhD Traineeship for Transfusion Science (2005-2006)Operating grants: CIHR International Opportunities Planning Grant ($24,800 for 1 year)CRC Royal Brompton and Harefield Clinical Sciences Pilot Graft (£50,000 for 1 year)British Heart Foundation International Collaborations Grant (£3,000 for 3 months)131


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