Characterization of the

Fgl2-Prothrombinase Binding

Jemi fer AM Crookshank

A thesis submitted in coiiformity with the requirements

for the degree of Master of Science,

Graduate Department of Laboratory Medicine and Pathobiology.

University of Toronto

6Copyright by Jemifer Ann Crookshank 2001 The author has granted a non- L'auteur a accordé une licence non exclusive licence dowing the exclusive permettant a la National Li* of Canada to BibliothQue nationale du Canada de reprduce, Ioan, dismiute or reproduire, prêter, disûibuer ou copies of this thesis in inicroform, vendre des copies de cette thése sous paper or electronic formats. la forme de rnicrofiche/fih, de reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propridte du copyright in this thesis. Neither the &oit d'auteur qui protège cette thèse. thesis nor substantial extracts kom it Ni la thèse ni des extraits substantiels may be printed or otherwise de ceiie-ci ne doivent être imprimes reproduced without the author's ou autrement reproduits sans son pemiissian. autozisation. Characterization of the Fgl2-Binding Protein

Master of Science 2001

Jenni fer C rookshank

Department of Laboratory Medicine and Pathobiology

University of Toronto

Abstract

The inflammatory cascade and the system are intimately comected;

several coagulation have been demonstrated to regulate inflammatory events.

Studies have shown that is able to promote cellular adhesion and

transendothelial migration through interactions with ICAM-l and Mac- 1, prompting

speculation that related proteins may have similar roles. Fg12 prothrombinase shares

significant homology to the fibrinogen-y chah; in particular the PI domain of fi brinogen.

which is responsible for interactions with Mac-1, is highly conserved in Fg12. We

hypothesized that soluble Fg12 has an immunornodulatory eflect through interactions with

a cel1-surface receptor. We further speculated that Fg12 interacts with this putative

receptor through its fibrinogen-related domain, and may bind to Mac4 through the conserved Pl region. We found that Fg12 binds to RAW 264.7 cells in a specific and dose-dependent marner (&= 185 nM), and that this binding is not dependent on Mac- 1. Acknowledgements

The people who have contributed to this work are too numerous to count, but 1 would especially like to thank the following:

My supervisor, Dr. Gary Levy, for his encouragement, guidance and financial

support;

The memben of my advisory cornmittee, Dr. Myron Cybulsky, Dr. M. James

Phillips and Dr. Li Zhang for their helpful cornments and direction;

Laisum Fung for his insights and assistance;

The members of the Levy lab, for sharing both their scientifc knowledge and

their friendship;

Carnie Chan for her experimental contributions and her willingness to answer

countless questions;

The members of the CIHR Transplant Research Group for their willingness to

share reagents and expertise;

Morn and Dad, Steph, Clint, Mike, Jack, Diane, Mark and Janet for their love

and support;

nie "Gang for helping me stay sane;

Adam, my "knight in shining armor", for more than 1 can possibly Say.

iii Contents

1.1 : INFLAMMA'TION...... I

1.2. ROLEOF FIBRINOGENAND RELATEDPROTEINS IN INFLAMMATION ...... 5

1.3. FGL~PROTHROMBINASE ...... 9

3m MATERIALS AND METHODS ~~~~~~~~~~~~~a~~~~~~~~a~~~~a~a~oamaa~~~~amaaaaaaaoaaaaaa19

3.1 : RECOMBINANTVIRUS PRODUCTION...... 19

3.2. RECOMBINANTPROTEIN PRODUCTION ...... 20

3.3. B~OTINYLATIONOF kL2. BSA AND FIBRINOGEN ...... 21

3.4. CELLS...... 21

3.5. FLOW CYTOMETRYANALYSIS ...... 24

3.6. INHIBI-~ION STUDIES...... 24

3.7. ASSOCIATIONOF FGLS WITH RAW 264.7 CELLS OVER TIME ...... 25

4 RESULTS aaa*a*aaa*a*maa*aaaaaaam**aaaa**aaaa*a***aa~aaa*aa***aa*a*a***a***m*a-.aoaa*~aa*aa*~*~*a~a****aa***a~*~~aam~*aa**a*a*aaa*a26 iv 4.1 : HYPOTHESIS:FGL~ PROTHROMBWASE ACTS TO MEDIATE ADHESION BETWEEN

LEUKOCYTES AND ENDOTHELlAL CELLS. AND DOES SO THROUGH INTERACTIONS

WlTH THE INTEGRIN MAC-I AND [CAM-I ...... 26

4.2. CHARACTERIZATIONOF THE FGL:! BINDING PROTEIN ...... 39

FUTURE DIRECTIONS...... 49 List of Tables

TABLE1: CONSERVATIONOF THE P 1 DOMAININ FIBRINOGEN-RELATEDPROTEINS ...... 8

TABLE2: COMPARISONOF THE CALCULATED K, VALUE FOR THE INTERACTION OF FGL~

PROTHOMBINASE WlTH THE FGL~-BINDING PROTEIN AND THE REPORTED Ko VALUES

FOR OTHER LIGAND-RECEPTOR INTERACTIONS ...... 46 List of Figures

FIGURE 1: BINDING OF BIOTINYLATEDFGLS PROTEIN TO MACROPHAGEAND RAW

264.7 CELLS ...... 27

FIGURE2: DOSE-DEPENDENTBINDING OF BIOTINYLATED FGL~PROTEIN TO RAw 264.7

CELLS ...... 29

FIGURE3: EFFECT OF LPS STIMULATION ON THE BlNDlNG OF BIOIINYLATED FGL~

PROTEIN AND BIOTINYLATED FIBRINOGENTO fb4~264.7 CELLS ...... 3 1

FIGURE4: BINDINGOF BIOTINYLATED FGL~PROTEIN TO SVEc4-I O CELLS ...... 33

FIGURES:BINDING OF BIOTINYLATED FGL~PROTEIN IS INHIBITED BY WLABELLED

FGL~,BUT NOT FIBRINOGEN OR ANTI-MAC-I ANTIBODIES ...... 35

FIGURE 6: BINDING OF BIOTINYLATED FIBRINOGEN IS INHIBITED BY UNLABELLED

FIBRINOGEN, ANTI-CD 1 1 B ANTlBODY AND ANTI-CD 18 ANTIBODY ...... 36

FIGURE7: EFFECT OF CHELATORS ON THE BINDING OF BIOTINYLATED FGL~PROTEIN TO

RA W 264.7 CELLS, SVEC~-1 O CELLS AND FlBROBLAST CELLS ...... ,..38

FIGURE8: BINDINGOF BIOTINYLATED FGL~TO VARIOUS CELL TYPES ...... 40

FIGURE9: ASSOCIATIONOF BIOTMYLATED FGL~PROTEIN WlTH RAW 364.7 CELLS OVER

vii FIGURE10: BlNDING OF BIO'TiNYLATED FGL~PROTEiN T0 RAW 264.7 CELLS IS

SATURABLE AND SPEClFlC ...... 42

FIGUREf 1: NON-LCNEAR REGRESSION ANALYSIS OF SPEClFlC BlNDlNG OF BIOTINYLATED

FGL~TO MW264.7 CELLS ...... 43

viii List of Abbreviations

Adult T-ceIl leukemia ATL

Amencan Type Culture Collection ATCC

Autographa californica multiple nuclear polyhedrosis virus AcMNPV

D-Biotinyl-E-arninocaproicacid-N-hydroxy-succinimide ester Biotin-7-NHS

Bovine Serum Albumin BSA

Complementary Deoxyribonucleic Acid cDNA

Concanavalin A ConA

Deoxyribonucleic Acid DNA

Dulbecco's Modifed Eagle's Medium DMEM

Dulbecco's Phosphate-Buffered Saline DPBS

Effector Cell Protease- 1 EPR-1

Ethylenediaminetetraacetic Acid EDTA

Fetal Bovine Sem FBS

Glycosylphosphatidylinositol GPI

Granulocyte-MacrophageColonyStimulating Factor GM-CSF

High Molecular Weight Kininogen Hka Human T-ce11 Leukemia virus type 1 HTLV-I

Inserted Domain 1 domain

Intercellular Adhesion Molecule- 1 ICAM- 1

Interferon-y IFN-y interleukin IL

Leukocyte Adhesion Deficiency LAD

Lipopolysaccharide LPS

Lymphocyte Function-Associated Antigen LFA- 1

Mean Fluorescence lntensity MF1

Messenger Ribonucieic Acid mRNA

Modified Eagle's medium MEM

Monoclonal Anti bodies mAbs

Mouse Hepatitis Virus-3 MHV-3

Natural Ki1 ter NK

N uc leocapsid N

Phosphate-Buffered Saline PBS

Platelet-Endothelial Ce11 Adhesion Molecule- 1 PECAM-1

Polymerase Chain Reaction PCR

Procoagulant activi ty PCA

Protease-Activated Receptor- 1 PAR- 1

P-Selectin Glycoprotein Ligand-1 PSGL- 1 Stre pavidin-Phycoerythrin SA-PE

Transfotming Growth Factor-P TGF-P

Tumor Necrosis Factor-a TNF-a

Urokinase Type Plasminogen Activator uP A

Urokinase Ueceptor uPAR

Vascular Ce11 Adhesion Molecule- 1 VCAM- 1

Very Late Antigen VLA Chapter 1

Introduction

1.1: Inflammation

Inflammation is a critical component of the host defense against invading pathogens

and tissue injury. However this innate response can result in significant damage to the

host when unregulated or inappropriately invoked, as demonstrated in auto-immune

diseases such as rheumatoid arthritis and multiple sclerosis. The inflammatory response

also presents a significant barrier to successful transplantation, as it is significantly

involved in the rejection response (1 ).

Several events occur on a ceIluIar level to trigger the idammatory cascade. Fint, endothelial cells become activated in response to tissue injury. This results in increûsed expression of P-selectin on the endothelial ce11 surface. P-selectin in turn interacts with its ligand, P-selectin glycoprotein ligand4 (PSGL-1), a molecule found on the surface of , , natural killer (NK) cells and memory T lymphocytes (2). The CHAPTER 1: MTRODUCTION 2 interaction of P-selectin with its ligand instigates leukocyte rolling and tethering, of the leukocytes dong the activated endothelial ce11 layer.

The activated endothelial cells release chemokines that interact locally with their receptors (members of the seven-transmembrane-spanning,G-protein-coupled receptor family) on the surface of the rolling leukocytes (3), trigget-ing an inside-out signaling pathway that leads to integrin activation (4). Firm adhesion is mediated by interactions between the leukocyte integrins and members of the immunoglobulin superfamily. such as intercellular adhesion molecule-1 (ICAM-1) (5). Leukocyte adhesion is followed by the transendothelial cell migration of effector cells into the site of tissue injury. While the molecular mechanism of transendothelial migration is still being investigated, it has been demonstrated it is rnarked by a disruption of endothelial ce11 adherens junctions, specifically the dissociation of katenin, p120lp100, and plakoglobin fiom VE-cadherin

(6).

1.1.2: Role of Mac-1 in inflammation

Integrins are a large family of heterodimeric molecules that have been implicated in a wide range of cellular functions, including signal transduction and adhesion to the extracellular matrix. Attachent of leukocytes to the endothelial ce11 layer during inflammation is dependent on interactions between membea of the P 1, P2 and P7 subfamilies of integrins and membea of the immunoglobulin superfamily. Leukocyte

Adhesion Deficiency (LAD) syndrome is caused by a lack or greatly reduced expression CHAPTER 1: DJTRODUCTION 3

of the P2 integrins, underscoring the importance of this integrin subclass in infiammation

(7). Four P2 integrins (also known as the leukocyte integrins, due to their restricted

cellular distribution) have been identified to date: LFA- 1, Mac-1, CD 1 1&Dl8 and the

recently discovered CD 1 1&CD 18. In particular, Mac-1 has been shown to be critical to

the inflammatory process.

Mac- 1, also known as CD 1 1 b/CD 1 8, is perhaps the best-characterized member of the

integrin family. Mac- 1 is a highly promiscuous receptor, whose ligands include ICAM- 1

(8), high molecular weight kininogen (Hka) (9), urokinase receptor (uPAR) (IO), C3bi

(1 1) and fibrinogen (12). Mac4 also interacts with a wide variety of microbial ligands

(13). Many of these ligands bind to the Mac-1 inserted (1) domain, a 200 amino acid

region that is found in several proteins, including the remainder of the P2 integrins, Very

Late Antigen-1 (VLA-1), VLA-2 and aEP7 integrin (14). Both the Mac-1 and LFA-1 1-

domain contain a rnetal iondependent adhesion site (MIDAS), indicating that ligand

binding is sornewhat dependent on the presence of divalent cations. Studies have shown

that Mac- 1 mediated-leukocyte adhesion is positively regulated by divalent cations, in

particular by ~n''( 15).

A variety of studies have demonstrated that, in addition to its ability to recognize a varîety of diverse ligands, Mac-1 is able to associate with severd glycosylphosphatidylinositol (GP1)-Iinked recepton, including FcyRiIIB, uPAR, and

CD14 (16). These interactions provide a means by which the GPI-linked receptors may participate in transmembrane signaling, despite the lack of an intracellular domain (1 7). CHAPTER 1: INTRODUCTION 4

Interactions between Mac- 1 and uPAR have been extensively discussed in the literature

and there is increasing evidence that the actions of the pl and 82 integrin families arc

coordinated through the actions of uPAR.

Similar to Mac-1, uPAR is a promiscuous receptor; its ligands include urokinase type

plasminogen activator (uPA)and vitronectin (17). uPAR was first shown to interact with

Mac4 in 1996, and it was demonstrated that this association resulted in an increase in

cellular adhesive properties (18); further studies have shown that uPAR clustering

initiates activation and increases Mac- l expression (19). Recently, a pathway

has been elucidated by which uPAR mediates activation of Mac-1 and LFA-1 by the

a4P1 integrin (VLA-4). Briefly, the clustering of a4pl integrin is able to induce

adhesion through the activation of both Mac-1 and LFA-1; this reaction was inhibited by

treatment with an anti-uPAR antibody (20). The result of this study suggests that uPAR

plays a critical role in regulating cellular adhesion, in addition to its other roles.

1.1.3: The link between inflammation and coagulation

The infiammation and coagulation cascades are intimately connected, with cross-talk occurring between the two pathways. lnflammatory molecules are known to increase expression of procoagulants on the surface of monocytes and , while coagulation proteins can in turn regulate inflammation. has been shown to activate endothelial cells~to increase P-selectin expression (2), and to induce the expression of ICAM-1 and Vascular Cell Adhesion Molecule-1 (VCAM-1) on the CHAPTER 1: INTRODUCTION 5

endothelial ce11 surface (21). Thrombin is thought induce these effects through a

Protease-Activated Receptor- l (PAR4 )-mediated pathway (22).

Other coagulation proteins are also involved in the regulation of the inflamrnatory

response. has been demonstrated to inhibit leukocyte-endothelial interactions,

(23) while its detivative, factor Xa, has been demonstrated to increase cytokine

production and adhesion molecule expression on endothelial cells via interactions with

Effector Ce11 Protease-l (EPR-1) (24). has been show to promote

inflammation in conjunction with factor VIla (25) and to play a role in the regulation of

the inflamrnatory response by promoting the reverse transmigration of mononuclear

(26). Tissue factor as has also been implicated in the activation of PAR-2

(27), a senne protease believed to be involved in early inflammatory events.

1.2: Rote of Fibrinogen and Related Proteins in Inflammation

Fibrinogen is multi-functional protein with several important biological roles. In addition to its well-characterized role in coagulation, fibrinogen has also been demonstrated to mediate ce11 adhesion, transendothelial migration, and to increase cell proliferation. Mac-l was first identified as a fibrinogen-receptor in 1988. Studies by

Wright and colleagues (1 2) demonstrated that, when coated ont0 plates. fibrinogen could mediate the binding and spreading of polymorphonuclear leukocytes and that this could be blocked using antibodies against the a and P chahs of Mac-1. ICAM-1 was later identified as a second fibrinogen-receptor (28) and studies have since demonstrated that CHAPTER 1 : MTRODUCTION 6

fibrinogen mediates interactions between Mac-1 and ICAM-1 (29). Further research has

locaiized the sites of fibrinogen-Mac4 interaction to amino acid residues 190-202 (Pl)

(30) and residues 377-395 (P2) (3 1) of the fibrinogen y chain and the Mac4 1-domain

(32). Fibrinogen-ICAM- 1 interactions occur between amino acid residues 1 1 7-1 33 of the

fibrinogen y chain and the first immunoglobulin-like domain of ICAM-I (33; 29; 34).

These interactions have been shown to promote both cell-cell adherence and

transendothelial migration (28; 29).

The role of fibrinogen in promoting ce11 adhesion has prompted speculation that

related proteins may also be involved in odhesion and migration. Tenascin, which

contains a fibrinogen-related domain at the C-terminus, is an extracellular matrix protein

that is mainly expressed during embryo morphogenesis; it has also been implicated during

turnor growth, inflammation and tissue repair. Studies have shown that this molecule to

have immunoregulatory fictions in that it can inhibit integrin-dependent adhesion of

monocytes to fibronectin, induce Epstein-Barr virus-transformed B cell aggregation, and

alter some pathways of T ceIl activation (35). It has also been dernonstrated that tenascin

interacts with endothelial cells (36) and aortic smooth muscle cells (37) through its

fibrinogen-related domain. Further studies have shown that endothelial attachent to tenascin is mediated through the integrins a2pl and uvB3 (38). Interestingly, the avB3 integrin also binds to the Pl site and to amino acid residues 246-358 of the fibrinogen y chain (39). CHAPTER 1 : iNTRODUCTION 7

M-ficolin is also a member of the fibrinogen-related family of proteins, and contains a fibrinogen-related domain at the C-terminus. The Pl region, responsible for mediating interactions between fibrinogen and Mac-1, is conserved in this protein (table 1). M- ficolin is found on peripheral blood monocytes but not on -derived dendritic cells and at low levels on monocyte-derived cells. Its tùnction on circulating monocytes has yet to be fully elucidated, however a recent report has shown that M- ficolin is able to mediate adhesion of the monocyte-denved ce11 line U937 to immobilized

ab')^ Fragments of an anti-fibrinogen antibody and thus may be involved in monocyte migration (40). CHAPTER 1 : INTRODUCTION

Table 1: Conservation of the Pl Domain in Fibrinogen-related Proteins Protein Function Integrins Conserved P 1 1 YO l Interactions Fibrinogen Coagulation aMP2, protein, aVP3 immunoregulation Angiogenesis, GWTVIQHREDGSL 71% anti-apoptotic factor 1 Angiopoietin-2 Angiopoeitin- I GWTVIQHREDGSV 77 % antagonist M-Ficolin Unknown; may GWTVFQRRVDGSL 77% play role in cellular adhesion Tenascin-C Em bryogenesis, Not conserved wound healing inflammation Fgl2 Procoagulant with GWTVLQARLDGST 7 7 % prothrombinase roles inviral hepatitis, fetal loss, gr& reiection

Both angiopoietin- 1, and its antagonist angiopoietin-2, contain fibrinogen-related domains at the C-terminus and thus are memben of the fibrinogen-related family of proteins. As with M-ficolin, the Pl region is conserved (table 1). Studies have shown that angiopoietin-1 is an apopiosis survival factor, and that it inhibits the apoptotic processes through interactions with the Tie-2 receptor (4 1). Binding of angiopoietin-l to

Tie-2 has ken localized to the fibrinogen-related domain (42). A receni study has shown that angiopoietin-1 inhibits endothelial ce11 pemeability by promoting the recruitment of

Platelet-Endothelid Cell Adhesion Molecule-1 (PECAM-1) to cellular junctions. In CHAPTER 1 : iNTRODUCTION 9

addition, angiopoietin-1 expression inhibited E-selectin expression on the surface of

endotheliai cells, and prevented Tumor Necrosis Factor-a (TNF-a)-induced

transmigration of leukocytes (43). Other studies have shown that angiopoietin-1, and to a

lesser degree angiopoietin-2, binds to endotheliai cells through interactions with avp5

integrin and memben of the p 1 integrin subfarnily (44). This suggests that in addition to

its role in angiogenesis and as an anti-apoptotic factor, angiopoietin-1 also functions as an

anti-inflamrnatory molecule and may be involved in cellular adhesion.

1.3: Fgl2 Prothrombinase

The fibrinogen-related protein Fg12 prothrombinase (fibroleukin; pT49 product;

(rnus)/alp protein) has a molecular weight of 70 kilodaltons (kDa) and shares 36%

homology with the fibrinogen y chain. The gene was first isolated from a T-ce11 minus B- cell subtractive complementary deoxyribonucleic acid (cDNA) library (45) and has been localized to the proximal region of 5 in mice (46) and to chromosomal region 7q11.23 in hurnans (47). Constitutive expression of this protein has been reported by both CD4+ and CD8+ cells (48) and cm be induced on macrophages by treatment with Interferon-y (LFN-y) (49). In addition, Fg12 messenger ribonucleic acid (mRNA) transcripts have been detected in mouse endothe1 id cells fol lowing infection wi th Mouse

Hepatitis Virus3 (MHV3), suggesting a wider cellular and tissue distribution (50). To date the functions of this protein have not been Mly elucidated, however studies have shown Fg12 to have a role in MHV-3 irifection in mice (5 1) fulminant hepatitis in humans CHAPTER 1 : INTRODUCTION 10

(52) and spontaneous abortion (53). There are indications that Fg12 may also be involved

in transplant rejection (54) and adult leukemia (55).

1.3.1: Rote of Fgi2 prothrombinase in viral hepatitis

The majority of individuals who contract acute hepatitis recover completely.

However a small percentage (less than 0.1%) go on to develop fulminant hepatitis, a

condition characterized by an influx of inflammatory cells resulting in extensive liver

necrosis. Previous studies have utilized MHV-3 infection in susceptible mouse strains to

investigate the pathogenesis of hlminant hepatitis.

MHV-3 is a member of Coronaviridae, a family of positive, single-stranded RNA

viruses. The outcome of infection with this virus in mice is strain-dependent; A/J and

SJL strains are fully resistant to the disease, while the Balb/cJ and C57BL/6J stains are

susceptible and inevitably die of fulminant hepatic failure (56). The disparity in the

manifestation of the disease cannot be accounted for by differences in virus susceptibility

as both susceptible and resistant strains have similar viral loads (57). Rather, variable

stimulation of the immune coagulation system, specifically the induction of macrophage

procoagulant activity, by MHVJ appears to be the key factor in the development of

fulminant hepatitis (58).

A panel of monoclonal antibodies (mAbs), directed against the MHVJ-induced procoagulant activity (PCA), was produced in order to investigate the role of immune coagulants in fulminant hepatitis. The mAb 3D4.3 reacted against a 70 kDa protein by CHAPTER 1 : INTRODUCTION 11

Western blot analysis and was shown to inhibit PCA expression in a one-stage clotting assay and to inhibit the conversion of prothrombin to thrombin in vitro (59). Further studies demonstrated mAb 3D4.3 increased the survival of susceptible mice when used in vivo (60). In order to identify the gene encoding the protein responsible for the MHV-3- induced PC A activi ty, a cDN A 1i brary was prepared fiom MHV-3 -in fected macrophages and screened using mAb 3D4.3 (5 1). A reactive clone was identified; sequencing of this cloned showed that it was identical to a portion of exon 2 of the previously identified musfbp gene (45).

The identification of the gene encoding the MHV-3-induced PCA protein (Fg12 prothrombinase) allowed for investigation into the molecular pathogenesis of fulminant hepatitis. Studies demonstrated that Fg12 mRNA transcripts were not detectable in the liver, spleen, lungs kidneys or brain prior to MHV-3 infection, nor was Fg12 protein expressed (50). Following MHV-3 infection' Fg12 mRNA transcnpts were detected in the liver (8 hours pst-infection), spleen and lungs (6 houn post-infection), but not in the kidneys or the brain. Fgl2 transcnpts were Merlocalized to nonparenchymal cells.

Recent studies have shown that the MHV-3 Nucleocapsid (N) protein is able to induce transcription of the Fg12 gene, and investigations have been undertaken to identim elements in the Fg12 gene prornoter that are responsive to the N protein (6 1). CHAPTER 1: iNTRODUCTION

1.3.2: Role of FgU prothrombinase in Fetal Loss Syndrome

The successful pregnancy represents an immunologicai paradox; as a composite of

both matemal and patemal antigens, it should be recognized as 'foreign' by the matemal

immune system, and thus be rejected. However, while a number of embryos are lost

throughout gestation, a high proportion are cmied through until term; the question

therefore arises as to how these immunologically foreign embryos survive. The utems

constitutes a unique immune environment that is adapted to supporting pregnancy while

maintaining its role as a mucosal barrier to infection (62). Several variables within this

environment have been identified as factors contributing to the success or failure of a

fetus, however the role of T cells and their associated cytokine profiles is of particular

interest. Studies have shown that a Th2 response is associated with a successful

pregnancy, while a Th1 cytokine profile contributes to the loss of a fetus.

CD4+ celis can be grouped into several subsets based on their function. Th1 cells

secrete cytokines that induce cell-mediated imrnunity, such as interleukin-2 (IL-2) and

IFN-y. Th2 cells induce B ce11 proliferation and antibody production primarily through

the interaction of CD40 on B cells and CMOL on T cells (63) and also produce

cytokines, such as IL04 and IL-IO, which enhance antibody production. The recently

described Th3 ce11 subset (64) is thought to induce tolerance via the secretion of

Transforming Growth Factor-B (TGF-P). The factors that cause T ce11 differentiation

have yet to be hlly elucidated, however there is evidence that cytokines secreted by antigen-presenting cells such as dendntic cells, B cells and macrophages play a critical CHAPTER 1 : INTRODUCTION 13

role (65; 66). ThIlTh2 paradigm is now known to affect the outcome of several

pathological conditions, such as tuberculosis, and MHVJ infection (56). Typically, a

Th1 response is associated with a resistant phenotype and a Th2 response is associated

with a susceptible or tolerant phenotype.

Cytokine-mediated abortion has been investigated using the CBA x DBN2 mouse

model of fetal loss, in which fetal rejection is believed to be mediated by activated NK

cells and macrophages (67) and the cytokines TNF-a and IFN-y. Fetal resorption is

marked by and hemorrhage in this model, suggesting a role for coagulation

proteins in fetal loss. Fg12 was first implicated as an important mediatw of fetal rejection

by a study that demonstrated that treatment with an anti-Fgl2 antibody significantly

reduced fetal rejection in the CBA x DBN2 afler cytokine treatment (53). In sitir

hybridization studies demonstrated that Fg12 rnRNA expression was increased at the junction of the decidua and myometrium and in trophoblast after cytokine treatment with

TNF-a and IFN-y as compareci to control mice (68). Similar levels of Fg12 mRNA

expression were observed in human decidua and trophoblast in cases of recurrent

spontaneous abortion with normal fetal karyotypes (68). A recent study has shown that

the effects of Fg12 in the decidua and trophoblast may be countered by the molecule OX-

2, suggesting a mechanism by which the uterus regulates fetal success (69). CHAPTER 1: INTRODUCTION

15.3: Otber roles of Fgl2 prothrombinase

Currently Fg12 has been identified as an important factor in viral hepatitis and fetal

loss syndrome; however the biologicai role of this protein has yet to be fÙlly understood.

Several studies have been undertaken to gain a more complete picture as to the total

functionality of this protein, including its role in adult T-cell leukemia and transplant

rejection.

Adult T-ce11 leukemia (ATL) is one of several diseases caused by the human

retrovirus Hurnan T-ce11 Leukemia virus type 1 (HTLV-1). It exhibits a wide spectrum of

clinical features, and thus can be funher classified into acute, lymphomatous, chronic and

smoldering types (70). ATL cells are characterized by several unique features, including

their ability to infiltrate into a variety of organs, including the liver, lung, spleen and

gastrointestinal tract; this is made possible by interactions between the leukemic cells and

the vascular endothelium. OX-40 and its ligand pg34 have been identified as the major

mediators of interactions between ATL leukemic cells and the endothelium (71 ). A

recent study has shown that Fg12 expression is decreased in chronic and acute ATL (55);

it is suggested by the authors that this may be partially responsible for the rnalignant

features of afflicted cells.

Transplantation is now widely utilized as an effective treatment for end-stage organ failure. General immunosuppressive agents, such as cyclosporin A, have been demonstrated to greatly enhance pst-transplant survival, however the use of these drugs also increases the gr& recipients risk of bacterial and viral infection. Thus current CHAPTER 1: INTRODUCTION

research efforts have focused on dissecting the mechanism of rejection at both a

rnolecular and irnmunological level in order to determine a more specific method of

promoting gr& swival.

Graft rejection can be sub-divided into three types: hyperacute rejection, acute

rejection and chronic rejection. Hypenicute rejection occurs rapidly following transplant, and is characterized by neutrophil accumulation, the deposition of pre-existing antibodies along the vesse1 walls, and the activation of the complement and coagulation cascades.

Acute rejection occurs somewhat later than hyperacute rejection, and is mediated by T cells. Long-term organ loss following transplantation is referred to as chronic rejection, and is mediated by both cellular and humoral mechanisms (72). Several studies are currently king undertaken to examine the role of Fg12 in both allograft and xenogrd loss. Prelirninary studies have show that Fg12 prothrombinase expression is increased during allograft rejection, and have localized expression to endothelial cells, CD3+ T cells and CD I I b+ macrophages. Fg12 expression was correlated with thrombin formation and fibrin deposition within the gr& (54). Studies using an Fg12 knockout mouse have ken undertaken to detemine whether Fg12 prothrombinase constitutes the key pathway of thrombin formation and fibrin deposition during transplantation.

In addition to its role as a membrane bound procoagulant, Fg12 prothrombinase may have additional functions as an immune-modulator. Recently Maraei and colleagues

(73) reported that fibroleukin (also known as Fg12 prothrombinase) was secreted as a 250-

300 Dacomplex fiom fieshly isolated peripheral blood mononucleat cells and from CHAPTER 1 : iNTRODUCTION

puified T lymphocyte; the authors speculated that this soluble form of Fg12 may have

immuno-modulatory activity. Work in our laboratory has demonstrated that Fg12 prothrombinase inhibits T ce11 proliferation, and promotes a Th2 cytokine response (74). suggesting that Fg12 does have immuno-regdatory activity.

The question remains as to how a protein can be both a membrane-bound procoagulant and a secreted immune modulator. Studies are currently being performed in our laboratory to resolve this paradigm. Analysis of the DNA sequence has revealed the presence of multiple Kozak consensus sequences. These translation start sites are often preceded by transcription start sites, thus raising the possibility that multiple mRNA species exist. This hypothesis is currently king investigated using S'RACE (75). We suspect that analysis will reveal the presence of at least hvo mRNA species, leading to the production of both a membrane-bound Fg12 molecule, and a secreted Fg12 molecule that lacks the N-terminal transmcmbrane domain. Chapter 2

Rationale and Statement of Hypothesis

Fg12 prothrombinase has been shown to be a unique procoagulant that is able to

directly cleave prolhrombin to thrombin. The role of this protein in both fulminant

hepatitis and fetal loss syndrome has been characterized and a role in transplant rejection

is currently being investigated. Both membrane-bound and soluble foms of this protein

have been reported (59; 48: 73), suggesting that, in addition to its function as a

procoagulant, Fg12 may act as an immune modulator as well. We therefore hypothesize

that soluble Fg12 binds to a ce11 surface receptor, and that this ligand-receptor interaction

causes an immuno-modulatory effect.

Fibrinogen and several members of the fibrinogen-like family of proteins have been show to mediate adhesive events, such as leukocyte adhesion and transendothelial migration. Fibrinogen has been show to mediate interactions between Mac- 1 and

ICAM- I to promote cellular adhesion and migration (28; 29). Fibrinogen-Mac- 1 interactions occur in part through the Pl domain of the fibrinogen y chah (30); this site CHAPTER 2. RATIONALE AND STATEMENT OF HYPOTHESIS 18 has also been implicated in fibrinogensvp3 interactions (39). Analysis of the amino acid sequence has shown that this region is highly consewed in the Fg12, and several other fibrinogen-related proteins (table 1). Therefore, we hypothesize that Fg12 may regulate leukocyte-endothelid interactions in a similar manner to fibrinogen and other fibrinogen- related proteins. Furthemure, we suspect that Fg12 may directly interact with Mac4 and

ICAM- I through the conservd P 1 domain.

We therefore hypothesize that:

Soluble Fg12 binds to a cell-surface receptor, and is an immune modulator in

this form

Binding of Fg12 to its receptor is mediated through the fibrinogen-related

domain

Fg12 binds to both Mac4 and ICAM-1, and acts as a regulator of the

inflarnmatory response Chapter 3

Materials and Methods

3.1: Recombinant Virus Production

A vector containing the fg12 pne under control of the polyhedrin promoter was

constructed using the pBlueBacHis2a vector (Invitrogen, Carlsbad, California). A 1.4

kilobase cDNA was amplifieci by Polyrnerase Chain Reaction (PCR) using the forward

primer 5'-TGCCGCACTGGATCCATGAGGCTTCCTGGT-3'and the reverse primer

5'-AAGGCTGAAGGAATTCATCGTCC-3'. A total of 25 amplification cycles were

perfiormed (2 min at 96 OC, 2 min at 55 OC and 3 min at 72 OC). The resulting PCR

product was cloned into the EcoRl and the BamHl sites of the pBlueBacHis2a vector.

A recombinant baculovirus was generated by CO-transfectingSf9 cells with 2 pg of the pBlueBacHis2a vector containing the fgi2 insert and 1 pg linearized Aurogrupha californica multiple nuclear polyhedrosis virus (AcWPY))DNA using the Bac-N-Blue transfection kit (Invitrogen). Three rounds of plaque purification were then performed; CHAPTER 3. MATERIALS AND METHODS 20

plaques containing putative recombinant viruses were analyzed by PCR, and the resulting

product was sequenced using an automated DNA sequencer (mode1 377, PE Applied

Biosystems, Foster City, California).

3.2: Recombinant Protein Production

Recombinant Fg12 prothrombinase was produced under denaturing conditions using

the xpressm Baculovirus Expression System (Invitrogen) according to the manufacturers

directions. HighFive cells (Invitrogen) were infected with the recombinant Baculovirus

containing the Fg12 gene under the control of the polyhedrin promoter. Ce11 pellets were

harvested at 3 days post-infection and lysed in guanidinium lysis buffer (6M guanidinr

hydrochloride, 20 mM sodium phosphate, 500 mM sodium chloride, pH 7.8) by repeated

passage through an 18-gauge needle. Cellular debris was removed by centrifugation, and

the ce11 lysate was applied to a ProBond resin nickel column (Invitrogen) and incubated at

4OC for I hour with gentle rotation. The column was then washed 2X with binding buffer

(8 M Urea, 20 rnM sodium phosphate, 500 mM sodium chloride, pH 7.8), 2X with wash

buffer (8 M Urea, 20 mM sodium phosphate, 500 mM sodium chloride, pH 6.0) and 2X

with wash buffer (8 M Urea, 20 mM sodium phosphate, 500 mM sodium chloride, pH

5.3). The protein was then eluted fiom the column with elution buffer (8 M Urea, 20 mM

sodium phosphate, 500 mM sodium chloride, pH 4.0) and dialyzed against decreasing concentrations of urea (6M,4M, 2M, IM, pH 7.8) for 2 days. The protein solution was

then dialyzed against 10 mM Tns, pH 7.8 to remove traces of urea. CHAPTER 3. MATERIALS AND METHODS

3.3: Biotinylation of Fgl2, BSA and Fibrinogen

A 1 mg/dsolution of purified Fg12 and immunoglobulin-fiee bovine serum albumin

(BSA) (Sigma, Oakville Ontario) were incubated with a 1: 10 molar reaction mixture of

D-Biotinyl-E-arninocaproic acid-N-hydroxy-succinimide ester (Biotin-FNHS) (Roche

Diagnostics, Laval Quebec) for 1 hour at room temperature with gentle stimng. The reaction mixture was then applied to a Sephadex G-25 column (Roche Diagnostics) and washed with 1.5 mL of PBS, and eluted with 3.5 mL of PBS. The elutant was collected in 0.5 mL aliquots and the protein concentration was determined using a BCA assay

(BioLynx, Brockville Ontario). Murine fi brinogen (Sigma) was labeled in a similar manner, however the molar reaction mixture was 1: 1 00.

3.4.1: Preparation of Macrophages

Macrophage production in Balblc mice was stimulated by intraperitoneal administration of 1.5 mL of a 5% thioglycolate solution. Cells were harvested afier 4 days, and washed twice with senun-fiee RPMI (Life Technologies, Burlington Ontario).

The cells were then stimulated with 20 pg/mL lipopolysaccharide (LPS) (Sigma) for 6 hours in RPMI media supplemented with 10% fetal bovine sem (FBS) (Life

Technologies) at 3TC. CHAPTER 3. MATERIALS AND METHODS

3.42: Preparation of B cells and T cells

Splenic B cells and T cells were collected fiom Balb/c mice as follows: the spleen

was minced and passed through a nylon mesh filter to obtain a ce11 suspension. The cells

were then washed with serum-fkee a-modified Eagle's medium (MEM) media, and

resuspended in a hypotonic ammonium chloride solution. The red blood cells were lysed

by incubating the cells with the ammonium chlonde solution for 3 minutes at 37OC. The

cells were then washed with a-MEM media, and applied to a nylon wool colurnn (76).

The non-adherent T cells were removed by washing the colurnn with a-MEM media afier

a 1 hour incubation at 37°C; the adherent B cells were then collected by gently squeezing

the nylon wool and collecting the media. In some experiments, the cells were then

cultured in a-MEM with 10% fetal bovine serum (Life Technologies) and 2% Penicillin-

Streptomycin (Life Technologies) at 37°C and 5% COz. T cells were stimulated with 2

pg/mL Concanavalin (ConA) (Sigma) for 72 hours, and B cells were stimulated with 20

pghL of LPS (Sigma) for 48 hours.

3.4.3: Preparation of Dendritic cells

Dendritic cells were prepared as described elsewhere (77; 78). Bone marrow cells

were isolated fiom Balbk mice by flushing a-MEM media through the extracted femur

bone using a 22-gauge needle. The cells were then collected by centrifugation, and resuspended in a hypotonic ammonium ciiloride solution. The red blood cells were then

Iysed by incubating the cells with the ammonium chloride solution for 3 minutes at 37°C . CHAPTER 3. MATEWSAND METHODS 23

The cells were then wash with a-MEM,and incubated with anti-CD4 antibody

(Cedarlane Laboratones, Homby Ontario) on ice for one hour. The cells were then washed with a-MEM and incubated with rabbit anti-mouse complement for one hour at

37OC and 5% CO2. The cells were then washed with a-MEM supplemented with 10% fetal bovine senun and 2% Penicillin Streptomycin, and cultured with IL-4 (10 ng/mL)

(Cedarlane Laboratories) and -Macrophage-Colony Stimulating Factor (GM-

CSF) (10 ng/mL) (Cedarlane Laboratories) for 6 days, with media exchange occumng every 48 hours. The mature dendritic cells were obtained by stimulating the culture with

20 pg/mL for 2 days. The cells were confirmed to be dendritic cells based on morphology and ceIl surface staining for CD80 as assessed by flow cytometry.

3.4.4: Cell lines

The murine macrophage-like ceIl line RAW 264.7 (79) was obtained from the

American Type Culture Collection (ATCC) and were cultured in Dulbecco's modified

Eagle's medium (DMEM) with 10% fetal bovine serum and 2% Penicillin-Streptomycin at 37°C and 5% COz. For some expenments the cells were stimulated with 10 pg/mL of

LPS for 12 hours. The murine endothelid-like ce11 line SVEC4-IO (80) was obtained from the ATCC and were cultured in DMEM with 10% fetal bovine serum and 2%

Penicillin-Streptomycin at 37°C and 5% C02. Murine fibroblast cells were a kind gift of

Dr. Andres Kapus (University of Toronto, CIHR Transplantation Research Group) and CHAPTER 3. MATERIALS AND METHODS 24 were cultured in DMEM with 10% fetal bovine senun and 2% Penicillin-Streptomycin at

37OC and 5% COI.

3.5: Flow Cytometry Analysis

Cells were washed twice with Dulbecco's phosphate-buffered saline (DPBS) (Li fe

Technologies), and then blocked for 5 minutes with 5% mouse serum (Cedarlane

Laboratories) at room temperature. The cells (1 *lob) were then incubated with biotin- labeled Fg12, biotin-labeled immunoglobulin-free BSA (Sigma) or biotin-labeled fibrinogen (Sigma) for 30 minutes at 4OC. The cells were washed again, and incubated with a Strepavidin- phycoerythrin (SA-PE) conjugate (BD Pharmingen, Mississauga,

Ontario) for 20-30 minutes at 4°C. ExtrAvidin-phycoerythrin conjugate (Sigma) was used in some experiments. AAer an additional two washes, the cells were analyzed by flow cytometry.

3.6: Inhibition Studies

Cells were washed twice with DPBS and blocked for 5 minutes with 5% mouse serum at room temperature. The cells (1 * lob) were then incubated with 10X molar excess of unlabelled fibrinogen or fibronectin, anti-CD18 antibody or anti-CDllb antibody or IOX molar excess of unlabelled Fg12 protein for 30 minutes at 4OC. Cells were then washed and stained according to the above protocol. In control experiments, cells were incubated with 10X molar excess of unlabelled fibrinogen, anti-CD18 antibody CHAPTER 3. MATERIALS AND METHODS 35 or anti-CD1 1b antibody for 30 minutes at 4OC and then incubated biotinylated fibrinogen for 30 minutes at 4OC. In some experiments cells were pre-treated with lOmM EDTA

(Sigma) for 30 minutes at 4°C prior to king stained in accordance with the above protocol.

3.7: Association of Fgl2 with RAW 264.7 cells over time

RAW 264.67 cells were washed twice with DPBS and blocked for 5 minutes with 5% mouse senim at room temperature. The cells (2*10') were thrn incubated biotinylated

Fg12 protein (1 19 nM) for increasing amounts of time (10 minutes-210 minutes) ai 4"C.

The cells were then washed, and stained for flow cytometry analysis as described.

3.8: Saturation Binding Curve

RAW 264.7 cells were washed twice with DPBS and blocked for 5 minutes with 5% mouse serum at room temperature. The cells (2'10') were then incubated with increasing concentrations of biotinylated Fg12 protein (23.8 nM-476.2 nM) for 2.5 hours at 4OC. The cells were then washed and analyzed by flow cytometry as described previously. Non- specific binding was assessed in the presence unlabelled Fg12 protein (2.38 FM). Specific binding was calculated by subtracting non-specific binding from total binding. The & value was determined by non-linear regression analysis, using the GraphPad Prism

Software version 3.02 (GraphPad Software, San Diego California). Chapter 4

Results

4.1: Hypothesis: Fg12 prothrombinase acts to mediate adhesion behveen

leukocytes and endothelial cells, and does so through interactions

with the integrin Mac-1 and ICAM-L

4.1.1: FgU binding to Macrophages und Raw 264.7 cells

In order to investigate the possible alternative role of Fg12 prothrombinase as an

adhesion molecule, we first examined the binding of Fg12 to macrophages. Cells were

harvested from Bdbk mice 4 days pst-injection of 5% thioglycolate, and stimulated

with LPS for 6 hours. The cells were then incubated with biotinylated Fg12, stained with strepavidin-PE and analyzed by flow cytornetry. An increase in the fluorescent signal was apparent in cells that had ken stained with biotin-Fgl2 compared to cells stained with Strepavidin-PE alone or with biotinylated BSA (tigure 1). CHAPTER 4. RESULTS

Log Fluorescence Macrophage cells RAW cells

Figure la) Murine macrophages were harvested 4 days afier the intraperitoneal injection of 1.5 mL of thioglycolate; 10' cells were incubated with biotinylated Fg12 protein (840 nM) for 30 minutes on ice; the cells were then stained with 1 pg of SA-PE and analyzed by flow cytometry. (b) RAW 264.7 cells were cultured in DMEM+lO% FBS and 0.2% penicillin-streptomycin; the cells were harvested at 95% confluency, and analyzed as with macrophage cells. CHAPTER 4. RESULTS 28

In addition, the macrophage-like ce11 line RAW 264.7 was also found to bind biotinylated Fg12 in a similar marner to the thioglycolate-elicited macrophage cells

(figure 1). This binding was dose-dependent (figure 2). These results suggested the presence of an Fgl2-binding protein on the surface of macrophage and macrophage-like cells. CHAPTER 4. RESULTS

Log Fluorescence

Figure 2) 106 RAW cells were incubated on ice with increasing concentrations of biotinylated Fg12 protein (a)42 nM, b)84 nM, c) 420 nM, d) 820 RM, e) 1260 nM, f) 168 1 nM,g) 2521 nM) for 30 min; cells were then stained with 1 pg of SA-PE and analyzed by flow cytometry. Cells incubated with biotinylated Fg12 and SA-PE show in black; cells incubated with SA-PE alone shown in grey. CHAPTER 4. RESULTS 30

The effect of LPS stimulation on Fg12 binding to RAW 264.7 cells was then investigated.

Cells were either unstimulated or stimulated with 10 pg/mL of LPS for 12 hours, and incubated with biotinylated Fg12 for analysis by flow cytometry. Analysis showed that binding of biotinylated Fg12 was decreased in cells treated with LPS as compared to untreated cells. In contrast, binding of biotinylated fibrinogen to MW 264.7 cells increased with LPS stimulation (figure 3). CHAPTER 4. RESULTS

Log Fluorescence Fg12 Prothrombinase Fibrinogen

Figure 3) Effect of LPS Stimulation on (a) Fg12 and (b) Fibrinogen binding to RAW cells: RAW 264.7 celfs were cultured in DMEM + 10% FBS and 0.02% Penicillin- Streptomycin, and in some cases stimulated with 10 ug/d LPS for 12 hours. Cells were then either incubated with biotinylated Fg12 protein (420 nM) or biotinylated fibrinogen protein (170 nM) for 30 minutes on ice and analyzed as described by flow cytometry . CHAPTER 4. RESULTS

4.1.2: Binding of FgU to SVEC4-10 ceUs

The endothelial-like ce11 line SVEC4-10 is derived from BALBIc mice and does not express ICAM-1. This ce11 line was therefore used to investigate the binding of Fg12 to endothelial cells and to determine whether this binding was ICAM-1 dependent. Cells were harvested by treatment with 2 mM EDTA, washed and stained for flow cytometry analysis as described. An increase in fluorescent signal was apparent for cells stained with biotinylated Fg12 as compared to cells that had been stained with biotinylated BSA or strepavidin-PE alone (figure 4a), suggesting that Fg12 binds to endothelial cells, and that this binding is independent of ICAM-1. Binding occurred in a dose-dependent marner (figure 4b). CHAPTER 4. RESULTS

Log Fluorescence

Figure 1) Fg12 binding to SVECCIO cells: a) cells were cultured in DMEM + 10% FBS and 0.2% penicillin-streptomycin until 95% confluent; cells were harvested by treatment with 2mM EDTA in PBS for several minutes at room temperature and detached by gentle pipetting; cells were then stained with various concentrations of biotinylated Fg12 and analyzed as described previously; (b) binding was dose-dependent. CHAPTER 4. RESULTS

4.1.3: Fgl2 binding to RAW 264.7 ceUs is inhibited by pre-treatment rith unlabelled

FgU, but not by known Mac-l ligands or by anti-Mac4 antibody.

In order to investigate the role of Mac-1 as a potential Fgl2-binding protein. a series of inhibition studies were carried out using known Mac4 ligands and anti-Mac-1 antibody. Cells were pre-treated with unlabelled Fg12, unlabelled fibrinogen or unlabelled anti-Mac4 antibody for 30 minutes at 4OC. The cells were then stained as described previously for flow cytometry analysis. Cells treated with unlabelled Fg12 showed a dose-dependent inhibition of biotinylated Fg12 binding, whereas cells treated with unlabelled fibrinogen or unlabelled anti-CD1 lb antibody showed no inhibition

(figure 5); in contrast, binding of biotinylated fibrinogen was inhibited by pretreatment with unlabelled fibrinogen, anti-CD1 lb antibody and anti-CD18 antibody (figure 6).

These results suggested that the Fg12 binding site on macrophages is therefore unique cornpared to that of fibrinogen and may be a protein other than Mac- 1.

In order to investigate the role of other integrins as Fg12 binding proteins, a series of inhibition studies were undertaken using the extracellular matrix protein fibronectin nnd anti-CD 18 antibody. Cells were pretreated with unlabelled fibronectin or anti-CD I 8 for

30 minutes on ice and then stained for flow cytometry analysis as described above.

Pretreated cells failed to show any inhibition of biotinylated Fg12 binding as compared to untreated cells, suggesting that the Fgl2-binding protein is distinct fiom the fibronectin receptors, and is not a member of the P2 integrin family (figure 5). CHAPTER 4. RESULTS

Inhibition of Fgl2 binding

Figure 5) Inhibition of Fg12 binding to RAW cells: RAW cells were cultured and harvested as described previously; 106 cells were incubated with an excess of unlabelled Fgl2, fibrinogen, fibronectin, Mac-1 antibody or CD18 antibody for 30 minutes on ice; the cells were then washed and incubated with 420 nM biotinylated Fg12 and analyzed by flow cytometry as described previously. The MF1 value for each sample was compared to non-preireaied cells. Results were statistically analyzed using the students T test. CHAPTER 4. RESULTS

Inhibition of Fibrinogen Binding

rn Fibinogen

r Fbgn + antiCDl lb

-- 1-9. k ,/A nn i

Figure 6) Inhibition of Fibrinogen binding to RAW 264.7 cells: RAW cells were cultured and harvested as described previously; 106 cells were incubated with an excas of unlabelled fibrinogen, antiCDl lb antibody or antiCDl8 antibody for 30 minutes on ice; the cells were then washed and incubated with biotinylated fibrinogen (210 nM) and analyzed by flow cytometry as described previously. The MF1 was for each sample was compared to the non-pretreated cells. Results were analyzed using the student's T-test. CHAPTER 4. RESULTS

4.1.4: Chetation of divalent cations inhibits Fgi2 binding to RAW 264.7 cells, but not to SVEC4-10 cells

As the binding of ligands to members of the integrin family is known to be cation dependent, the effect of pretreatment with EDTA on Fg12 binding to RAW 264.7 cells and SVEC4-10 cells was investigated. Cells were washed with PBS without divalent cations. The cells were pre-treated with 10 mM EDTA for 30 minutes at 4'C, and were then stained as described previously for analysis by flow cytometry. RAW 264.7 cells that had been pre-treated with EDTA demonstrated inhibited Fg12 binding compared to cells that had not been pre-treated with EDTA. In contrast, SVECS-IOcells that had been pretreated with EDTA did not demonstrate inhibited Fg12 binding (figure 7). Therefore the dependency of Fg12 binding on the presence of divalent cations varies with cell type. a) Cation Oepndmcy of FgO bhding to b) Catbn Dopndency of Fgl2 binding to RAW 264.7 ceib SMC4-10 celh

.Foa n FgÎ 10 mM EDTA

c) Calon Oepondoncy of FgO bhding to

Figure 7) Cells were cultured, harvested and analyzed as described previously with the following exception: cells were pre-treated (30 minutes on ice) and analyzed in the presence of 10 mM EDTA in PBS. Results were statistically analyzed using the students T test. CHAPTER 4. RESULTS

-

4.2: Characterization of the Fgl2 binding protein

4.2.1: Distribution of the FgU-binding protein

A number of ce11 types were investigated to determine the cellular distribution of the

Fgl2-binding protein. Macrophages, RAW 264.7 cells, SVEC4-IO cells, murine

fibroblasts, T cells, B cells, and dendritic cells were isolated and cultured as described

previously. Cells were then analyzed by flow cytometry as described. The results of

these studies indicated that the Fgl2-binding protein is expressed constitutively on

macrophages and macrophage derived ceils (RAW 264.7 cells), SVEC4-IO cells.

fibroblast cells, and dendritic cells. Binding of Fg12 prothrombinase was not evident on

unstimulated B cells, but was demonstrated der LPS-stimulation. Similarly, Fg12 did

not bind to unstimulated T ceils, but a population of T cells demonstrated Fg12 binding

after Con. stimulation. (figure 8). CHAPTER 4. RESULTS

SVE œlts Muine Fbrobhsi ails

Unstimutated B cells

Log Fluorescence

Figure 8) Cellular Distribution of Fgl2-binding protein: Cells were cultured, harvested and washed as descnbed. Cells were then incubated with biotinylated Fg12 protein (840 nM) for 30 minutes on ice, and then analyzed by flow cytometry as described. CHAPTER 4. RESULTS

4.2.2: Association of Fgi2 prothrombinase with RAW 264.7 cells over time

The association of Fg12 prothrombinase with RAW 264.7 cells over time was

investigated. Cells were incubated a constant concentration (1 19 nM) of biotinyloted

Fg12 protein for increasing arnounts of time, and then analyzed by flow cytometry.

Maximum binding of Fg12 to RAW 264.7 cells was achieved at 150 minutes at 4OC. This

time point was used for subsequent investigation of binding atrinity (figure 9).

hociation of FglZ Prothrombinase with RAW 264.7 cells over tirne

O 50 1O0 150 200 250 Time (min)

Figure 9) Association Kinetics: Cells were harvested and blocked as described previously. 2* 1o5 cells were incubated with biotinylated Fg12 protein (1 19 nM) for increasing time intervals up to 21 0 minutes on ice. The cells were then analyzed by flow cytometry as descri bed (n>/=3). CHAPTER 4. RESULTS

4.23: Sahiration Curve and Assessment of Affinity

The binding of Fg12 prothrombinase to RAW 264.7 cells was assessed. Cells were

incubated with increasing concentrations of Fg12 for 2.5 hours on ice; non-specific

binding was assessed in the presence of an excess of unlabelled Fg12 protein. Binding of

the Fg12 protein to RAW 264.7 cells was saturable and specific (figure 10). The Kdvalue

was detemined by non-linear regression aaalysis using the GraphPad Prism software

version 3.02, and calculated 185 nM (figure 9).

Saturation Curve: Fgl2 binding to RAW 264.7 ce Ils

Total Binding Nomspecif ic Binding Specf ic Binding

O 20 40 60 Fgl2 added (prn OIS)

Figure 10) RAW cells were cultured and harvested as described previously; 2*105 cells were incubated with increasing concentrations of biotinylated Fg12 at 4OC for 2.5 hours on ice; for non-specific binding, cells were incubated with an excess of unlabelled Fg12. Specific binding was calculated by subtracting non-specific binding fiom total binding (n=4). CHAPTER 4. RESULTS

Non-li near Regression analysis of Fg12 prothrombinase binding to RAW 264.7 cells

Figure 11) Non-linear Regression Analysis of Fg12 prothrombinase binding to RAW 264.7 cells: Nonlinear regression analysis of specific binding in MF1 units was perfomed using GraphPad Pnsm Software version 3.02. The Kd value was determined to be 185 nM. Chapter 5

Discussion and Future Directions

The relationship between coagulation and inflammation has been well characterized.

Darnage to the endothelial ce11 layer triggers both the coagulation and the inflammatory

pathways, resulting in the cleavage of coagulation zymogens to an active state, and the

recruitment of leukocytes to the site of tissue damage. It is therefore not surprising that

coagulation factors and proinflarnrnatory molecules, such as adhesion factors, are able to

reciprocally regulate each other. Several recent publications have investigated the

relationship between inflammation and coagulation, and found that many coagulation

factors, including factor Xa. thrombin and tissue factor are important inflammatory

mediators. Moreover, there is increasing evidence that serine protease receptors, such as

the PAR family of receptors and EPR-1, are the bridge that spans the divide between

coagulation and inflammation (8 1).

The diverse biological functions of fibrinogen have been well documented; in addition to its role as a coagulation factor, fibrinogen exerts pro-adhesive and pro- CHAPTER 5. DISCUSSION 45

migratory effects through interactions with the integrin Mac- 1 and the immunoglobulin

superf'arnily member ICAM- 1 (28; 29). and participates in avp3-mediated clot retraction

(39). Several other fibrinogen-related proteins, such as tenascin and M-ticolin, have

recently been shown to mediate cellular adhesion (36,37; 40). In addition, angiopoietin- 1

has been demonstrated to inhibit endothelial permeability and leukocyte transendothelial ce11 migration (43). This raises the possibility that other fibrinogen-related proteins may also act to regulate adhesion and migration, and may be important immune regulators.

Fg12 prothrombinase was originally identified in cytotoxic T lymphocytes. Studies have shown a pivotal role for Fg12 prothrombinase in the pathogenesis of fulminant hepatitis, and have also demonstrated its role in fetal loss syndrome. Current research is focused on examining the role of Fg12 in both allo- and xenotransplantion, and prelirninary results suggest that it may also mediate graft rejection. A secreted fom of the molecule has also been reported, leading to speculation that Fg12 may act as an immuno-regulatory molecule, however no work has ken published to support this possibility. Amino acid sequence analysis has demonstrated that Fg12 shares 36% homology with the fibrinogen y chain, and that the PI site is highly conserved. This site has been shown mediate interactions between fibrinogen and Mac-1, and to promote cellular adhesion and migration. We therefore hypothesized that soluble Fg12 rnay regulate cellular adhesion and migration by binding to Mac-l on lcukocytes and ICAM-1 on endothelial cells. CHAPTER 5. DISCUSSION 46

True l igand-receptor interactions are c haracterized by both saturability and spec ificity

(82). We therefore investigated the binding of Fg12 to RAW 264.7 cells, a macrophage ce11 line, to determine whether or not this interaction met the criteria of a true ligand- receptor relationship. We found that Fg12 prothrombinase fully associated with the putative receptor in 150 minutes on RAW 264.7 cells at 4OC. Fg12 binds with high affinity to its putative receptor (Kd = 185 nM). This is comparable to severai other ligand-receptor interactions (see table 2). Therefore we conclude that the binding of FglZ prothrombinase on RAW 264.7 cells does represent a true ligand-receptor interaction.

Table 2: Comparison of the calculated Kdvalue for the interaction of Fg12 prothombinase with the Fgl2-binding protein and the reported Kd values for other ligand-receptor interactions.

Ligand Receptor or Cell Type Kd Reference Fi brinogen Monocytes High affhity site: 4 nM 83 1 Low affinity site: 1.3 pM 1 1 Fi brinogen Neutrophils 170 nM 84 Vitronectin UPAR 30 nM 85 Angiopoetin- 1 Tie-2 3.7 nM 86 HKa Mac- 1 62 nM 9 Fibronectin 300 nM 87 Fg12 Prothrombinase Unidenti fied receptor 185 nM on RAW 264.7 cells

In order to test Our hypothesis regarding the identity of the Fgl2-binding protein, we first investigated whether Fg12 was able to bind to ceil types known to express the Mac-1 molecule. Monocytes and macrophages are known to express Mac-1, and studies have shown that fibrinogen binds to monocytes in a Mac-l dependent fashion (88). We have demonstrated that Fg12 is able to bind to peritoneal macrophages and to the macrophage- CHAPTER 5. DISCUSSION 47

like ce11 line RA W 264.7 in a dose-dependent and specific manner. Furthemore, we

have shown that binding of Fg12 to RAW 264.7 cells is dependent on the presence of

divalent cations, a cornrnon requirement of integnns for ligand binding.

To examine the possibility that Mac-1 is the receptor for Fg12, we perfonned a series

of inhibition experirnents using fibrinogen and antibodies for the Mac-l a and P subunits.

The rationale for these experiments was that if Fg12 was binding to Mac-1 through the P1 region then pre-treatment of the cells with fibrinogen should inhibit binding of Fgl2, as should pre-treatment with the Mac4 antibodies. However, our results dernonstrated that

Fg12 binding was not be inhibited by pre-treatment with fibrinogen or with antibodies to the Mac-l a and P subunits. Furthemore. we have demonstrated that Fg12 binding to

RAW 264.7 cells is decreased when the cells are stimulated with LPS, while binding of fibrinogen is increased. Therefore, we have concluded that Fg12 prothrombinase binds to

RAW 264.7 cells in a Mac-l-independent fashion. Furthermore, the failure of the anti-

CD1 8 antibody to inhibit Fg12 binding while inhibiting the binding of fibrinogen suggests that none of the P2 integrins are involved in the binding of Fg12 to RAW 264.7 cells.

Several other integins have been show to be important inflanmatory mediators.

The nine pl integrins fùnction primarily to mediate cellsell adhesion, and cell- extracellular matrix adhesion; some members have been show to be involved in T cell signaling (89). Our data indicated that binding of Fg12 to RAW 264.7 cells was cation dependent, suggesting that the Fg12 bhding protein shares properties with members of the integrin family. We therefore attempted to Fg12 binding to RAW 264.7 cells with CHAPTER 5. DISCUSSION 48

fibronectin, a common ligand for several members of the pl integrin farnily. We found

that pretreatment of RAW 264.7 cells with fibronectin did not inhibit Fg12 binding,

suggesting that Fg12 and fibronectin do not share a common cellular binding site and that

the Fg12 binding protein may not be a member of the integrin family.

We concurrently investigated binding of Fg12 to the endothelial-like ce11 line SVEC4-

10. Fibrinogen has been shown to facilitate leukocyte adhesion and migration by binding

to both Mac-1 and ICAM-1 and by promoting interactions between these two molecules.

We therefore hypothesized that Fg12 may also bind to ICAM-1 on endothelial cells. The

SVEC4-IO ce11 line has been previously shown to not express ICAM-1 in either a

constitutive or inducible fashion (80), and we therefore used this ce11 line as a preliminary

means to test Our hypothesis. We demonstrated that Fg12 bound to the SVEC4- 1O cells in

a dose-dependent manner. We have also shown that this binding is not dependent on the

presence of divalent cations. Therefore, we can conclude that binding of Fg12 prothrombinase to SVEC4- I O cells is ICAM- 1 independent.

Having established that Fg12 prothrombinase did not bind to Mac-1 on macrophage and macrophage-like cells, and that Fg12 did not bind to ICAM-1 on endothelial cells, we then examiwd the cellular distribution of the Fg12 receptor. We chose to look at the distribution of the binding protein on both leukocytes, and fibroblast cells. We found that the Fg12 receptor is expressed on several ceIl types. It is expressed constitutively on macrophages and denciritic cells; while not constitutively expressed lymphocytes, it can be induced on both LPS-stimulated B cells and population of ConA activated T cells. It CHAPTER 5. DISCUSSION is also expressed on endothelial and fibroblast cells; Fg12 binding to these cells is cation- independent.

Future Directions

Although we have determined that Fg12 does not bind to Mac4 or ICAM- 1, the cellular distribution on leukocytes, endothelial cells and fibroblast cells does suggest that

Fg12 prothrombinase may be involved in the regulation of the immune system. As stated previously, coagulation proteins have been show to have a broad specm of activities in addition to their primary function as procoagulants. These functions range from promoting adhesion and migration and increasing the expression of pro-intlammatory cytokines to inhi biting the interactions between leukocytes and endothelial cells. In addition, related proteins such as tenascin, M-ficolin and angiopoietin- 1 have been show to have immuno-modulatory effects; this strongly suggests that Fg12 is involved in immune regulation.

Several recent findings in our laboratory support this hypothesis. Ongoing studies have demonstrated thot Fg12 prothrombinase promotes a Th2 cytokine response in vitro, inhibits T ceIl proliferation and suppresses ce11 surface expression of the CO-stimulatory molecules CD80 and CD86 (74), suggesting that Fg12 negatively regulates the T ce11 response. An anti-invasive role for Fg2 is suggested by recent studies utilizing anfgl2 -/- mouse as the donor animal in a heterotropic heart xenotransplant mode1 (90). Animals lacking the fgl2 gene demonstrated an increase in the infiltration of mononuclear cells as CHAPTER S. DISCUSSION compared to the control animal. Although the mechanism has yet to be elucidated, one possible suggestion is that Fg12 inhibits cellular adhesion andor migration through the endothelial cell layer, and that its absence results in increased permeation. This hypothesis is supported by the fïnding that expression of Fg12 is decreased in ATL leukemic cells, which have increased adhesive properties. Also, Fg12 shares a high degree of homology with angiopoietin-1, which was recently shown to be an anti-invasive molecule (43).

However, in order to fully elucidate the mechanism by which Fg12 causes these effects, the identity of the Fgl2-binding protein must be established. There are several methods that could be utilized to realize this goal. The conventionai approach for determining protein-protein interactions is the yeast two-hybrid system (91). In this system, a known gene is cloned into vector to create a "bait" protein, where the protein of interest is linked to a DNA binding domain. This protein is then used to screen a library of "prey" proteins, in which unknown proteins are linked to an activation domain. When the DNA binding domain and activation domain are brought together by the interactions of the "bait" and 'prey" proteins, a reporter gene is transcribed.

Several more traditional, biochemical methods exist for determining protein-protein interactions, such as phot~~nityor chernical crosslinking and immunoprecipitation.

Photoaffinity crosslinking has been used for the characterization of many receptor-ligand interactions, including the mapping of the integrin avp3-ligand interface (92). in this method, a photactivatable molecule is chemically linked to the ligand of interest. ne CHAPTER 5. DISCUSSION 5 1 crosslinker is activated in the presence of ultraviolet light, causing the ligand to be irreversibly bound to the receptor molecule (93). The ligand-receptor complex can then be analyzed on a sodium dodecyl-sulfate (SDS) gel, either as a crude cellular lysate or as a purified complex.

The field of proteomics has been defined as "the study of protein properties.. .on a large scale to obtain a global, integrated view of diseases processes, cellular function and networks at the protein level" (94). This emerging field illuminates protein structure and fùnction, and identifies protein-protein interactions using complex, computer-based algorithrns. Wr are currently investigating this method in Our laboratos, as a means of identifying potential FglZ-prothrombinase receptors. We will then test the target molecules to determine if they bind purified Fg12. Once the receptor has been identified, we will also carry out more extensive kinetic studies using labeled protein.

Our data has also indicated that Fg12 prothrombinase has a variable requirement for divalent cations when binding to its receptor. We have shown that Fg12 binding to RAW

264.7 cells is significantly reduced in the presence of EDTA, while binding to SVEC4- 1O ceUs and fibroblast ceiis is not aEected. This suggests that Fg12 binds to iwo distinct recepton. More work must be done to investigate this possibility. Other leukocyte cell types should be investigated to determine if they require the presence of divalent cations to bind Fg12 prothrombinase. Furthermore, the kinetics of Fg12 binding to SVEC4-IO cells should be investigaied. If the results of these expenments support the theory that CHAPTER 5. DISCUSSION 52

Fg12 prothrombinase binds to two receptors, the second receptor will also have to be

identi fied.

Following the identification of the receptor molecule, the site of interaction must then

be identified. We have hypothesized that the binding of Fg12 prothrombinase to its

receptor is rnediated by the conserved Pl site; this hypothesis could be tested by using a

syntheiic Pl peptide to block Fg12 binding to RAW 264.7 cells. If interactions with the

receptor are not mediated by the PI site, truncated proteins could be utilized to determine

which region of Fg12 prothrombinase mediates interactions with the Fgl2-binding protein.

Once the binding of Fg12 prothrombinase to its receptor is fully characterized, functional

assays cm be performed to further elucidate the biological role of this protein in vivo.

In summary, we have demonstrated that Fgl2 does bind to a specific ceIl surface

receptor on RAW 264.7 cells. Our data indicates that this molecule is not the Mac-1

integrin. We have also detemined that Fg12 binding to SVECClO cells in a dose- dependent manner, but that the putative receptor on these cells is not ICAM-1. We have determined that Fg12-binding protein is ex pressed on several leukocyte ceIl types, and also on endothelial cells and fibroblast cells. In the future, studies will be carried to detemine the identity of the Fg12 receptor, to localize the site of interaction between Fg12 and its receptor, and to explore the biological functions of this molecule. References

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