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1.101 EEl{ VER\A.ryOER WORD NIE . Universiteit Vrystaat
PHAGE DISPLAY SELECTION OF PEPTIDE
INHIBITORS OF FVlla AND THEIR
FUNCTIONAL CHARACTERISATION
by
Catharina Elizabeth Roets
Submitted in fulfilment of the requirements for the degree
Philosophiae Doctor (Ph.D)
In the Faculty of Health Sciences
Department of Haematology and Cell Biology
at the University of the Free State
Bloemfontein
South Africa
June 2002
II Promoter: Dr. S.M. Meiring
Declaration
Hereby I declare that the script submitted towards a Ph.D. degree at the
University of the Free State is my original and independent work and has never
been submitted to any other university or faculty for degree purposes.
All the sources I have made use of or quoted have been acknowledged by
complete references.
----·~~iJ---'-5------------_.
e.E. Roets
June 2002
Dedicated to my two beautiful children, Lize-Louise and Carlize
Table of Contents
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ABBREVIATIONS
LIST OF FIGURES IV
LIST OF TABLES V
CHAPTER 1
INTRODUCTION 1
CHAPTER 2
LITERATURE REVIEW 4
2.1 Blood CoagulatiOn 4
2.2 Role of platelets in thrombosis and coagulation 6
2.3 Factor VII 8
2.3.1 Genetics and Structure 8
2.4 Tissue Factor 12
2.4.1 Genetics and Structure 12
2.5 Factor Vila/Tissue Factor complex 14
2.6 Inhibitors of the factor Vila/tissue factor complex 19
2.6.1 Tissue Factor Pathway Inhibitor 19
2.6.2 Antithrombin III 22
2.6.3 Nematode Anticoagulant Proteins 23
2.6.4 Active site inhibited factor Vila 23
2.6.5 Peptide inhibitors 24
2.7 Phage display 26
2.7.1 Introduction \" 26
2.7.2 Structure and genome of the filamentaus phage 27
2.7.3 Life cycle of M13 phage 29
2.7.4 Phage display of peptides and proteins 31
2.7.5 Phage display systems 32
2.7.6 Applications of phage display 36
CHAPTER 3
MATERIAL AND METHODS 39
3.1 Phage display 39
3.1.1 Phage display peptide libraries 39
3.1.2 Biotinylation of factor VI/a 40
3.1.3 Biopanning 41
3.1.4 Global ELISA 43
3.1.5 Growing and amplification of single colonies 44
3.1.6 ELISA to identify FVlla binding phage colonies 44
3.1.6.1 Dilution ELISA 45
3.1.6.2 Inhibition ELISA 45
3. 1.7 Prothrombin time '. 46
3.1.8 Sequencing of FVI/a-inhibitory phages 46
3.1.8.1 DNA preparation 46
3.1.8.2 Polymerase chain reaction 47
3.2 Tests performed on the synthesised peptide 48
3.2.1 Prothrombin time and thrombin time 48
3.2.2 Perfusion studies with endothelial cells 48
3.2.3 Perfusion studies with collagen 50
3.2.4 Perfusion studies with tissue factor 50
3.2.5 Kinetic assay 51
CHAPTER 4
RESULTS 53
4.1 Biotinylation of FVlla and biopanning of biotinylated FVlla 53
4.2 Biopanning of FVlla coated directly'to the tube 53
4.3 Sequences of the phage colonies 57
4.4 Effect of peptide on prothrombin and thrombin times 57
4.5 Perfusion studies with endothelial cells 59
4.6 Perfusion studies with collagen 61
4.7 Perfusion studies with tissue factor 61
4.8 Kinetic assay 65
CHAPTER 5
DISCUSSION 68
CHAPTER 6
ABSTRACT 78
CHAPTER 6
ABSTRAK 80
REFERENCES 82
ACKNOWLEDGEMENTS 99
-,
Abbreviations
'êé9 MM jfuQ tê;@@f('¥'!!@\'iffi#Wiï!YRf9\ ,iAA""agM;:remIW Jt:Qt!..t;tfAWiWiilwWCêbdiS¥¥¥NS4 i'l,l@#'; wat;! S?i§§i§@ttW&1t''1' *,"*,'WbaibRMMWdhR9&'
ADP adenosi ne-5' -diphosphate
Apo(a) apolipoprotein a
Arg arginine
Asp aspartic acid
ATIII antithrombin III
Ca2+ calcium
Cys cysteine
DNA deoxyribonucleic acid
E coli Escherichia coli
EDTA ethylenediaminetetra-acetic acid
EGF epidermal growth factor
ELISA enzyme-linked immuno-sorbent assay
FITC fluorescein isothiocyanate
FVII factor VII
FVlla activated factor VII
Gla y-carboxy glutamic acid
Gp glycoprotein
HEPES N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)
His histidine
HMEC-1 human microvascular endothelial cells, type 1
HRP horseradish peroxidase
H202 peroxidase
H2S04 sulphuric acid
lie interleucin
IPTG isopropyl-0-D-thiogalactoside
KDa kilodalton \'_
Ki inhibition constant
Km Michaelis-Menten constant
LB lubria broth
LOL low density lipoprotein
LMWH low molecular weight heparin
Lp(a) lipoprotein a
Lys lysine
Met methionine
M molar
NaCI sodium chloride
Nal sodium iodide
NaOH sodium hydroxide
NAP nematode anticoagulant protein
OD optical density
PAR protease activated receptor
PAI-1 plasminogen activator inhibitor-1
PBS phosphate buffered saline
PCR polymerase chain reaction
PE phycoerythri n
PEG polyethyleneglycol
PT prothrombin time
RF replicative form
Ser serine
SM skimmed milk
SV40 simian virus 40
TF tissue factor
TFPI tissue factor pathway inhibitor
tPA tissue plasminogen activator
Tris tris(hydroxymethyl)aminomethane
TSP-1 thrombospondin-1
TT thrombin time
Vmax maximum velocity
Il
Vo initial velocity
vWF von Willebrand factor
Xgal 5-bromo-4-chloro-3-indonyl-S-D-galactoside
-,
III
List of Figures
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Figure 2.1 A schematic representation of the coagulation cascade 5
Figure 2.2 Schematic presentation of FVII 9
Figure 2.3 Secondary structure of Gla-domainless human FVII 10
Figure 2.4 Secondary structure of human TF 14
Figure 2.5 Secondary structure of factor Vlla/TF complex 16
Figure 2.6 Structure of TFPI 20
Figure 2.7 Complex formation of the FVlla/TF/FXa/TFPI complex 22
Figure 2.8 Fitamentous phage structure 29
Figure 2.9 Life cycle of M13 phage 30
Figure 2.10 Types of phage display systems 35
Figure 4.1 Dilution ELISA of 7-mere and 12-mere colonies 54
Figure 4.2 Inhibition ELISA of cyclic 7-mere sequence at TF concentrations 55
Figure 4.3 PT's with increasing concentrations of linear 12-mere sequence 56
Figure 4.4 PT with increasing concentrations of cyclic 7-mere sequence 56
Figure 4.5 Prolongation of PT in human plasma 58
Figure 4.6 Prolongation of TT in human plasma 58
Figure 4.7 Platelet adhesion at shear rates (A) 200 S·1 and (B) 1000 s -1 60
Figure 4.8 Platelet adhesion on TF coated coverslips at shear rates 63
(A) 200 S·1 and (B) 650 S·1
Figure 4.9 Platelet adhesion on TF coated coverslips at shears 1300 S·1 64
Figure 4.10 Different substrate concentrations at 0 J-lMpeptide 65
Figure 4.11 Michaelis-Menten kinetics of the inhibition of FVlla by the peptide 66
Figure 4.12 Lineweaver-Burk plot 67
Figure 4.13 The Km value plotted against the peptide concentrations 67
iv
List of Tables
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Table 4.1 Percentage coverage on cover slips at shears of 200 S·1 and 1000 S·1 59
Table 4.2 Percentage of coverage on TF-coated coverslips 61
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v
CHAPTER 1
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INTRODUCTION
Blood coagulation is initiated when injured blood vessels expose blood to
tissue factor (TF) in the subendothelium (fig. 2.1). Coagulation factor VII
(FVII) or activated factor VII (FVlla) present in plasma binds to TF to form the
FVlla/TF complex. This complex activates factor X which on its turn activates
thrombin. Thrombin is responsible for the formation of fibrin and activation of
platelets to form a thrombus.
Despite many years of research using various strategies only two
anticoagulants are in widespread clinical use (Hirsh and Weitz, 1999). These
are coumarins and heparins (Hirsh et aI., 1994; Weitz, 1997). Coumarins
impair the function of the vitamin K-dependent proteins including both
procoagulants (thrombin, factor Xa, factor IXa' and factor Vila) and
anticoagulants (activated protein C and protein S) whereas heparin enhance
the inhibition of thrombin and factor Xa by antithrombin Ill. The non-selective
mode of inhibition of both of these anticoagulants probably accounts for their
therapeutic limitations in maintaining the balance between thrombosis and
haemostasis (Hirsh and Weitz, 1999).
Because it initiates the coagulation process, the FVllalTF complex represents
a good target for developing therapeutic anti-coagulants. Tissue factor
pathway inhibitor (TFPI) naturally inhibits human FVlla/TF. TFPI is a protein
containing three Kunitz domains and it inhibits the FVllalTF complex in a
factor Xa-dependent manner (Broze et aI., 1988).
The elaborate nature of the FVllalTF complex suggests additional approaches
that may impair its function (Higashi ana Iwanaga, 1998). FVII is a vitamin K
dependent glycoprotein, which circulates in blood as a single chain
glycoprotein of 406 amino acids (Kumar and Fair, 1993). Activation of factor
VII by factors IX and X involves the cleavage of an Arg152-lIe153 bond. The
activated factor VII (FVlla) then circulates as a two-chain glycoprotein
composed of a heavy and light chain. The light chain of FVlla consists of the
N-terminus Gla-domain followed by two EGF-like domains. The heavy chain
is the catalytic domain and disulphide bonds link the two chains (Eigenbrot,
2002; Persson, 2000; Banner et al. 1996). FVII makes extended contact to TF
in the FVllarTF complex. The Gla-domain, the EGF-1 domain and the
catalytic domain are involved in the interaction with TF.
TF is a small transmembrane cell surface receptor with an extracellular
domain, a transmembrane domain and a cytoplasmic domain (Spicer et al.,
1987). The extracellular domain consists of two fibronectin type III domains,
TF1 and TF2. The main binding site for FVlla is located at the interface
between the TF1 and TF2 domains (Banner et al., 1996).
Compounds that block the association of TF with FVlla can prevent the
activation of the macromolecular substrate. FXa, and therefore inhibit
coagulation. We designed a study to develop peptide inhibitors of FVlla using
phage display technology.
The major thrust these days is to develop inhibitors against factor X and
thrombin. The fact that FVlla acts higher up in the coagulation cascade than
these two factors, made us speculate that less inhibitor might be needed to
inhibit FVlla. We thus decided on investigating the possibility of developing
inhibitors against FVlla. Although there are no FVlla inhibitors commercially
available as anticoagulants yet, studies on this have been reported (Dennis et
al. 2000; DeCristofaro, 2002; Roberge et al. 2002; Dennis and Lazarus,
1994b).
We selected possible inhibitors of FVlla using phage display technology. The
technique of phage display allows for larqe numbers of phage clones to be
screened. Greater than 109 different sequences can be screened which gives
phage display a major advantage over other methods (New England Biolabs,
2
2000). Different single phage colonies were picked and grown and then their
ability to bind to and inhibit FVlla were tested. We sequenced the FVlla-
inhibitory colonies and decided on one sequence to synthesise. A 7-mere
cyclic peptide was synthesised and characterised by performing prothrombin
times and thrombin times. We also tested the effect of this peptide kinetically
on FVlla-inhibition and also on platelet adhesion to endothelial cells and
tissue factor. We selected a small peptide since small peptides have the
advantage of being non-immunogenic (Markwardt, 1990).
-,
3
CHAPTER 2
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LITERATURE REVIEW
This study focuses on the selection and characterisation of novel FVlla
inhibitory peptides. It is therefore necessary to commence the literature
review with a brief discussion on the mechanisms of blood coagulation.
2.1 Blood Coagulation
Blood coagulation in concert with platelet activation and fibrinolysis is part of
the haemostatic response to injury and serves to maintain the integrity of the
vascular system. It also helps to prevent excessive blood loss through
platelet-fibrin formation. Blood coagulation is initiated when blood vessels are
injured, exposing blood to tissue factor (TF) in the exposed subendothelium
(figure 2.1). TF is produced constitutively by cells beneath the endothelium,
as well as rnonocytes, macrophages, brain -, lung - and placental cells.
Coagulation factor VII (FVII) or activated factor VII (FVlla) present in plasma
binds to tissue factor forming a FVlla/TF complex which activates limited
quantities of factor X (Xa) and factor IX (IXa). TF acts as a cofactor for FVII
activation and enhances the proteolytic activity of FVlla towards its substrates,
factors IX and X (Broze, 1992; Ruf, 1998). Factor Xa in turn activates
prothrombin to form thrombin (Tuddenham, 1996). Tissue factor pathway
inhibitor (TFPI) almost immediately inhibits the FVlla/TF complex after
triggering the coagulation cascade (Figure 2.1). Thrombin accelerates its own
production by activating platelets to provide coagulation surfaces where the
prothrombinase complex, an enzyme complex activating prothrombin in
plasma, assembles in a Ca2+ - dependent reaction. Thrombin also activates
factors Vand VIII to provide the cofactors for factor Xa in the prothrombinase
complex, and for factor X activation by factor IXa respectively. Even when
\.
factor X activation is initiated by tissue factor, efficient propagation of factor X
activation is critically dependent on the factors IXa and Villa enzyme complex
(TENASE complex) (Ofosu et al. 1996). The FVllalTF complex is the
4
essential initiator of blood coagulation, due to activation of FX and FIX not
being detectable in the absence of FVlla or TF (Butenas and Mann, 2002;
Butenas et al. 2000; Davie et al. 1991; Nemerson, 1986).
Extrinsic TENase complex
Factor Vila
Tissue factor Factor IX
Factor X "Phospholipid"
.....u------ Calcium
Factor IXa
1 ................T..F..PI VIntrinsic TENase complexFactor IXa
Factor Villa
"Phospholipid"
Calcium
Prothrombinase
Factor Xa
Factor Va
"Phospholipid"
Calcium Factor XI _____. Factor Xla
Prothrombin --_Jl> Thrombin _,t
1. Fibrinoqen-e-Fibrin
2. Platelet activation
3. Activation of factors Vand VIII
Figure 2.1 A schematic representation of the coagulation cascade. The
boxes represent the components of the vitamin K-dependent complexes. The
top component of each complex is the vitamin K-dependant serine protease
and the second its required cofactor protein. "Phospholipid" represents the
appropriate membrane surface required for precise protein assembly and is
mainly supplied by platelets. Calcium ions stabilise the interactions (Broze,
1992; Meiring, 1996).
5
2.2 Role of platelets in thrombosis and coagulation
Since this study focuses on the characterisation of a FVlla inhibitory peptide,
studying its effect on platelet adhesion, necessitates discussion of the role of
platelets in thrombosis and haemostasis.
Platelets play a major role in haemostatic plug formation following injury to
blood vessels where normally they do not adhere. Several components of the
subendothelium are exposed with vascular injury. They are fibrillar and non-
fibrillar collagens, elastin, proteoglycans, laminin, thrombospondin, fibronectin,
vWF and TF. Platelets are localised to the injury site and adhere directly to
the exposed collagen through their Gplallla receptor, and indirectly via
circulating von Willebrand factor (vWF). Von Willebrand Factor (vWF)
circulating in the blood binds to the exposed collagen following damage to the
endothelium. The vWF subsequently undergoes a conformational change
whereafter platelets can bind to it through their membrane glycoprotein IbN/IX
receptor (GplbN/IX). vWF thus forms a bridge between the exposed collagen
and the receptor.
The tethering of platelets to vWF slows down the movement of the platelets.
These tethered cells roll on the damaged surface and eventually the platelets
become adhered through binding of other recepters. The binding of vWF to
platelets activates the platelets, resulting in a conformational change in
platelet membrane glycoprotein, Gpllb/llla. The conformational change in
Gpllb/llla enables fibrinogen and vWF to bind to it. The fibrinogen and vWF
can then bind to adjacent platelets and this is called platelet aggregation
(Jackson et al. 2000; George, 2000; Ofosu, 2002).
The activated platelet also releases the contents of its CJ.- and dense granules.
These contents help to reinforce platelet activation. The dense granules
release ADP and calcium. ADP is responsible for aggregation of platelets,
and platelets have transmembrane receptors for ADP. Fibrinogen, factor XI,
factor IX, factor V, factor XIII and vWF are also secreted by the activated
6
platelets as well as other adhesive proteins such as fibronectin,
thrombospondin and vitronectin (Ofosu, 2002; George, 2000; Gachet, 2001).
Activated platelets generate thrombin on their surface and the thrombin binds
to transmembrane receptors on the platelets. This leads to further platelet
activation and also aggregation. Thrombin is described as the most potent
activator of platelets and the principle receptor for thrombin is PAR-1 (Ofosu,
2002). Thrombin also activates fibrinogen to fibrin polymers, which stabilises
the haemostatic plug. Activated platelets provide a negatively charged
surface for the coagulation enzyme complexes of the coagulation cascade.
Calcium released from the granules of activated platelets is thus also involved
in these platelet surface coagulation reactions (Ofosu, 2002).
TF is also present on microparticles in circulating blood and in thrombi. This
circulating TF is potentially thrombogenic because thrombus formation was
reduced by the addition of a TF inhibitor to native human blood (Rauch and
Nemerson, 2000). Polymorphonuclear leukocytes and monocytes contain TF-
positive microparticles. These mieroparticles adhere to platelet thrombi.
Leukocytes and monocytes can transfer these TF microparticles to platelets
via an interaction between the cell adhesion molecule CD15 on the leukocytes
and monocytes and P-selectin on platelets. P-selectin is an a-granule-derived
adhesion molecule present on activated platelets and is also known as
CD62P (0sterud, 2001; Giesen et al. 1999; Rauch et al. 2000; Rauch and
Nemerson, 2000). Under normal conditions, most of the TF activity on the
Iymphocyte and monocyte cell membrane is latent or encrypted, implying that
the TF binds to FVlla, but is not capable to initiate coagulation. Platelets play
an important role in the decryption of monocyte TF through the interaction
between CD15 and P-selectin. A mixture of leukocytes and platelets
generates more TF activity than either of these cells alone (0sterud, 2001;
Giesen and Nemerson, 2000). The transfer of TF microparticles to platelets
thus results in platelets being more capable of triggering and also propagating
thrombus growth.
7
2.3 Factor VII
Factor VII (FVII) was first purified in 1975 as a single chain inactive form
(Radcliffe and Nemerson, 1975). The single chain FVII is cleaved by FXa and
thrombin into a double chain active form (FVlla) (Radcliffe and Nemerson,
1975). The total isolation of human FVII was also reported in 1981 (Bajaj et al.
1981; Kisiel and McMullen, 1981).
The essential role of FVII and activated FVII (FVlla) in plasma is to bind to
tissue factor forming the factor Vlla/TF complex. Activated FVII present in
plasma fails to show appreciable enzyme activity until it is brought into contact
with TF (Banner, 1997). FVII is a vitamin K-dependent glycoprotein and
circulates in the blood as a zymogen at a concentration of about 10nM.
Approximately 1% of FVII is present in the activated form (FVlla) (Orninq et al.
1997). Of all the coagulation factors FVII has the shortest lifetime, which is
about 4 to 5 hours. The activated form of FVII (FVlla) circulates for about 21f2
hours (Nemerson, 1988).
2.3.1 Genetics and Structure
The FVII gene has 9 exons on the long arm of chromosome 13, and is located
adjacent to the FX gene (Hutton et al. 1999). FVII is synthesised and secreted
by the liver and circulates as a single chain zymogen of 406 amino acid
residue (Kumar and Fair, 1993). It is synthesised with a pre-pro-leader
sequence of 38 amino acids (see figure 2.2). For the mature protein to be
produced, an arginyl-alanine bond of the pre-pro-leader sequence must be
cleaved (Hagen et al. 1986; Tuddenham and Cooper, 1994).
The mature protein consists of an amino terminus y-carboxy glutamic acid rich
(Gla) domain, a short signal peptide, two epidermal growth factor-like (EGF)
domains, a short activation peptide ane a catalytic carboxyl serine protease
domain (see figure 2.2) (Banner et al. 1996; Hutton et al. 1999). This
zymogen is post-translationally modified to produce 10 Gla residues (Chang
8
et al. 1995). The Gla-domain extends from residue 1 to 35. It mediates
calcium ion binding, which induces a conformational change leading to the
expression of membrane and cofactor binding properties. Calcium ions are
required for the stabilisation of the FVllfTF complex. Residues 37 to 46 form
the short signal peptide, and it consists of an amphipathic helix region,
followed by the two EGF-domains. The EGF domains mediate protein-protein
interactions and are necessary for binding to TF. The short activation peptide
following the EGF-domains is a connecting region containing an arginyl-
isoleucine cleavage site for FXa at residue 152/153. Finally, the catalytic
carboxyl serine protease domain is homologous to trypsin and contains a
catalytic triad consisting of residues His-193, Asp-242, and Ser-344 (Higashi
and Iwanaga, 1998; Broze, 1992; Hagen et al. 1986; Tuddenham and Cooper,
1994). This serine protease domain has two a-helices, and the ends of these
two helices form a concave surface covering a part of the amino-terminus of
TF. In the first helix Arg276 is the main contact residue and the contact
residues are Met306 and Asp309 in the second helix. The Asp309 residue
precedes the Cys310-Cys329 loop (Ruf, 1998; Banner et al. 1996).
activation
pro-peptide signal peptide peptide
'""DLi__ I::L::L /\'---Y.J...-j-"'Il---+-I OD2~~1~t-'---r'-----'
pre-pro- ~ .==;=. ~
leader Gla-domain
sequence two EGF- catalytic-domain
domains (serine protease)
Figure 2.2 Schematic presentation of the domain structures of FVII.
EGF=epidermal growth factor; Gla=y-carboxy glutamic acid (Hutton et al.
1999)
Both factors Xa and IXa activate FVII. FXa, however, is approximately 800
times more efficient than FIXa. Ca2+ and phospholipids are essential for the
9
activation (Masys et al. 1982). Thrombin or FXlla can also activate FVII in the
absence of cofactors (Broze and Majerus, 1981).
serine protease
domain EGF domains
calcium ion
Figure 2.3 Secondary structure of Gla-domainless human FVII. The purple
represents the two EGF-domains and the yellow represents the serine
protease domain. The green represents a calcium ion (Pike et al. 1999)
FXa activates FVII by splicing the peptide bond between Arg 152 and lie 153
of the activation peptide. This results in the formation of a light chain of 152
residues and a heavy chain of 254 residues (Eigenbrat, 2002; Banner et al.
1996). The light chain consists of the amino-terminus Gla-domain followed by
two EGF domains. The heavy chain contains the catalytic domain, which has
a core structure common to all serine proteases of the thrombin/trypsin family.
It is an arginine specific serine protease, therefore it cuts its substrates
proximal to an arginine residue (Banner, 1997; Banner et al. 1996). The heavy
chain is linked to the light chain via a disulphide bridge between Cys-135 and
Cys-262 (Higashi and Iwanaga, 1998). Three different binding regions are
10
contained within the catalytic domain of FVlla. These three binding regions
are: a TF binding region, active site binding region, and a macromolecular
substrate binding region, all three these regions being important for proteolytic
activity (Persson, 2000; Ruf and Dickinson, 1998). Activation of the trypsin-
type serine proteases like FVlla results in formation of a salt bridge between
the amino-terminus a-amino group and the ~-carboxyl group of the aspartic
acid residue adjacent to the active serine. This bridge is essential for the
catalytic activity since it stabilises the active conformational states of the
protease domain. In FVlla this salt bridge is formed between the amino-
terminus a-amino group of Ile-153 and the ~-carboxyl group of Asp-343. It is
important to note that this bridge is not completely formed in FVlla unless it is
bound to TF (Higashi and Iwanaga, 1998).
Mutagenesis showed that trace amounts of FVlla could activate FVII bound to
TF. This auto-activation might be more important for the initial initiation of
blood coagulation than previously considered (Nakagaki et al. 1991;
Neuenschwander and Morrissey, 1992; Thomas, 1947). The conformational
change that FVII undergoes when it binds to TF makes it susceptible to
activation by trace amounts of FVlla as well as other proteases (Nakagaki et
al. 1991). ATIII had no apparent effect on activation of FVII by FVlla, while it
could completely block FXa dependent activation of FVII under the same
conditions. FVlla subjected to auto-activation could generate FXa in the same
way as otherwise activated FVlla (Yamamoto et al. 1992).
FVlla can trigger signalling events in cells via the protease activated G
protein-coupled receptor-2 (PAR2) only in the presence of TF. The other
PAR's, PARi, PAR3, and PAR4 are all activated by thrombin. The factor
VllalTF generated FXa can also cleave PAR2 to trigger transmembrane
signalling (Camerer et al. 2000; Riewald and Ruf, 2001).
11
2.4 Tissue factor
Tissue factor (TF) is a small transmembrane cell surface receptor that triggers
the coagulation cascade. It does not require proteolytic modification to fully
express its activity, thereby making it the primary initiator of coagulation
(Edgington et al. 1991; Tuddenham and Cooper, 1994). Cells normally in
contact with plasma - the blood cells and endothelium - do not express TF
without activation. TF is present in brain, lung and placenta and in the media
and adventitia of blood vessels. It is also found in the bronchial mucosa and
alveolar epithelial in the lung (Ruf and Edgington, 1994). Normally,
monocytes and endothelial cells do not express TF, but these cells are
stimulated to express tissue factor on their surfaces by endotoxin, interleukin-
1, tumour necrosis factor, cytokines and platelets. In the cellular immune
response, monocytes express TF after stimulation by T-helper cells.
Monocytes also express TF in vivo in certain pathological conditions
associated with intravascular coagulation and thrombosis such as
meningococcal infection and peritonitis (Broze, 1992; McVey, 1994; Hutton et
al. 1999; Ruf and Edgington, 1994; Miller et al. 1981; 0sterud and Flaeqstad,
1983; Bajaj et al. 2001). TF is expressed by vascular adventitial cells,
neuroglia, vascular smooth muscle and epidermal cells (McVey, 1994) TF,
therefore forming a protective envelope around blood vessels and organs in
order to initiate coagulation as soon as is necessary (Morrisey, 2001).
2.4.1 Genetics and structure
The gene that encodes TF has six exons and is located on the short arm of
chromosome 1 (Hutton et al. 1999; Edgington et al. 1991). The mature
protein consists of 263 residues and is preceded by a 32-residue signal
peptide (Fisher et al. 1987; Spicer et al. 1987). The TF molecule has an
extracellular domain, a transmembrane domain, and a cytoplasmic domain
(see figure 2.4) (Banner et al. 1996; Spieer et al. 1987). The extracellular
domain consists of two fibronectin type III domains, TF1 and TF2 connected
end to end to form an angle of about 1200 (Morrissey et al. 1997). Both TF1
12
and TF2 domains consist of 219 amino acids. The main binding site for FVlla
is located at the interface between the TF1 and TF2 domains (Banner et al.
1996). The transmembrane domain consists of a 23-residue domain and the
cytoplasmic domain of a 21-residue domain. This domain is crucial for the
anchoring of TF to the membrane and therefore localises the catalytic
initiation of coagulation (Ruf et al. 1991).
Two adjacent lysine residues in TF, Lys165and Lys166,are important for FX
activation (Kelley et al. 1997). These two residues interact directly with the
Gla-domain of FX and are also required for the accelerated inhibition of the
FVlla/TF complex by TFPI mediated by FXa (Huang et al. 1996; Roy et al.
1991; Rao and Ruf, 1995). The structure of the FVllalTF complex will be
discussed in more detail in the next section. TF accelerates the activation of
FVII by FXa, which makes it a bifunctional coagulation cofactor by enhancing
the activity of FVII as a cofactor and as a substrate. TF is thus required for
activation of FX by FVlla and the acceleration of FVII activation by FXa
(Nemerson and Repke, 1985). The primary function of TF is however to.
anchor the complex to the membrane surface by its transmembrane domain
(Krishnaswamy, 1992).
Tissue factor therefore plays a key role in the initiation of blood coagulation
during physiological haemostasis, but it may also be responsible for
thrombotic disorders. It is also involved in processes other than coagulation,
such as intracellular signalling, metastasis, angiogenesis and inflammation.
Tissue factor plays an important role in arteriosclerosis and arterial and
venous thrombosis. TF is also the dominant procoagulant in many tumour
cells ( Pendurthi, 2002; Kirchhofer, 2001; Semeraro and Colucci, 1997).
13
TF2 domain
TF1 domain
Figure 2.4 Secondary structure of the extracellular domain of human tissue
factor. The red represents the TF2 domain and the TF1 domain is
represented by the purple (Huang et al. 1998)
2.5. Factor VllalTF complex
TF and FVlla form a 1:1 complex where TF acts as a cell-surface receptor for
FVlla (Nemerson, 1988). FVlla can only reach its full catalytic potential when
it is in complex with TF in the presence of calcium ions (Bom and Bertina,
1990; Banner et al. 1996). Free FVlla does not recognise factors IX and X.
The binding of TF to FVlla enhances the enzymatic activity of FVlla several
thousand-fold (Broze, 1992). When bound to TF, FVlla undergoes a
conformational change, creating recognition sites for factors X and IX
(Nemerson, 1988). It could be said that FVlla on its own is "zymogen-like";
and when it is in complex with TF it becomes more "active-enzyme-like"
(Banner, 1997). It was thus proposed that FVlla exists in equilibrium and the
binding to TF leads to a shift in this equilibrium towards the active state
14
(Pendurthi, 2002; Higashi and Iwanaga, 1998). TF binds to FVlla with a high
affinity (binding constant (Kb) of about 3nM) and complex formation is thus
very rapid within a wound site (Tuddenham, 1996). Three characteristics are
responsible for the dramatic enhancement of the catalytic function of FVlla.
These are a) facilitation of interaction with FX by localising the reaction to the
phospholipid surface, b) allosteric activation of FVlla, and c) the alignment of
specific regions of FVlla and TF to form a surface for recognition of its
substrate, FX (Ruf, 1998). This will be discussed in some detail hereafter.
FVlla adopts a stretched-out conformation along the elongated TF in the
FVlla/TF complex (Ruf, 1998) (See figure 2.5). Two modules on the light
chain of FVlla make distinct contact with TF. This is the amino-terminus Gla-
domain of FVlla interacting with the carboxy-terminus of TF through
hydrophobic contacts because deletion of the Gla domain of FVlla results in a
decreased affinity for TF, and the Gla-domain is essential for calcium ions,
which in turn is essential for TF binding (Edgington et al. 1997; Banner et al.
1996; Ruf, 1998; Higashi et al. 1996; Nemerson. 1988). The other. module on
the light chain of FVlla in the EGF1 domain packs into a cleft formed in the TF
molecule where the amino- and carboxy-terminals collide. This contact area
is the largest in the complex and accounts for more than 50% of the free
energy of complex formation (Edgington et al. 1997; Ruf, 1998). Arg79, IIe69,
and Phe-71 in the EGF-1 domain are primarily involved in the high affinity
binding to TF. A recent study showed the role of the EGF-1 domain of FVlla
in the conformational change of the active site, following activation of FVII
forming an allosteric linkage between EGF-1 and the active site (Leonard et
a/.2000).
The catalytic (serine protease) domain of FVlla and its tightly associated EGF-
2 domain forms a continuous interface with the amino-terminus module of TF,
and this contact is responsible for the allosteric activation of FVlla's catalytic
function (Edgington et al. 1997; Ruf, 1,998). The catalytic (serine protease)
domain has a low affinity calcium-binding site that is also necessary for
interaction with TF (Wildgoose et al. 1993). Arg304 in the catalytic domain is
IS
implicated in the binding of FVlla to TF, while Arg290 and Lys341 appear to
be critical for proteolytic function and substrate specificity (Ruf, 1994;
Neuenschwander and Morrissey, 1995). The protease domain of FVII and
FVlla docks similarly with TF and the structures of the FVllalTF and FVIIITF
complexes thus are similar (Dickinson and Ruf, 1997).
The interaction between TF and FVlla forms a salt bridge between lIe-153 and
Asp-343 of FVlla, and is part of the acceleration of the catalytic activity of
FVlla by TF. The formation of this salt bridge can also protect the a-amino
group of FVlla from carbamylation (Owenius, 2001; Higashi et al. 1994;
Higashi and Iwanaga, 1998).
Tissue factor
FVlllight
chain
FVII heavy
chain
Figure 2.5 Secondary structure of the complex of active site inhibited human
FVlla with human recombinant soluble tissue factor. The light purple
represents the light chain of FVlla, the yellow represents the heavy chain of
FVlla. The blue and darker purple represents TF (Banner et al. 1996)
16
The importance of the presence of Ca'" and phospholipids in the FVllalTF
complex is that it is responsible for a 15 million-fold increase in the catalytic
efficiency of FX activation (Bom and Bertina, 1990). TF can however form a
complex with FVlla on membranes that only contain neutral phospholipids, but
when acidic phospholipids are also present, the function of the complex is
dramatically enhanced (Mann et al. 1990).
One mechanism, by which TF regulates the activity of FVlla, is to properly
align the active sites of the enzymes above the membrane surface and
therefore phospholipid surfaces. The catalytic activity of FVlla in complex with
TF is enhanced by reducing the conformational mobility, thus a loss of
rotational and transitional freedoms (McCallum et al. 1996; Banner, 1997;
Higashi et al. 1994; Higashi and Iwanaga, 1998). The reactivity of the active
site of FVlla is enhanced by reorientation of the amino-terminus, a
conformational change in the catalytic triad to facilitate hydrolysis of the ester
substrate (Rapaport and Rao, 1995; Higashi et al. 1992).
Another mechanism by which TF regulates the activity of FVlla, is by initiating
allosteric cross talk between the three regions in the catalytic domain of FVlla
in the FVllalTF complex (Ruf and Dickinson, 1998). A short a-helix is situated
at residues 307-312 in the substrate-binding region of the catalytic domain of
FVlla and this helix is distorted in free FVlla. Free FVlla contains a Met-
residue in the 306 position. This differs from the other serine proteases. This
Met-306-residue causes an unstable helix but stabilises after binding to TF,
thus preventing the expression of FVlla activity in the absence of TF.
Stabilising by TF thus optimises substrate binding to the altered FVlla
(Kemball-Cook et al. 1999; Pike et al. 1999). This helix is attached to the
protein body through a Cys31 0-Cys329 disulphide bridge (Pike et al. 1999).
Furthermore, the second ~-strand in the B2-barrel of the FVII zymogen shows
a different registration with the ~-strana in the A2-barrel. In the FVllalTF
complex however, this ~-strand (B2-barrel) has shifted three residues toward
the carboxy-terminus. This shift results in a shorter activation domain loop as
17
in the FVlla enzyme (Eigenbrat et al. 2001; Eigenbrot and Kirchhofer, 2002).
This is another mechanism by which TF regulates the activity of FVlla.
Studies on mouse models have proved that inactivation of the TF gene and
therefore total TF deficiency, resulted in lethality at embryonic stage. This
appeared to be due to a defect in the vascular integrity of the yolk sac.
However, FVII deficiency was proved not to be lethal to the embryo, but the
neonates with FVII deficiency died from haemorrhage within days after their
birth (Rosen et al. 1997; Chan, 2001; Mackman, 2001; Aasrum and Prydz,
2002). It is also known that mice expressing a mutant form of TFPI, in which
the first Kunitz domain is deleted, die between embryonic day and birth. The
few mice that were born with this deficiency died shortly after birth.
Interestingly in this study they showed that diminishing FVII activity in these
mice resulted in rescuing them from intrauterine death. They developed
normally in utero and survived birth. However, postnatal they also died from
bleeding events (Chan et al. 1999; Chan, 2001).
',
18
2.6 Inhibitors of the factor Vlla/TF complex
2.6.1 Tissue factor pathway inhibitor (TFPI)
TFPI is the natural inhibitor of factor Xa and the factor Vlla/TF complex. It
also inhibits the FVllalTF complex in a FXa dependent manner by producing a
feedback inhibition (Wun et al. 1988; Broze et al. 1988; Broze, 1995; Broze
and Miletich, 1987a; Broze and Miletich, 1987b; Broze, 1987).
As early as 1947 TFPI was found in serum, inhibiting coagulation in the
presence of Ca2+ (Thomas, 1947; Schneider, 1947). This inhibitor of the
FVII/TF complex was recognised in 1957 (Hjort, 1957). TFPI inhibits the
enzymatic activity of the FVllalTF complex and not just the activity of TF alone
(Rao and Rapapert. 1987). It was cloned in 1988 (Wun et al. 1988).
TFPI is composed of three Kunitz type domains (see figure 2.6), which are
intervened by linker regions. The linker regions are less structured than the
domains, but is important because the isolated Kunitz domains are less potent
inhibitors compared to the intact full-length TFPI. TFPI has an acidic amino-
terminus region and a basic carboxy-terminal region (see figure 2.5) (Braze,
1995; Wun et al. 1988; Bajaj et al. 2001).
TFPI is synthesised mainly by the endothelial cells under normal conditions
(Bajaj et al. 2001). It is present in three pools in blood. About three percent
of the circulating TFPI is carried in platelets, and platelets release the TFPI
after stimulation by thrombin (Braze, 1992). Ten percent of the TFPI circulates
in plasma in association with lipoproteins. A small amount of full-length TFPI
circulates in free form. The major form of TFPI in association with lipoproteins
is associated with low-density lipoproteins (LOL) and has a molecular weight
of 34kDa. This form of TFPI lacks the distal portion of the full-length TFPI.
This distal portion includes the third Kunitz domain and this form of TFPI is
carboxy-terminally truncated. TFPI associated with high-density lipoproteins
has a molecular weight of 41 kOA and is also carboxy-terminally truncated.
19
The carboxy-terminally-truncated form of TFPI is not as efficient an
anticoagulant as full-length TFPI. The highest percentage of TFPI, 80-85%, is
the full-length TFPI and is associated with the endothelial cell surface by
binding to glycosaminoglycan (Broze, 1995; Bajaj et al. 2001; Broze et al.
1994). TFPI binds specifically and saturably to thrombospondin-1 (TSP-i), a
protein in the a-granules of platelets. TSP-1 is secreted from activated
platelets at sites of vascular injury. The binding between TFPI and TSP-1
thus causes TFPI to efficiently down-regulate coagulation at vascular injury
sites (Mast et al. 2000).
It was shown that lipoprotein a (Lp(a)) binds to TFPI and this binding
inactivates TFPI. Lp(a) is a complex of low-density lipoprotein (LOL) and
apolipoprotein a (apo(a)). The apo(a) portion is most likely involved in TFPI
binding (Caplice et al. 2001).
Figure 2.6 Structure of TFPI with the three Kunitz-type domains (Braze,
1995).
\.
20
The first Kunitz domain binds to the active site of FVlla. The second Kunitz
domain is responsible for the inhibition of FXa, although other parts are also
involved in the interaction with FXa. The carboxy-terminus is required for
optimal inhibition of FXa (Wesselschmidt et al. 1992). The function of the third
Kunitz domain is still unknown. The full-length TFPI had a tenfold higher rate
constant for binding FXa than variants with a truncated carboxy-terminus
(Lindhout et al. 1995). TFPI forms a 1:1 complex with the active site of FXa
and it does not need Ca2+ (see figure 2.7) (Broze, 1992; Rapaport, 1991).
Inhibition of the FVllalTF complex involves the formation of a quaternary
complex (see figure 2.7). This complex contains FXa-TFPI-TF/FVlla and its
formation requires Ca2+ (Rapaport, 1991). The TF and FVlla can be released
from the quaternary complex by decalcification, this thus being a reversible
action (Rapaport, 1991; Broze, 1995; Novotny, 1994; Higashi and Iwanaga,
1998; Bajaj et al. 2001).
TFPI is a slow, tight binding, competitive and reversible inhibitor, and inhibits
as follows:
k, k3
Xa + TFPI <=> Xa/TFPI <=> XaITFPI*
Factor XalTFPI is the final complex and the inhibition constant (Ki) value of
XalTFPI is 30nM (Bajaj et al. 2001).
\
21
.. Kunitz domain
Indentations: active sites of FVlla and FXa
Figure 2.7 Schematic presentation of the proposed mechanism for complex
formation of the FVllaffF/FXafTFPI complex (Girard et al. 1989)
2.6.2 Antithrombin III (ATIII)
Antithrombin III is the natural inhibitor of thrombin. Although antithrombin III is
not an inhibitor of FVlla, the interaction of FVlla with TF, however, enhances
its susceptibility to inhibition by ATIII (McVey, 1994). This inhibition, in turn, is
enhanced by heparin and is irreversible (Broze et al. 1993). The inhibition of
the FVllfTF complex by AT III involves accelerated dissociation of FVlla from
the FVllafTF complex due to destabilisation of the salt bridge in FVlla when
ATIII binds to it. The circulating FVlla-ATIII complexes are then unable to
bind to new cell surface TF receptors (Higashi and Iwanaga, 1998; Rao et al.
22
1995). However, in the presence of sufficient amounts of FVlla, ATIII is not
an efficient inhibitor (Higashi and Iwanaga, 1998).
2.6.3 Nematode anticoagulant proteins
The nematode anticoagulant proteins (NAPs) derived from the
hematophagous nematode Ancylostoma caninum can be used as inhibitors of
blood coagulation. Their use has been based on the observation that
infection by human hookworm can result in significant blood loss. NAP5 and
NAP6 inhibit FXa by binding to its active site. NAPc2, on the other hand,
binds to an exosite, distinct from the active site of FXa, and this binary
complex inhibits the TF/FVlla complex (Johnson and Hung, 1998; Duggan et
al. 1999; Bergum et al. 2001; Stanssens et al. 1996). NAPc2 is functionally
similar to TFPI, but differs in that it binds to an exosite on FXa (Johnson and
Hung, 1998),
2.6.4 Active site inhibited factor Vila
It is interestingly to note that by blocking the active site of FVlla its affinity for
TF is enhanced. This was done by the incorporation of a small peptide
inhibitor (Phe-Phe-Arg chloromethylketone) into the active site. The reason
for this could be that the incorporation of the inhibitor stabilises the protease
domain and increases the number of residues in contact with TF (Sorensen et
al. 1997). There are definite differences between active site inhibited FVlla
and FVlla in their recycling and intracellular compartmentalisation. A fraction
of both recycles back to the cell surface, but the percentage of recycled active
site inhibited FVlla is much higher than FVlla. This means that more FVlla
are accumulated intracellularly (Iakhiaev et al. 2001).
Inactivated active site FVlla was used in studies of thrombus formation on
immobilised TF in a perfusion chamber. v, The results showed that inactivated
active site FVlla did inhibit thrombus formation and the inhibition was dose-
dependent. It also showed that the antithrombotic efficacy of the inactivated
23
active site FVlla depended on shear rate, the efficacy was best at a shear rate
of 650 S·1 and represents arterial blood flow (0rvim et al. 1997).
Inhibition by active site inhibited FVlla was capable of eliminating vascular
thrombus formation at sites of mechanical vascular injury in baboons and rats,
without having an effect on the bleeding time (Harker et al. 1996; Soderstrom
et al. 2001).
The antithrombotic effect of active site inhibited FVlla was also tested in a
rabbit model and it was shown that the effect was prolonged and even
persisted after the active site inhibited FVlla plasma levels were almost
baseline (Golino et al. 1998).
Studies in rats showed that the anti-thrombotic effect of active site inhibited
FVlla can be totally reversed by administration of an equal dose of
recombinant FVlla (Ghrib et al. 2001).
2.6.5 Peptide inhibitors
Kunitz domain variants displayed on the surface of filamentaus phage was
used to select potent active-site inhibitors of the FVlla/TF complex. The
selection was directed against FVlla bound to TF. The inhibitor found differed
from TFPI and ATIII and was named TF71-C. This peptide inhibited the
FVllalTF complex with a Ki of 1.9nM. This Ki represented an increase in
binding affinity of more than 150-fold compared to TFPI (Dennis and Lazarus,
1994a; Dennis and Lazarus, 1994b).
Peptide inhibitors of FVlla have been selected by using the technique of
phage display. Naive polyvalent phage libraries of 20-residues were used.
One selected inhibitor, E-76, is an 18-residue peptide that binds to a distinct
and functionally relevant exosite on the TF/FVlla complex. E-76 binds to the
140s loop of FVlla. This loop stretches from residues 142-153, and its binding
causes a conformational change in the 140s loop. It is a potent inhibitor of FX
24
activation with a median inhibitory concentration of 1nM. Although E-76 does
not bind to the active site of FVlla, it was shown to inhibit amidolytic activity
towards a chromogenic substrate and the inhibition is TF-dependent. Kinetic
analysis indicated that E-76 is a non-competitive inhibitor of FX, reducing the
maximum reaction velocity (Vmax), but showing no effect on the Michaelis
constant (Km) (Dennis et al. 2000; DeCristofaro, 2002).
Another inhibitor A-183 is a peptide that also binds to an exosite of the
protease domain of FVlla. It binds to the 60s loop. This peptide is a partial
mixed-type inhibitor of FX activation with a Ki of 200pM (DeCristofaro, 2002;
Dennis et al. 2001; Roberge et al. 2001).
These two peptides, E-76 and A-183, were linked to create a bifunctional
peptide. Compared to their individual inhibition activity, the combined
peptides showed stronger inhibition of TF-dependant coagulation. A
combination of the two peptides in a 1:1 molar ratio showed complete
inhibition of FX activation as compared to 78% and 92% of the two peptides
separately (Roberge et al. 2002).
Another potent inhibitor was developed against loop I (91-102) of the second
EGF-domain of FVlla. This is a cyclic peptide that disrupts the essential
interaction between the second EGF-domain and the catalytic domain of
FVlla. This peptide was named PN7051 and is a dose-dependent inhibitor of
FX activation by factor Vlla/TF complex with an ICsDvalue of 1O~lM. The
inhibition of this peptide on thrombus formation was also tested in an ex vivo
experiment with a perfusion chamber. The peptide showed inhibition of fibrin
deposition, platelet/fibrin adhesion, platelet/thrombus volume, and thrombin
activation. The overall ICsD in this case was 0.5 mM. This peptide thus
proves to be capable of inhibiting complete thrombus formation at sufficiently
high concentrations (Órninq et al. 1997; Orning et al. 2002).
25
2.7 Phage display
2.7.1 Introduction
The technique of phage display is used in this study, and for purposes of
clarity I will discuss this technique in some detail.
Phage display technology describes an in vitro selection technique
(biopanning) in which a peptide or protein is genetically fused to a coat protein
of a bacteriophage. This results in the display of the fused peptide or protein
on the exterior of the phage, while the DNA encoding the fusion resides in the
virion (Smith, 1985). The technique allows for large numbers of phage clones
greater than 109 different displayed sequences to be screened, this being the
major advantage over other methods (Smith, 1985). The selection technique,
biopanning, is carried out by incubating the pool of phage-displayed variants
with the target protein to select phage clones that bind .or inhibit the target
protein. Phage display is therefore a very powerful technique since it links
peptide or protein display with genetic information, i.e. the displayed peptide
or protein allows rapid selection and the genetic information allows reliable
amplification of the phages.
Phage display technology is used for a variety of purposes, which include
mapping of epitopes, identification of antagonists and agonists for target
molecules, engineering of human antibodies, optimising of antibody
specificities, and creation of novel binding activities (Kayand Hoess, 1996). A
number of applications in which phage display has been used, have been
described including epitape mapping (Scott and Smith, 1990), mapping
protein-protein contacts (Hong and Boulanger, 1995), and the identification of
peptide mimics of non-peptide ligands (Devlin et al. 1990). Peptides have
been identified by either panning against an immobilised purified receptor or
against intact cells (Doorbar and Winter, 1994). Larger proteins such as
antibodies (Barbas et al. 1991), hormones (Lowman et al. 1991), protease
inhibitors (Roberts et al. 1992) and DNA binding proteins (Soumillion et al.
26
1994) have been displayed on phages and resulted in isolation of variants
with altered specificity or affinity.
2.7.2 Structure and genome of the filamentous phage
The filamentous phages M13 and fd are mostly used for phage display, mainly
because they are not lysogenic towards their hosts (Messing, 1983). The
M13, fd, and f1 phages are all part of the Ff class of filamentous phages
(Webster, 2001). The phages have a filamentous shape containing a single
stranded closed circular molecule of DNA (Arza and Félez, 1998).
M13-filamentous and fd-phages infect Escherichia coli (E.coli) containing the
F-conjugate plasmid, as it codes for the extracellular F-pili which serves as
receptors for the phage (Rodi and Makowski, 1999). M13 is the best-studied
member of this class and therefore I will concentrate on this phage type in
further discussion (Kornberg, 1980). M13 phage is approximately 6.5 nm in
diameter and 930 nm in length. The length is dependent on the length of the
enclosed genome (Rodi and Makowski, 1999; Webster, 2001). The genome
of the M13 phage is a single strand covalently closed DNA molecule, which
consists of about 6400 nucleotides. A flexible protein cylinder encases the
DNA molecule (Webster, 2001). This DNA molecule codes for 11 genes.
Genes Ill, VI, VII, and IX code for the minor coat proteins, while gene VIII
codes for the major structural protein (Webster, 2001) (see figure 2.7). Genes
I, IV, and XI code for the assembly of the phage and genes II and X for DNA
replication (Webster, 2001).
Coat protein gplll, a 406 amino acid polypeptide, is located at the proximal
end of the virion. Four to five copies are present. (Figure 2.7) This protein is
necessary for the infection of bacterial cells (Smith, 1985; Makowski, 1994).
In phage display technology, the amino-terminus of gplll is used for the fusion
of short peptides. All of the 5 copies of..gplll can be fused to short peptides;
therefore these peptides are present at low valency, 1-5 copies per virion
(Wilson and Finlay, 1998; New England Biolabs, 2000).
27
Coat protein VI is also located at the proximal end of the virion. Four to five
copies of minor coat protein VI are present (Wilson and Finlay, 1998). This
coat protein consists of 113 amino acids and in this case the carboxy-terminus
is exposed (Makowski, 1994). Gp VI is required for the attachment of gplll to
the phage.
The major coat protein VIII (gpVIII) forms a thick flexible cylinder around the
single-stranded viral genome and there are ± 2800 copies present (see figure
2.7) (Makowski, 1994; Webster, 1996). It is a 50 amino acid protein and
about 10% of the 2800 copies can be fused to short peptides at the amino-
terminus (Makowski, 1994; New England Biolabs, 2000). The display of the
peptides or proteins in M13 can have different valencies of copies. This
valency is dependent on the site of display and type of vector (Armstrong et
al. 1996).
Minor coat proteins, gp VII and gp IX, are situated on the distal end of the
virion. Four to five copies of each of gpVl1 (a 33 residue protein) and gplX (a
32 residue protein) are present (Webster, 2001). These proteins are
responsible for the maintenance of the phage stability and have not yet been
employed for phage display (Rodi and Makowski, 1999; Wilson and Finlay,
1998).
28
Distal end ...
~ gplX
I gpVII
single stranded
genome °gplX
0gpVII
gpVIII
°gpVI
~ gplll
Proximal end _.
Figure 2.8 Filamentaus phage structure. The single-stranded circular
genome is surrounded by gpVIII. (Wilson and Finlay, 1998)
2.7.3 Life cycle of M13 phage
The life cycle of the M13 phage starts by infecting Ecoli. cells. This is done
by binding of the amino-terminus of gplll to the F-pilus of the Eeoli. cells. The
phage then penetrates the F-pilus of the Ecoli. cell and in this process gpVIII
is stripped off in the cell membrane. The minor coat protein, gplll, remains
attached and guides the phage in the infection process. The replicating
enzymes of the host cell convert the single-stranded DNA of the phage into a
double-stranded circular form (Figure 2.9), the so-called replicative form (RF
form). No viral product is synthesised during this phase. The viral DNA is
29
always indicated as the (+) strand. The complementary strand, the (-) strand,
contains the coding information.
0 host enzymes <0 replicationIll> Ill> <0
infecting ss viral duplex DNA
genome replicative form <
e l
0
gene II nicks
(+) strand
gene II nicks
completed
(+) strand
... c~C. »)<::) ...circularisationof completed I~ rolling circle ©(+) strand replication
Figure 2.9 Life cycle of M13 phage (Blaber, 1998)
The next phase of the life cycle is the replication phase where transcription
starts and it always occurs in the same direction. Two terminators for
transcription are located at the ends of genes IV and VIII. Transcription
occurs in a cascade form. Gene VIII is transcribed frequently, whereas gene
III is transcribed in small amounts (Messing, 1983; Blaber, 1998).
In the next phase, the gene II product is synthesised after completion of the
synthesis of the complementary strand. This protein is responsible for
introducing a "nick" in the (+) strand and therefore initiating the (+) strand
synthesis. DNA polymerase is responsible for extending the (+) strand at the
3'-OH end. The complementary strand is now used as template to synthesise
a new copy of the (+) strand. After the (+) strand synthesis has made one trip
around the (-) strand, gene II introduces another "nick" in the (+) strand. This
separates the parental (+) strand from the newly synthesised (+) strand. The
(-) strand continues to serve as template for the synthesis of new (+) strands.
30
The parental (+) strand is then circularised and can be converted again to the
RF form. Gene V protein levels increase as the RF molecules accumulates in
the cell. This protein binds to the (+) strand and prevents conversion of the
newly synthesised single (+) strands to the RF form. This leads to the build-
up of circular single stranded (+) DNA, the M13 genome. This complex of
single stranded (+) DNA and gene V protein now moves to the membrane
where the gene V protein is replaced and gpVIII covers the phage DNA again
as it is being extruded out. The release of the phage does not require the F-
pilus (Messing, 1983; Blaber, 1998).
2.7.4 Phage display of peptides and proteins
The gplll coat protein is most commonly used for phage display. The M13
phage vector is modified and carries the lacZ gene. When the lacZ gene is
present, phage plaques appear blue when grown on agar plates containing
bromo-4-chloro-3-indoyl-~-D-galactoside (XGal). The presence of white
plaques suggests contamination. The lac Z gene is used to distinguish
phages foreign inserts from those without inserts (Armstrong et al. 1996).
Foreign peptides are fused to the chosen structural coat protein, mostly gplll
of the phage, by inserting the corresponding DNA sequence into the gene
coding for the coat protein. The expression of the fusion protein and the
subsequent incorporation into the mature phage particle will result in the
display of the foreign peptide on the surface of the phage (see figure 2.9).
Phage libraries are constructed by inserting random oligonucleotide
sequences in the phage genome. This results in the display of random
peptides on the phage surface and therefore allows selection of peptides with
specific affinities or activities (Makowski, 1994) (Figure 2.8). The most
common type of phage libraries are random peptide libraries. In these
libraries the DNA inserts are derived from degenerate oligonucleotides with a
NNK sequence where N is an equal mixture of A, G, C and T. K is an equal
mixture of G and T. The degenerate oligonucleotides are synthesised
chemically by adding mixtures of nucleotides. Each NNK is therefore a
31
mixture of 32 triplets that include codons for all 20 natural amino acids (Smith
and Petrenko, 1997).
The M13 phages are used for the display of not only foreign peptides, but also
proteins (Makowski, 1994). Phage display has made engineering of protein
properties possible, without a detailed knowledge of the structure-function
relationship (Katz, 1997). Phage display is also being used to identify highly
active substrates by including a "tether" sequence recognised by monoclonal
antibodies (Smith et al. 1995).
2.7.5 Phage display systems
The different phage display systems can be classified according to the
arrangement of their coat protein genes. The display sites, which are most
commonly used, are within genes III and VIII, however gene VI has also been
used for display (Armstrong et al. 1996).
The phage display systems are differentiated on the basis of the coat protein
used for display, whether fusion can be to all copies or only a fraction, and
whether the fusion is encoded on the phage genome or on a separate
genome. This classification was made by George Smith (1993). The valency
of the display can vary between one and thousands of copies and is
dependent on the vector type.
The type 3 and type 8 vectors are the simplest phage display vectors. The
basic functions of these vectors have not been changed with the display of
peptides/proteins. A single genome bears a single gene III in type 3 vectors
and this single genome accepts foreign DNA inserts and therefore encodes a
single type of pili molecule. The same applies for type 8 vectors, however
short peptides and a variety of proteins can be displayed at the N-terminus of
pili, but pV1I1 can display only short peptides. This is called multivalent
display, because the phage contains multiple copies of the inserted
peptide/protein and is used for the selection of phage with relatively low target
32
affinity. One limitation of multivalent display is that only small peptides can be
displayed because larger inserts can interfere with the function of the coat
protein. This leads to a poorly infecting phage (Lowman et al. 1991; Alien et
al. 1995, Armstrong et al., 1996; Phizicky and Fields, 1995).
Some of the sequences are not displayed well enough on the type 3 and 8
systems which leads to the development of two other systems, types 33 and
88 (Smith and Petrenko, 1997).
In these vector types, 33 and 88, the genome bears two different copies of
gene III or VIII and this encodes two different types of coat proteins. Only one
of these gpVIII or III is recombinant and bears the DNA insert, the other coat
protein is the wild-type.
In another vector type, 3+3 and 8+8, there are also two different copies of the
genes, but in this case the two genes are on different genomes. The wild-type
version is on a phage and the recombinant version is on a phagemid. The
wild-type version is also called a helper phage. In these tYPE?susually only a
single copy of the peptide-bearing protein is displayed, thus both the helper
phage and the phagemid will have only one copy of the peptide. A major
advantage of monovalent display is that apart from the mutant coat protein
provided by the phagemid, the helper phage then supplies a large excess of
the wild-type coat protein, resulting in phages with good infectivity (Smith,
1997; Armstrong et al. 1996). In monovalent phage display, where only one
copy of the inserted peptide/protein is displayed on the phage, the conditions
can be designed for the selection of high-affinity interactions. The different
display systems are shown in figure 2.10 (Lowman et al. 1991; Alien et al.
1995, Armstrong et al., 1996; Phizicky and Fields, 1995).
Foreign peptides have also been fused to pVI. This fusion however, takes
place at the C-terminus of coat protein VI (Jespers et al., 1995). A major
advantage of pVI display is that pVI is not involved in phage infection and the
C-terminus of the coat protein is exposed on the surface, therefore the
presence of stop codons will not prevent the display of proteins (Hufton et al.
33
1999; Amery et al. 2001). Jespers et al. (1995) and Hutton et al. (1999) found
that pVI is suitable for the cDNA expression. There are also the different
types of pVI display. The types are type 6, 66 and 6+6 (Smith and Petrenko,
1997).
Proteins are also displayed on A phage. Peptides or proteins are fused to the
amino terminus of the 0 protein (11 kDa) of the A capsid. The fusions then
assemble onto the viral capsid. The A phage display system has the
advantage of efficient construction and maintenance of very large libraries. A
unique feature of the A system is that the displayed peptides/proteins does not
need to be secreted across the membrane because the virus assembles
intracellularly prior to the release of particles (Maruyama et al. 1994;.
Sternberg and Hoess, 1995).
34
Type 3 [:ISE:::I::~
Type8 mn c::::J gene IIIc::J foreign DNA
_ gene VIII
[:IZE::::r::t o displayedType 33 peptide
• pVIII
l:::w.::2$I:l ", piliType 88
helper phage
[::e:::::z::t ['---I'1-1-) ~+Type 3+3
+
Type 8+8 l:::w.:::ê:::J::l
Figure 2.10 Types of phage display systems (Smith and Petrenko, 1997)
35
2.7.6 Applications of phage display
In the field of thrombosis and haemostasis, phage display has been used in
the isolation of a peptide antagonist to the thrombin receptor (Doorbar and
Winter, 1994). A constrained peptide library was used to isolate ligands of the
allb~3 integrin, the platelet receptor for fibrinogen. This was done by flanking a
library of hexapeptides by cysteine residues to introduce a degree of
conformational constraint into random peptides (O'Neil et al. 1992). A random
cyclic heptapeptide library was used to characterise the peptide binding
specificity of the a5~1 integrin, the fibronectin receptor on platelets (Koivunen
et al. 1994). Furthermore a phage display library of plasminogen activator
inhibitor (PAl-I) mutants was used to determine the interactive sites between
PAl-I and thrombin, and also between PAl-I and the tissue-type plasminogen
activator (tPA) variable region 1 (Van Meijer et al. 1996). Three APPI Kunitz
domain libraries were used to select potent active-site inhibitors of human
FVllalTF complex as well as competitive phage selection on the same three
libraries. The selection conditions were altered in this competitive phage
selection technique. FXla, plasma kallikrein, and plasmin was included in the
selection, and Kunitz domain phage that specifically binds to immobilised
TF/FVlla complex were selected and enriched, and the phage that binds to
the other proteases were eliminated (Dennis and Lazarus, 1994a; Dennis and
Lazarus, 1994b).
A phage-epitope library was used to identify a ligand peptide mimicking the
conformation dependent epitope for a monoclonal antibody (mAS 5.5). This
antibody is directed against the ligand-binding site of the nicotinic
acetylcholine receptor (Balass et al. 1993). Phage display was also used to
localise the epitopes of monoclonal antibodies that were raised against human
pro-enkephalin (Sottger et al. 1995). Epitopes were also localised in
polyclonal antibodies (Dybwad et al. 1995). Winthrop et al. (2000) describes
gene libraries, which can be used for antibody-based binding peptide
modules.
36
There are several examples of studies where phage display technology was
used to identify peptide ligands for recepters. Ligands were identified for the
antigen-binding site of the surface immunoglobulin receptor of the human
Burkitt lymphoma cell line SUPB8. Potent ligands were identified for the
human urokinase receptor and phage display was also used to localise
epitapes for the binding protein, somatostatin (Renschier et al. 1994; Goodson
et al. 1994; Wright et al. 1995).
Furthermore phage display was also successfully used to isolate three
peptides that interacted with the HIV-1 nucleo-capsid protein (NCp7) (Lener et
al. 1995). All these examples show the broad spectrum of applications for the
phage display technique
Phage display selection has also been performed against whole cells such as
blood leukocytes, CHO cells to isolate antibodies against antigens on the cells
(De Kruif et al. 1995; Hoogenboom et al. 1999). and insect cells. Successful
selection of peptides using phage display against mouse melanoma cells has
also been described (Szardenings, 1997). Pasqualini and Ruoslahti (1996)
reported on the approach of organ-selective targeting based on in vivo
screening of random peptide sequences. A new phage display technology
has also been described, this is phage selection using ligand identification via
expression (LIVE). This technique combines phage biology with functional
selection of altered cell function through gene transfer and could be a
powerful tool for identifying naturalligands (Larocca and Baird, 2001). Finally,
phage display is now a well-established tool for research (Cortese et al. 1995)
and it shows potential in the discovery of new drugs as well as the
development of new vaccines (Arza and Félez, 1998; Wittrup, 1999; Sidhu,
2000).
Sometimes the recombinant libraries have a poor display of some peptides or
poor production of phage clones displaying peptides. Other disadvantages
are the fact that some of the binding peptides could be missed because of
overpanning. Phage display also has the disadvantage that it is not suitable
37
for the selection of protein that requires posttranslation modification (Scott,
2000; Silverman, 2000).
Another method that can be used to identify protein-protein interactions is the
yeast two-hybrid system. This method, however, also has its limitations.
Proteins that cannot fold correctly in the cytoplasm may not be suitable for use
in this system while it has been described that proteins can fold correctly on
phage. This occurs when phage is expressed in E.coli that are severely
defective in disulphide bond formation. Interactions such as glycosylation and
disulfide bond formation might not occur in the two-hybrid system because the
proteins that are generated are targeted to the nucleus. It was also found that
some hybrid genes could be harmful or lethal when they are expressed in
yeast (Alien et al. 1995; Bardweil et al. 1991). In 2000 a bacterial two-hybrid
selection system was described for the studying of protein-DNA and protein-
protein interactions. This system has advantages over the hybrid system in
that it can analyse libraries larger than 108 in size and it also has a faster
growth rate (Jqung et al. 2000).
38
CHAPTER 3
¥tiff€t@ ItQéa>QhlcW;;.414JQ!k'ihFim~4;*$i?·*S'Ijf&M#-m :r -@'.Wtê¥%I4a'w,,@ MI§" ·Ii ;a#lêiZfNjI'tM2&Mll@$ @:BWI!§## '7' m
MATERIALS AND METHODS
American Diagnostica Incorporated, Greenwich, CT, USA kindly supplied the
recombinant human FVlla (cat # 407rec/N), tissue factor, murine monoclonal
antibody against human FVlla and the chromogenic substrate, Spectrozyme
FVII as part of our collaboration agreement with them. General laboratory
reagents were of analytical grade and purchased from various suppliers as
outlined in the text.
3.1 Phage display
3.1.1 Phage Display Peptide Libraries
We used two peptide libraries, a cyclic 7-mere Phage Display Peptide Library
Kit (Ph.D.-C7C cat. # 8120) and a linear 12-mere Phage Display Peptide
Library Kit (Ph.D.-12 cat. # 8110). The kits were purchased from New England
Biolabs, Beverly, MA, USA. Both libraries are gplll fusions with 5 copies of
the fused peptide per phage. The cloning vector for both is M13KE (New
England Biolabs, 2000).
The Ph.D.-12 peptide library kit includes a combination library of random 12-
mere peptides fused to the minor coat protein, gplll of M13 phages. The 12-
mere peptides are expressed at the amino-terminus of gplll. This 12-mere
library contains approximately 2.7 x 109 electroporated sequences. These
sequences were amplified once to yield about 55 copies of each sequence in
1O~LoI f the phage library (New England Biolabs, 1998b).
\.
The Ph.D.-C7C peptide library kit includes random 7-mere peptide
sequences, which are flanked by a pair of cysteine residues. This cyclic 7-
39
mere library is therefore also a combination library of random 7-mere peptides
fused to gplll. The cysteine residues will spontaneously form a disulphide
cross-link under nonreducing conditions. This will result in the display of
cyclic peptides, in contrast to the linear peptides displayed in the 12-mere
Ph. 0.-12 library. The cyclic peptides are also displayed at the amino-terminus
of gplll. This library contains approximately 3.7 x 109 electroporated
sequences. These were also amplified once to yield about 50 copies of each
sequence in 1Oul of the phage library. An advantage of the cyclic library is
that binding is more tightly than in the linear library. It was also shown that the
cyclic libraries show more stringent sequence specificity than the linear ones
(New England Biolabs, 1998a; McLafferty et al., 1993).
The E.coli host strain ER2537 was supplied with the library kits. This strain is
a robust F+ strain with a rapid growth rate.
3.1.2 Biotinylation of FVlla
FVlla was biotinylated with sulfo-NHS-LC-biotin (74 ~Lgbiotin/1 00 ~LgFVlla)
(Pierce, Illinois, USA) for 2 hours at 4°C. To separate the biotinylated factor
Vila from the free biotin, the solution was run through a PO-10 sephadex
column (Whitehead Scientific, RSA). Twenty fractions of 500 ul each were
collected and the protein concentration of each fraction measured at 280 nm.
The peak protein fractions were pooled and the protein concentration
determined by using the Biorad method (Sambrook and RusselI, 2001).
The percentage biotinylation was measured with a calorimetric assay based
on displacement of the dye HABA [2-(4'-hydroxyazobenzene)benzoic acid]
according to the instructions of the manufacturer (Pierce, Illinois, USA cat. #
28010).
40
3.1.3 Biopanning
We used two methods of biopanning to select FVlla-binding phages
(biotinylated and direct method). In the first method we incubated 2.1011
phages of each library with 10 ~lgof biotinylated FVlla. By using streptavidine
magnetic beads, we could separate the FVlla-binding phages from the
unbound phages. In the second method we coated 20 ~lg FVlla to the inside
of an immune-tube.
The first method (biotinylation method) was done as follows:
Streptavidine magnetic beads (1.5 mg; Roche Molecular Biochemicais,
Mannheim, Germany) were first washed twice with 200 ~tI PBS. The beads
were blocked with 500 ul of 2% Skimmed milk (SM) (DIFCO Laboratories,
Detroit, USA) in PBS for one hour at room temperature (tube 1). After
blocking, 170 ~ll of this mixture was removed and placed in another tube to
which 2.1011 phages of each phage library (C7C or L12) were added in a final
concentration of 2% SM and incubated for an hour at room temperature while
rotating. Biotinylated FVlla (10 ~lg) and SM were added to the other tube in a
final concentration of 2% SM and incubated at room temperature for 30
minutes. The phage solution was then put on the magnet and the phages that
did not bind to the magnetic beads were added to the tube with the
biotinylated FVlla and rotated overnight at 4°C. In this way the non-specific
binding phages that bind only to the magnetic beads were removed. The next
day, the phage and FVlla solution were put on the magnet and the phages
that bind to FVlla will stay bound to the beads while the non-binding phages
were removed and kept as the "INPUT" phages. The beads were washed 10
times with 0.1% Tween-20 in PBS and the FVlla-binding phages were eluted
non-specifically with 0.5 ul of 0.2 M glycine (pH2) solution (a high acid
solution) for 15 minutes at room temperature. The tube was placed on the
magnet and the supernatant was added-. to another tube containing 125 ~tI of
Tris (pH 8) to neutralise the low pH. These phages we called the "OUTPUT"
phages, they were the phages that bound to FVlla.
41
In the second method, we coated 20 ~lg of FVlla to the inside of a Maxisorb
immune-tube (Nunc, IL, USA) by adding a solution of FVlla in PBS (20 ~lg
FVlla/1 ml PBS) to the immune-tube and rotated it for 2 hours after which we
incubated it overnight at 4°C. The next day we removed the non-binding
phages ("INPUT" phages) and eluted the FVlla-binding phages from the tube
non-specifically also with 0.5 ul of 0.2 M glycine (pH 2) for 15 minutes. To
neutralise the low pH, we also added 125 ~d Tris (pH 8) These are the
"OUTPUT" phages.
Simultaneously, a pre-culture of ER2537 Ecoli cells was diluted 1:100 in 40
ml Lubria Broth-medium (LB-medium, DIFCO Laboratories, Detroit, USA) and
incubated at 3YOCfor about 2 hours until the Ecoli cells were in the log-phase
of growth, i.e. an 00600 of between 0.6 and 1.
We made dilutions of the "INPUT" and "OUTPUT" phages and plated it out on
Lubria Broth/ isopropyl-~-D-thiogalactoside/5-bromo-4-chloro-3-indolyl-~-D-
. galactoside plates (LB/IPTG/XGal plates). This was done by diluting ten ul of
the "INPUT" and "OUTPUT" phages in LB-medium. The "INPUT" phages
were diluted up to 10-9 and the "OUTPUT" phages to 10-4. Two hundred ~LIof
the log-phase Ecoli cells were added to 10 ul of each of the last 4 dilutions of
"INPUT" and "OUTPUT" phages and plated out on LBIIPTG/XGal plates which
were incubated at 3YOCovernight. LBIIPTG/XGal plates were used to ensure
phage colonies that contain the random DNA inserts, were picked (in the next
section) because phages with inserts contains the lac Z gene and such
colonies colour blue in the presence of XGal.
The "OUTPUT" phages were amplified by infecting 40 ml of log-phase E coli
cells and incubating it overnight at 3YOC. The next day the infected cell
cultures were centrifuged at 23 000 g for 15 minutes at 4°C to remove the
Ecoli. The phages in the supernatant were precipitated on 20%
Polyethyleneglycollsodium chloride (PEG/NaCI, PEG, 0.03 M, NaCl, 2.5 M)
for two hours. Following centrifugation, the phage pellet was dissolved in 1 ml
42
PBS and precipitated again on 20% PEG/NaCI. The amplified phages were
finally dissolved in 0.5 ml PBS and the phage concentration determined by
reading the OD at 260 nm and calculating the phage concentration
(phages/ml) as follows:
Amount phages/m I = 00260 x dilution x constant
where the constant is 2.214.1011 since the 00260 of 2.214.1011 phages is
equal to 1. We used 2.1011 of the purified phages for the next round of
selection. Three rounds of selection were done all together. During the
second and third selection rounds, we eluted the FVlla binding phages
specifically with 10 ~lg of Murine antihuman FVlla, a monoclonal antibody
against human FVII/FVlla (ADI, Greenwich, CT, USA). This antibody binds to
the catalytic site of FVlla and blocks the assembly of the FVlla/TF complex
and it also blocks FVlla mediated plasma coagulation.
3.1.4 Global Elisa
A global ELISA was performed on the amplified phages of each round of
panning. The initial phage concentration on the ELISA was 5x1010 phages
and these phages were then diluted 1:2 until a concentration of 7.81 x108.
Half of an ELISA plate (48 wells) was coated overnight with 20 ~lg/ml FVlla at
4°C. The plate was blocked with 4% SM in PBS for 2 hours at room
temperature and washed with 3 times 0.1% Tween-20 in PBS. 5.1010 phages
of each round was added to the first well of each column of the coated and
non-coated part of the ELISA plate in a final concentration of 2% SM and
diluted 7 times 1:2 into the other wells of the columns. No phages were
added to the last well of each column. These wells served as a negative
control. We added the same amount of phages to the non-coated wells as to
the coated wells to make sure that we have not selected plastic-binding
phages instead of FVlla-binding phages. After incubation for 2 hours at room
temperature, the plate was washed 9 times with 0.1% Tween-20. A
horseradish peroxidase conjugated anti-M13 phage antibody (Amersham
Pharmacia Biotech, NJ, USA), was added and incubated for 1 hour. The
-D
substrate, orto-phenyldiamine-dihydrochloride (OPO) and peroxidase (H202)
was added after 12 wash steps and incubated at room temperature for 10
minutes to develop a colour reaction. The reaction was stopped with 1 M
H2S04. The optical density was read at 490 nm on an EL312e Microplate Bio-
Kinetics reader (Bio-tek instruments, Vermont, USA).
The global ELISA indicated that we have selected the highest concentration of
FVlla-binding phages in round 3 since the 00490 of the round 3 phages was
the highest. We thus grew single phage colonies of the third selection round
to search for displayed peptides that bind to and inhibit FVlla.
3.1.5 Growing and amplification of single colonies
We picked one hundred and fourty-four (3 times forty-eight) colonies of each
library from the IPTG/XGal plates of the third selection round. They were
grown in 2 ml of a 1:50 diluted pre-culture of E.coli cells in LB-medium in a
culture tube overnight at 3?OC. Half of each culture was centrifuged and the
supernatants containing the amplified phages were used for an ELISA to
determine which colonies bind to FVlla (binding ELISA). A cell stock was
prepared with the other half of the cultures. It was mixed with glycerol (35%)
and frozen for later use.
3.1.6 ELISA to identify FVlla binding phage colonies (Binding ELISA of
single colonies)
In order to distinguish between actual FVlla binders and "plastic" binders,
each colony was tested in a FVlla-coated and non-coated well. Half of an
ELISA plate (48 wells) was coated overnight with 20 ~lg/ml FVlla at 4°C. The
plate was blocked with 4% SM in PBS for 2 hours at room temperature and
washed 3 times with 0.1% Tween-20 in PBS. 100 ~I of the supernatant of
each grown colony of round 3 was added to a coated and non-coated well of
the ELISA plate in a final concentration of 2% SM, incubated for 2 hours at
room temperature and then washed 9 times with 0.1% Tween-20. A
44
horseradish peroxidase conjugated anti-M13 phage antibody (Amersham
Pharmacia Biotech, NJ, USA), was added and incubated for 1 hour. The
substrate, orto-phenyldiamine-dihydrochloride (OPD) and peroxidase (H202)
was added after 12 wash steps and incubated at room temperature for 10
minutes to develop a colour reaction. The reaction was stopped with 1 M
H2S04. The optical density was read at 490 nm on an EL312e Microplate Bio-
Kinetics reader (Bio-tek instruments, Vermont, USA). Wells with the most
intense colour indicate either the strongest binding phages or a high
concentration of weak FVlla-binding phages.
Since we only found 6 colonies of each library that had high colour intensity,
we decided to grow all of them up from the cell stock and tested them for
concentration dependant binding to FVlla in a dilution ELISA.
3.1.6.1 Dilution ELISA
We diluted (1:2 dilution) the 12 colonies (6 of each library) that was grown up
and tested them for concentration dependent binding to FVlla. The highest
phage concentration was 2.5.1010 and we diluted it 1:2 as in the global ELISA.
Each colony was also diluted on non-coated wells to distinguish between
FVlla-binding phages and non-specific plastic binding phages. The rest of the
ELISA was done the same way as the global ELISA. We repeated this ELISA
three times.
3.1.6.2 Inhibition ELISA
This ELISA was performed do determine whether TF was able to prevent the
12 strongest binding colonies from binding to FVlla. Different concentrations
of TF were added to FVlla coated wells. American Diagnostica Inc.
(Greenwich, CT, USA) kindly supplied TF. After incubation for 15 minutes,
5.1010 phages in 2% SM (off all 12 colonies) were added to each well of a
column and incubated for 2 hours. Bound phages were detected after 1 hour
of incubation with the anti-M13 antibody, visualisation done with OPD and
45
H202 and absorbency measured at 490 nm. Only one colony from the cyclic
7-mere library showed inhibition and we repeated the inhibition ELISA with
this colony three times.
3.1.7 Prothrombin time (PT)
The effect of a dilution series of phage colonies on the PT was determined.
Phages of the 12 strongest binding colonies (6 from each library) were added
to human plasma. For the control, PBS was added to the plasma. A negative
control was also performed where a non-binding phage was added to the
plasma. To prepare platelet poor plasma 5 ml of citrated blood was
centrifuged at 2000 g for 10 minutes and the plasma aspirated. The PT was
determined by incubating 100 ul of plasma with 501-11of different
concentrations of phages for 10 minutes, using PBS as control. Two hundred
~LI of Dade Innovin (Dade Behring, Marburg, Germany) was added and the
clotting time was measured on the STart®4coagulation timer (Diagnostica
Stago, Asnieres, France). Only one colony of each library showed
lengthening in PT. We then repeated the PT three times with each of these 2
colonies.
3.1.8 Sequencing of FVlla-inhibiting phages
3.1.8.1 DNA preparation
We sequenced both colonies that showed lengthening of the PT.
DNA was prepared to sequence the phages. Briefly, 100 ~LIof the amplified
phages were diluted in PBS and precipitated on 20% PEG/Nael for one hour.
The phages were then centrifuged for 10 minutes at 18000 g and the pellet
dissolved in 100 ~d of a sodium iodide buffer (10 mM Tris, 1 mM EDTA, 4 M
Nal), incubated for 10 minutes at room temperature and centrifuged again for
10 minutes. The pellet was washed with 70% ethanol and left to dry. The
DNA was rehydrated in 30 ul of distilled H20.
46
The DNA concentration was determined by preparing a 10x and 20x dilution
and reading the OD at 260 nm. The DNA concentration was calculated by a
standard method (Sarnbrook and Russeii, 2001).
3.1.8.2 Polymerase chain reaction (peR)
We used the DYEnamic ET Terminator Cycle Sequencing Premix Kit
(Amersham Pharmacia Biotec Inc, NJ, USA.) for sequencing reactions. A
PCR reaction mixture was prepared by using 8 ~LIpremix reagent, 0.5 ~LI
primer (5 pmol, 5'-CCC TCA TAG nA GCG TAA CG-3') and 2 ~lg DNA from
the inhibiting phages. The final volume of the reaction was 25 ~LI.The PCR
was performed on the Gene Amp PCR System (Applied Biosystems, CA,
USA). One cycle was performed at 94°C for 1 minute. Twenty-five cycles
were performed at, 94°C for 30 seconds, 45°C for 30 seconds and 60°C for 4
minutes.
After the PCR, the DNA was extracted by adding 2 ~LIof 3 M sodium acetate
and 76 ~LI100% ethanol to the PCR product and incubated at 4°C for 15
minutes. It was then centrifuged for 15 minutes, the pellet washed with 70%
ethanol and dried in a vacuum drier for 2 minutes at medium heat. Template
suppression reagent (TSR, 30 ul) (Applied Biosystems, CA, USA) was added
to the dried DNA. The DNA was then denatured at 95°C for 1 minute.
Sequencing was performed on the ABI Prism 310 Genetic Analyser (Applied
Biosystems, CA, USA). Interestingly the sequence from the cyclic 7-mere
phage fitted exactly into the middle of the sequence from the linear 12-mere
phage. We compared these sequences to the sequences of TF, FX and TFPI
and could not find any comparison. Unfortunately according to a contract of
agreement with the company ADI, who supplied the reagents for this study, it
will not be possible to disclose the peptide sequence. A cyclic heptapeptide
with a sequence similar to the peptide sequence displayed on the phage
,
colony form the cyclic library was synthesised. American Diagnostica Inc.
(ADI, Greenwich, CT, USA) kindly supplied the peptide. The purity of the
peptide was higher than 80%.
47
3.2 Tests performed on the synthesised peptide
3.2.1 Prothrombin times and thrombin times
The effect of the peptide on PT and thrombin time (TT) was measured in
normal pooled plasma. PT was measured as described (see 3.1.7). Final
peptide concentrations in the PT reaction were 1.17 mM, 0.58 mM, and 0.3
mM. As a control, we added PBS to plasma. For a negative control we
added a peptide that does not bind to FVlla.
TT was measured by incubating 100 ul of plasma with the same peptide
concentrations as for the PT, for 10 minutes. 50 ~Liof Dade thrombin reagent
(Dade Behring, Marburg, Germany) was added and the clotting time was
measured on the STart®4coagulation timer (Diagnostica Stag a, Asnieres,
France). Again we used PBS as a control and a peptide that does not bind to
FVlla as a negative control. We repeated the PT and TT three times.
3.2.2 Perfusion studies with endothelial cells
The antithrombotic effect of the peptide was tested in perfusion studies in the
laboratory of Prof. Jolan Harsfalvi from the University of Debrecen, Hungary.
The parallel flow chamber, described by Sakarfassen in 1983, was used
(Sakarfassen et al. 1983). Thermanox Plastic cover slips (Nunc, IL, USA)
were coated with human microvascular endothelial cells (HMEC-1) as follows:
The cover slips were sterilised in 80% ethanol. They were then dipped into
sterile 1% gelatine followed by dipping into 0.5% gluteraldehide. The cover
slips were then left to dry before coated with the endothelial cell suspension.
We coated cover slips with simian virus 40 (SV40)-immortalised human
microvascular endothelial cells (HMEC-1), a generous gift from E.W. Ades
and T.J. Lawley from the Centres for Disease Control and Prevention and
Emory University School of Medicine, Atlanta, GA. The cells were cultured as
described by Ades et a/.(1992) with slight modification. MCDB131 culture
medium (Sigma Chemical Co, St Louis, MO, USA) was supplemented with
48
15% heat denaturated human serum and 20 mg/ml gentamicine. The medium
was substituted every 2 days under sterile conditions. The cells were
detached from the flask surface by treatment with a mixture of 0.125% (w/v)-
Trypsin and 0.01% (w/v)-EDTA. After the treatment with trypsin, 105 cells in 1
ml culture medium were added to the culture wells containing the cover slips
and incubated at 37°C with 5% CO2. The medium was substituted after 4
hours and when the cover slips were completely covered, they were treated
with 1 mmol/L ammonia solution to expose the extracellular matrix (ECM) of
the endothelial cells. The ammonia solution damages the endothelial cell to
express TF among other proteins on the surface of the cover slips. The cover
slips were washed with PBS and stored at -4°C.
Ten ml of blood from normal humans was collected in 200 U/ml low-molecular
weight heparin (Clexane, Rhone-Poulene Rorer, France). The different final
concentrations of the peptide in whole blood were 2.34 ~LM,4.7 ~LM,and 9.35
~LM. The blood was incubated at 3rC for 10 minutes before starting the
experiment. The chamber was first rinsed with HEPES buffer (0.01 M HEPES,
0.15M NaCl, pH 7.35). The cover slips were placed in the perfusion chamber
perpendicularly to blood flow. Constant blood flow was obtained and
maintained with a peristaltic pump, and the blood was recirculated through the
chamber. The effect of the peptides on platelet adhesion onto the cover slips
was studied at 37°C at shear rates of 1000-5 and 200-5. Blood flow was
maintained for 5 minutes followed by rinsing the cover slips with HEPES
buffer. The cover slips were removed from the chamber, rinsed in HEPES
buffer and fixed in methanol. They were stained in May-Grunwald (diluted 1:1
in Sórensen buffer) for 10 minutes and in Giemsa stain (diluted 1:5 in
Sórensen buffer) for 30 minutes and finally washed in Sórensen buffer (32
mM KH2P04 and 40 mM Na2HP04).
Thirty fields per cover slip were analysed. The percentage coverage on each
field was determined with a Zeiss "Iight microscope connected to a
computerised image analyser (Virginia, MTAKFKI, Budapest).
49
The peptide was also tested for inhibition of platelet adhesion to collagen and
TF.
3.2.3 Perfusion studies with collagen
The Thermanox cover slips were coated with 100 ~lg/ml Harm Collagen
reagent (Nycomed, Oslo) for one hour at room temperature and left to dry
before use. Perfusion studies were performed as described in 3.2.2. We
used the same peptide concentrations and shear rates as in 3.2.2.
3.2.4 Perfusion studies with TF
Twenty ml of blood from normal humans was collected in 200 U/ml low-
molecular weight heparin (Clexane, Rh6ne-Poulenc Rarer, Port Elizabeth,
South Africa). We used Innovin (Dade Behring, Marburg, Germany) as a
source of tissue factor. The Thermanox cover slips were cleaned for 30
minutes in 5 parts of ethanol (98%. pure) and 1 part of 10 mM NaOH and
rinsed with ethanol. They were then coated with 1OO~d of Innovin and dried
for 45 minutes before use.
The same method of perfusion with platelet aggregates as in HMEC-1
covered slips were used as described in 3.2.2. Five fields per coverslip were
analysed. A photo of each field was taken with a Zeiss microscope connected
to an Olympus digital camera and the percentage coverage was determined
with an image analysing program. We used final peptide concentrations of
29.2 ~lM and 58.5 ~lM at shear rates of 200 S-1, 650 S-1 and 1300 S-1. Since
the inhibition effect was more pronounced at a shear of 1300 S-1, we did the
perfusion experiment at this shear with a higher peptide concentration of 117
~M as well.
50
3.2.5 Kinetic assay
The chromogenic substrate, Spectrozyme FVlla, that was used for the kinetic
studies were kindly supplied by American Diagnostica.
Chromogenic substrate hydrolysis was detected using the EL312e Microplate
Bio-Kineties reader (Bio-tek instruments, Vermont, USA) equipped with kinetic
module software. Chromogenic substrate hydrolysis was followed for 40
minutes at 405 nm. Every 5 minutes a reading was taken. The first kinetic
assays were done with different FVlla concentrations (800 nM, 400 nM, 200
nM, 100 nM and 50 nM) to determine the optimal FVlla concentration to use in
further assays. The optimal FVlla concentration was 400 nM. The next
assays were done with this FVlla concentration and different peptide
concentrations (0 mM, 0.057 mM, 0.114 mM, 0.171 mM, 0.228 mM and 0.456
mM) and different substrate concentrations (0 mM, 0.5625 mM, 1.125 mM,
2.25 mM, 4.5 mM and 9 mM) were used. The total reaction volume was 200
ul. All reagents were diluted in the reaction buffer (0.05 M Tris, 0.1 M NaCI
pH 8.4). We used Dade Innovin (Dade Behring, Marburg, Germany) as a
source of TF. All reactions contained 100 ul of Dade Innovin (Dade Behring,
Marburg, Germany). The reactions were started by the addition of different
substrate concentrations to a mixture of 400 nM FVlla and different peptide
concentrations.
We determined the initial velocity (Vo-value) of each reaction by dividing the
change in absorbency over the first 15 minutes by the time period (15
minutes). The reaction curves at 400 nm FVlla are straight lines over the first
15 minutes (see figure 4.10). We plotted the mean Vo-values against the
different substrate concentrations at the different peptide concentrations and
calculated the maximum velocity (Vm-value) and apparent Michaelis-Menten
constant (Km'-value) of each peptide concentration by using one-site binding
non-linear regression (r2>0.990). Each reaction was repeated 4 times. To
determine the type of inhibition of the peptide, we plotted the Lineweaver-Burk
plot (1No vs 1/substrate). We determined the inhibition constant (Ki-value) by
mu."'SD01'llll 51
plotting the apparent Km values against the peptide concentrations (see figure
4.13). The Ki-value is then measured at the x-axis intercept.
52
CHAPTER4
, t
-.4'&42-M:gW'# #Whts! WC ...... WSw" . W\wPUhU; t".EsPie,.
RESULTS
4.1 Biotinylation of FVlla and biopanning on biotinylated FVlla
The biotinylation of FVlla was successful because 14.6 molecules of biotin
were bound to a molecule of FVlla. The biotinylation was calculated
according to the instructions of the manufacturer of biotin (Pierce, Illinois,
USA). We however did not find any FVlla binding phages in either of the
three selection rounds as indicated by the global ELISA. Therefore we
decided to continue biopanning by coating FVlla directly to the immune-tube.
4.2 Biopanning of FVlla coated directly in immune-tubes
The concentration of the amplified phages increased after each round of
panning. The concentrations of the amplified phages were 1.99.1011 for round
1,4.95.1011 for round 2, and 1.5.1012 for round 3 for the cyclic 7-mere library.
For the linear 12-mere library the phage concentrations were 2.31.1011 for
round 1, 5.22.1011 for round 2 and 6.22.1011 for round 3.
The global ELISA indicated that we have selected the highest concentration of
FVlla-binding phages in round 3 since the optical density of these phages was
the highest. We therefore decided to use the phages from round 3 for binding
studies.
With the binding ELISA of the single colonies, we only found twelve phage
colonies that bind strongly to FVlla. Six colonies were from the linear 12-mere
library and the other six from the cyclic 7-mere library. Because we had
limited quantities of the very expensive FVlla, we did not search for more
binding colonies.
53
We performed a dilution ELISA with the twelve colonies. All the colonies of
each library showed about the same binding affinity to FVlla. To simplify the
graph, we only plotted the binding affinity of one colony of each library. These
results can be seen in figure 4.1. The 00490 increased with increasing phage
concentrations. These two colonies thus bind concentration dependently to
FVlla. We repeated this ELISA three times. The colonies of the cyclic 7-mere
library bind with a slightly higher affinity than the colonies of the linear 12-
mere library.
0.4 n=3
E 0.3 --- Cyclic 7-merec:
0
0)
oo:t 0.2
0 --- Linear 12 -mere
0
0.1
0.0
0.0 1.0 x1010 2.0x1010 3.0x1010
phages/ml
Figure 4.1 Dilution ELISA of both the cyclic 7-mere and linear 12-mere
colonies. The 00490 increased with increasing concentration of phages. The
cyclic 7-mere colony binds stronger to FVlla than the linear 12-mere one
(n=3).
An inhibition ELISA was also performed to determine if TF was able to prevent
the twelve colonies from binding to FVlla. No inhibition effect was seen with
the linear colonies. Only one colony (colony 38) of the cyclic library showed a
modest inhibition. This is shown in figure 4.2.
54
0.9
0.8
_ 0.7 n=3
~ 0.6 ~ l
~ 0.5 ---+-.1---------_,1 • Cyclic 7-mere
~ 0.4
o 0.3
0.2
0.1
0.0+---,----,-----,----,------,
0.00 0.01 0.02 0.03 0.04 0.05
TF (J..IM)
Figure 4.2 Inhibition ELISA of the cyclic 7-mere colony (colony 38) at different
TF concentrations. TF concentrations from 0 to 0.05 ~lMwere added to FVlla
coated wells and incubated for 15 minutes. 5.1010 phages of the cyclic 7-
mere colony were added to the wells and incubated for 2 hours where after
the FVlla bound phages were detected using an anti-phage antibody (n=3).
We also performed PT's on different concentrations of the 6 linear 12-mere
colonies and the 6 cyclic 7-mere colonies that showed binding in the binding
ELISA of single colonies. Only two colonies (one from the cyclic 7-mere
library and one from the linear 12-mere library) showed lengthening of the PT.
The colony from the cyclic library that lengthened the PT is the same colony
that showed inhibition in the inhibition ELISA. This was colony.38. Again we
only show the graphs of the two phage colonies that lengthened the PT.
Figure 4.3 shows the graph of the lengthening in the PT of the linear 12-mere
colony and figure 4.4 that of the cyclic 7-mere colony. For the control, PBS
was added to the plasma. A negative control was also performed where a
non-binding phage was added to the plasma.
55
n=3
22.5
- 20.0 ~ linear 12-mere(/)
....
c, 17.5
15.0
12.5
0.0 1.0x10 11 2.0x10 11 3.0x1011
phages/ml
Figure 4.3 PT's with increasing concentrations of the linear 12-mere colony.
Increasing concentrations of this phage colony were incubated for 10 minutes
with human plasma before adding the PT reagent (Dade Innovin). This phage
colony lengthened the PT concentration dependently. At the highest phage
concentration (2.3.1011 phages) the PT was prolonged with 5.4 seconds.
(n=3)
25
--- cyclic 7-mere
_ 20
(/)
Ic-,
15 n=3
10+-------~----~------~----~
0.0 2.0x10 11 4.0x10 11 6.0x10 11 8.0x10 11
phages/ml
Figure 4.4 PT's with increasing concentrations of the cyclic 7-mere colony.
Increasing concentrations of this phage colony were incubated for 10 minutes
with human plasma before adding the-PT reagent (Dade Innovin). The PT
was prolonged with 8.3 seconds at the highest phage colony concentration
56
(5.8.1011 phages). This phage colony also lengthened the PT concentration
dependently. (n=3)
4.3 Sequences of the phage colonies
We sequenced both the phage colonies (one from the cyclic 7-mere and one
from the linear 12-mere library) that showed lengthening in PT. Interestingly,
the sequence of the colony from the cyclic 7-mere library fitted exactly into the
middle of the sequence of the colony from the 12-mere library. We compared
these sequences to the sequences of TF, FX and TFPI and could not find any
comparison. Unfortunately, it is not possible to disclose the sequence
because of our agreement with American Diagnostics Incorporated (ADI).
Because the cyclic colony binds stronger to FVlla we decided to synthesise a
cyclic peptide with the sequence similar to the peptide sequence displayed on
this colony. Cyclic peptides are also more stabile than linear ones and more
resistant to proteolysis (Pearce, 2001).
4.4 Effect of peptide on prothrombin times and thrombin times
PT's were done on human plasma. High concentrations of the peptide were
needed to prolong the PT. The final peptide concentrations were 1.17 mM,
0.58 mM, and 0.3 mM. Figure 4.5 shows the prolongation of the PT with
human plasma. The PT was prolonged by 30 seconds at the highest final
peptide concentration of 1.17 mM. We repeated the experiment three times.
As a control, we added PBS to plasma. For a negative control we added a
peptide that does not bind to FVlla.
\
57
50
40
-en 30I--
a.. 20
n=3
10
O+-----~----~----~----~--~
0.00 025 050 OJ5 1~0 125
Peptide (final conc. mM)
Figure 4.5 Prolongation of PT in human plasma. Different peptide
concentrations were added to normal pooled human plasma and incubated for
10 minutes before the PT reagent (Dade Innovin) was added. At the highest
peptide concentration, 1.17 mM, the PT was prolonged with 30 seconds.
(n=3)
The TT also showed prolongation at high concentrations of the peptide.
Prolongation of the TT in human plasma, with the same peptide
concentrations as the PT's, are shown in figure 4.6.
60
en
I--- 50
I-- n=3
40+-----~----~----~----~--~
O~O 025 0.50 OJ5 1~0 125
Peptide (final concentration mM)
,
'"
Figure 4.6 Prolongation of TI in human plasma. Different peptide
concentrations were incubated with normal pooled human plasma for 10
58
minutes before the TT reagent (Dade thrombin) was added. The TT is
prolonged at the highest peptide concentration with 9.1 seconds. (n=3)
4.5 Perfusion studies with endothelial cells
We used different peptide concentrations (2.34 ~LM,4.7 ~LMand 9.35 ~LM)to
study the effect of this peptide on platelet adhesion to human microvascular
endothelial type 1 cells (HMEC-1 cells). We performed each peptide
concentration in triplicate and table 4.1 shows the mean values and the
standard deviation of the percentage of coverage. We used a low and a high
shear (200 S-1 and 1000 S-1) that simulate conditions for venous and arterial
blood flow.
Percentage coverage Percentage coverage
at a shear rate 200 S-1 at a shear rate 1000 S-1
Control 17.9±0.96 11.6±0.64
2.34J..lM 14.5±1.40 9.0±1.29
4.7 J..lM 8.1±0.20 6.1±2.24
9.35 J..lM 8.3±0.55 3.0±0.60
Table 4.1 Percentage of coverage on cover slips at a shear rate of 200 S-1
and 1000 S-1 (n=3).
The percentage coverage at both shears decreased with increasing peptide
concentrations. Photo's of platelet adhesion (50x enlarged) at both shear
rates can be seen in figure 4.7. The platelet adhesion of 9.35 ~LMand 4.7 ~lM
peptide at 200 S-1 are the same. I therefore show the effect of only the 4.7 ~lM
peptide at 200 S-1.
"\
59
A
ii
B
.vl.
- ."
Figure 4.7 Platelet adhesion to HMEC-1 cells at shear rates (A) 200 S-1 and
(B) 1000 s -1.
A: (i) Control, (ii) 2.34 f.lM peptide, (iii) 4.7 f.lM peptide
B: (i) Control, (ii) 2.34 f.lM, (iii) 4.7 f.lM and (iv) 9.35 f.lM peptide.
60
4.6 Perfusion studies with collagen
We also did perfusion studies to determine the effect of different
concentrations of the peptide on platelet adhesion to collagen. The peptide
however does not have any influence on platelet adhesion to collagen at
concentrations of 2.34 ~lM, 4.7 ~lM and 9.35 ~lM peptide. These results are
not shown.
4.7 Perfusion studies with TF
By coating the coverslips with Innovin (Dade Behring, Marburg, Germany), we
could study the effect of this peptide on platelet adhesion to TF because
Innovin is a source of TF. We performed each peptide concentration only in
duplicate because of limitations of the amount of peptide. Three different
shear rates (200 S-1, 650 S-1 and 1300 S-1) were used. The mean values of
platelet coverage at shears of 200 S-1, 650 S-1, and 1300 S-1, are summarised
in table 4.2. The photos (100x enlarged) of the platelet adhesion at 200 S-1
and 650 S-1 shear rates are shown by figure 4.8 and that of the 1300 S-1 shear
rate are shown by figure 4.9.
Percentage coverage Percentage coverage Percentage coverage
at a shear rate 200 S-1 at a shear rate 650 S-1 at a shear rate 1300 S-1
Control 6.1 11.4 22
29.21lM No effect No effect 10.4
58.51lM 4.1 4.7 2.9
117 IlM Not done Not done 3,3
Table 4.2 Percentage of coverage on TF-coated coverslips at shears 200 S-1,
650 S-1 and 1300 S-1 (n=2). "
",
A peptide concentration of 29.2 IlM had no effect on the percentage coverage
at shears 200 S-1 and 650 S-1 and those photos are not shown in figure 4.8.
61
This concentration however had an effect at the high shear of 1300 S-1.
Platelet adhesion to the TF-coated coverslips at 1300 S-1 was inhibited in a
dose-dependent manner by the peptide. At the lower peptide concentrations
the effect was not as pronounced as that at the high concentration.
62
A
ii
6.1% 4.1%
B
ii
11.4% 4.7%
Figure 4.8 Platelet adhesion on TF coated coverslips at shear rates (A) 200 S-1
and (B) 650 S-1. The percentage platelet coverage are shown beneath each
photo
A: (i) Control, (ii) 58.5 IlM peptide (200 S-1)
B: (i) Control, (ii) 58.5 IlM peptide (650 S-1)
63
ii
22% 10.4%
iii iv
2.9% 3.3%
Figure 4.9 Platelet adhesion on TF coated coverslips at shear 1300 S-1.
(i) Control, (ii) 29.2 f.lM peptide, (iii) 58.5 f.lM peptide, and (iv) 117 f.lM. The
percentage coverage is shown beneath each plot
64
Less coverage was observed at the low shear (200s-1) when compared to the
higher shears. This was expected, since thrombi that form at low shear rates
are platelet-poor and fibrin-rich (Sakarfassen et al. 2001; Roth, 1992). The
higher shears show more platelet adhesion in the control study. The peptide
also has a more pronounced effect on platelet adhesion at high shear. Low
peptide concentrations (29.2 ~lM) do not have any effect on platelet adhesion
at low shears. At high shears it however has a ± 50% decrease in platelet
adhesion. Concentrations of 58.5 ~lM peptide has an effect at all three shear
rates, but is more pronounced at the high shear rate.
4.8 Kinetic assay
The first step in the kinetic assay was to determine the optimal FVlla
concentration. This was determined at 400 nM. We then determined the Vo-
values of the reaction at different peptide and different substrate
concentrations. Figure 4.10 shows the change in optical density over time of
the control (0 mM peptide) at different substrate concentrations with 400 nM
FVlla. We included this figure to show that the reaction curves are straight
lines over the first 15 minutes, since we calculated the Vo-values as the
difference in optical density over the first 15 minutes over this time period.
Substrate concentrations
0.3
~9mM
--4.5mM
Ê
c 0.2 ---2.25 mM
LO ---1.125 mM
~ __._ 0.5625 mM
Cl
0 0.1 -<>-0 mM
0.0+----,-----,-------,--------,
o 10 20 30 40
lime (min)
Figure 4.10 Different substrate concentrations at 0 mM peptide.
65
Figure 4.11 shows the Michaelis-Menten kinetics of the inhibition of FVlla by
the different peptide concentrations.
Peptide concentrations
0.0075
c- D 0 mM
.- ..Q..).
Q) ::J 0.057 mM
C)c
~C·E- 0.0050 • 0.114 mM
J::-
CJ Q)
_CJ ·0.171mM
o c
>~ • 0.228 mM
~c ~ o 0.456 mM0 0.0025
Q) If)
2~
0.0000 -iF----,------r----,-----,
0.0 2.5 5.0 7.5 10.0
Substraat (mM)
Figure 4.11 Michaelis-Menten kinetics of the inhibition of FVlla by the peptide
(n=4).
According to figure .4.11 the Vm-values of the different peptide (inhibitor)
concentrations are unchanged but the Km-values increased with increasing
peptide concentrations. This could indicate that the peptide is a competitive
inhibitor. To confirm this, we plotted the 1No values against the 1/substrate
concentration (figure 4.12). This Lineweaver-Burk plot shows that the
inhibition of the peptide is competitive because the slope of the graphs
increased with increasing peptide (inhibitor) concentrations.
66
3000 Peptide concentrations
· 0 mM
2500 • 0.057 mM
T 0.114mM
• 0.171 mM
• 0.228 mM
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
1/substrate (mM)
Figure 4.12 Linewaever-Burk plot showing the effect of competitive inhibition.
To determine the inhibition constant (Ki) we plotted the apparent Km (Km')
values against the peptide concentrations in figure 4.13. We could fit a
straight line to the graph and determine the Ki value as the x-axis intercept at
0.1232mM.
20
18
16
14
- 12
~ 10
8
6
-0.0 0.1 0.2 0.3 0.4 0.5
-Ki = -O.1232mM Peptide (mM)
Figure 4.13 The Km value plotted aqainst the peptide concentrations. From
this graph the Ki could be determined by measuring the X-intercept of a linear
regression. (n=4)
67
CHAPTER 5
BbJi&4!ii @iii? '¥§Nft#idkMk*2 ·'ïSW>!8tW;l!laegt j,_ &44WMIDC§S&IGij]QIBM,tï4MENWWfMPél:i§!@1f!'5d¥ fMSi' "'?fhWbZiYS ¥MWMH FW
DISCUSSION
The importance of the FVllafTF complex for the initiation of not only
hemostasis, but also of thrombosis is now generally accepted (Davie et a/.,
1991). Several studies have shown the impact of this binary complex on
arterial thrombus formation (Barstad et al, 1995; 0rvim et al, 1994). Recently
it was confirmed by selective inhibition of the FVllafTF complex by active site
inactivated rFVlla (0rvim et al, 1997). In one study blockade of coagulation at
the level of the FVllalTF complex provided full antithrombotic protection
without abnormal bleeding (Harker et a/., 1996). Furthermore, although factor
X and thrombin are the most popular candidates against which inhibitors are
selected, one can argue that less inhibitor might be needed to inhibit FVlla,
since it is higher up in the coagulation cascade and is not responsible for its
own generation in the same magnitude than thrombin and FXa (Ofosu et al.
1996).
We selected peptide inhibitors to FVlla, because the advantage of smaller
peptides is that they are non-immunogenic. Markwardt (1990) showed that
small peptide inhibitors to thrombin are non-immunogenic and non-toxic.
We used the technique of phage display to select peptide inhibitors to FVlla.
Phage display describes a selection technique in which a peptide/protein is
expressed as a fusion with a coat protein of a bacteriophage, resulting in the
display of the fused protein on the surface of the virion, while the DNA
encoding the fusion resides within the virion. Therefore phage display
technology creates a physical linkage between a vast library of random
peptide sequences and the DNA encoding each sequence allowing rapid
identification of peptide ligands for ei" variety of target molecules. This
technique also allows for large numbers of phage clones (>109 different
sequences) to be screened which make it a very powerful technique (Smith,
68
1985). It has advantages over other methods for studying protein-protein
interactions in the fact that it allows proteins to fold correctly on the phage
surface (Allen et al., 1995; Bardweil et al., 1991).
We started the phage display selection process by using biotinylated FVlla.
The ability to use soluble antigen for selections imparts a greater degree of
control over the selection process (Smith et al., 1995). By using biotinylated
antigen, soluble selections can be achieved. Phages are allowed to bind the
biotinylated antigen and are recovered using streptavidine-coated magnetic
beads. Although the biotinylation of FVlla was successful, we could not find
any FVlla-binding phages. It could be that FVlla became inactive after
biotinylation. We therefore decided to continue biopanning in immune-tubes.
To achieve this, we coated the immune-tube with FVlla and incubated the
phage libraries with it. We used a murine monoclonal antibody against human
FVlla to elute the FVlla-binding phages specifically from the immune-tube in
the last two rounds of selection. This antibody binds to the active site of FVlla
and blocks the assembly of the FVllarrF complex. It also blocks FVlla-
mediated plasma coagulation. Since this antibody replaced the phages from
FVlla, it means that the selected phages bind to the catalytic domain of FVlla,
because this antibody inhibits FVlla in a two-stage chromogenic assay
involving the chromogenic substrate for FXa, spectrozyme Xa (Clarke et al.,
1992). We did three selection rounds and found that the twelve strongest
FVlla binding phage clones included six from the cyclic 7-mere library and six
from the linear 12-mere library. From the 12 strong-binding colonies, only 2
(one from each library) lengthened the PT in a concentration dependent
manner (fig. 4.3 and 4.4). Interestingly, the sequence from the cyclic 7-mere
library fitted exactly into the middle of the sequence from the linear 12-mere
library. No similarities were found between either of the cyclic and linear
sequences and those of TF, FX or TFPI. After the three rounds of enrichment
(biopanning), 12 phage colonies were tested for binding to FVlla. The phage
colonies from the cyclic heptapeptide library bind with a higher affinity than the
linear 12-mere sequence (figure 4.1). This is not surprising since it is known
that cyclic peptides bind more tightly to their ligands than the same linear
69
sequences due to improved binding entopy (Das and Meirovitch, 2001).
Furthermore, TF concentrations from 0 to 0.05 ~lM prevented the binding of
one of the colonies from the cyclic peptide library to FVlla. This suggests that
this cyclic heptapeptide sequence bind to the same binding site as TF to
FVlla. The effect is nevertheless modest, since TF does not prevent the
phages completely from binding to FVlla (figure 4.2). One phage colony from
each library lengthened the prothrombin time (PT) in a dose-dependent
manner (see figures 4.3 and 4.4). Unfortunately only limited numbers of
phages can be used in any assay because of the inherent size restrictions of
the phages. In view of this, no further results on inhibition could be obtained
by using the phages.
A cyclic peptide with the corresponding sequence as the peptide sequence
displayed on the cyclic 7-mere colony was synthesised. We cannot disclose
the sequence of the peptide since we have a secrecy contract with American
Diagnostica Incorporated (Greenwich, USA). There were two reasons why we
selected the cyclic peptide. First, it binds stronger to FVlla (see figure 4.1),
and second, cyclic peptides are more stable than those with linear sequences
(Fabiola et al., 2001). Furthermore, cyclic peptides are more resistant to
proteolysis (Pearce, 2001).
We characterised the peptide by studying its effect on platelet adhesion to
human vascular endothelial cell matrix (HMEC-1) in a parallel flow chamber,
under both arterial and venous shear stress conditions (shear rates of 1000 s
1 and 200 s' respectively). It is interesting to note that the percentage
coverage in the control study was higher at venous flow (shear rate of 200s-1)
than at arterial flow (shear rate of 1000 S-l). This is because minute levels of
TF are expressed on the endothelium which is a major initiator of venous
thrombosis (Salzman and Hirsh, 1993). The peptide inhibits platelet adhesion
in a dose-dependent manner at both shear stress conditions. The inhibiting
effect was more pronounced at arterial flow conditions (1000 s'), because the
highest peptide concentration (9.35 ~lM) inhibited coverage by approximately
74%. At the low shear rate (200 S-l) inhibition was approximately 54% at the
70
same peptide concentration (see table 4.1 and figure 4.7A). We cannot
explain this. We can however argue that the inhibition is more effective at
arterial flow conditions is because platelets are more involved in thrombi
formed at arterial shear whereas fibrin is more involved in thrombi formed at
venous shear (Sakarfassen et aI., 2001; Roth, 1992). This was also observed
in the perfusion studies where TF (Innovin)-coated coverslips were used. In
this case the control studies indicate that platelets adhere much more to the
TF -coated coverslips at high shear rates (1300 S-1), where the percentage
coverage was 22% (see table 4.2 and figure 4.9). At shear rates of 650 S-1
and 200 S-1, percentage coverage were 11.4% and 6% respectively. It is also
interesting that the fibrin network was highly visible at low shear rates (200 S-1)
(see table 4.2 and figure 4.8). It is known that TF in high concentrations
emerges as a potent inducer of both arterial and venous thrombosis. TF is
present in relatively high concentrations on the cover slips. We can explain
the higher platelet coverage at high shears to the fact that platelets are more
involved in arterial thrombogenesis (Sakarfassen et aI., 2001)
This cyclic peptide affected platelet adhesion to the TF-coated coverslips a
high shear rate of 1300 S-1 in a dose-dependent manner. We did not observe
a concentration-dependent effect at medium and low shears of 650 S-1 and
200 S-1 respectively because the percentage coverage was too low to observe
inhibitory effects. The peptide at a concentration of 56.5 ~lM however
inhibited platelet adhesion by 58% at 650 S-1. The effect at 200 S-1 could
again not be detected since the percentage coverage was too low. PN7051
also inhibits platelet adhesion to immobilised TF in a parallel-plate perfusion
chamber at a shear rate of 650 S-1. Fifty percent inhibition was observed at
500 ~lM. Our peptide inhibited platelet adhesion by 58% at 58.5 ~lM at the
same shear, i.e. it seems to have a stronger effect on platelet adhesion and
thrombus formation than PN7051 (Orniru; et al., 2002).
Furthermore, this peptide had no effecton collagen-coated coverslips in the
perfusion studies. It is known that other coagulation inhibitors such as hirudin
and PPACK also had no effect on platelet adhesion to collagen (Harker et al.,
71
1996). A possible explanation could be that there is no TF involved on the
collagen suriace and coagulation is not initiated by the FVllaffF complex, but
platelets are activated by vWF.
Thus, we can summarise the effect of the peptide on platelet adhesion at
different shears on the different matrixes in the periusion studies, as follows.
The peptide only has an effect on matrixes where TF is present, because
there was no effect on the collagen matrix. The amount of TF on the matrixes
also has an influence on the platelet adhesion at different shears, since at
minute levels of TF such as the HMEC-1 matrix, the platelet coverage is
higher at venous flow conditions. While at high TF concentrations such as the
TF matrix, the platelet coverage is much higher at arterial flow conditions. At
venous flow conditions, very low platelet coverage occurs on the TF matrix
and therefore we cannot compare the effect of the peptide on the TF matrix at
different shears. We can however compare the effect of the peptide at
different shears on the HMEC-1 matrix. Here it is clear that the peptide has a
stronger inhibition .effect at arterial conditions than at venous conditions,
especially at high peptide concentrations, although the platelet coverage is
better at venous conditions. We have no explanation for this.
lt is important to note that all the periusion studies were periarmed in
heparanised blood. It is known that LMWH does not block all coagulant
activity, but allow some thrombin formation and thus some fibrin deposition on
the suriace (Sakarissen et al., 2001). Fibrin networks can be clearly observed
on the TF matrix at low shear rates.
The kinetic data indicates that this peptide is a competitive inhibitor of FVlla.
Michaelis-Menten kinetics shows that the Vm-values stays the same with
increasing peptide concentrations, but the Km-values increased. Furthermore
the 1No versus 1/substrate curves (Lineweaver-Burk plots) of different
peptide concentrations cross-section on-the y-axis, which is also an indication
of competitive inhibition (see figures 4.11 and 4.12). This is because the
Lineweaver-Burk equation in the presence of a competitive inhibitor is,
72
1No = Km'Nm * 1/substrate concentration + 1Nm, and the slope of the plots
at different peptide concentrations increase with increasing peptide
concentrations. It is important to note that a competitive inhibitor does not
necessarily binds to the same binding site as the substrate. but can also bind
to a different binding site on the enzyme in such a way that the binding of
either one causes a conformational change in the enzyme which prevent s the
other from binding. This inhibition gives competitive kinetics (Dixon and
Webb, 1979).
The chromogenic substrate we used in the kinetic studies contains an arginine
sequence and binds to the catalytic site of FVlla. Since FVlla is a serine
protease, it cleaves the substrate at a site adjacent to an arginine residue.
The peptide does not contain an arginine residue and therefore we can accept
that the peptide does not bind to the same binding site as the substrate. The
peptide does however prevent the splicing of the colour reagent of the
chromogenic substrate. Therefore the binding of the inhibitor to FVlla may
cause -a conformation change to prevent the chromogenic substrate from
being spliced. Since we selected phages that binds to FVlla and eluted them
with an antibody that binds to the catalytic site of FVlla and prevents the
formation of the FVllalTF complex, we can argue that this peptide also binds
to the catalytic site and also prevent the binding of TF to FVlla. We know that
the catalytic domain (serine protease domain) contains three different binding
regions which are a TF binding region, active site binding region and a
macromolecular substrate binding region and all three of these regions are
involved in the proteolytic activity (Persson, 2000). Since free FVlla does not
recognise the substrate factors IX and X, the binding of TF to FVlla is
necessary for the binding of the substrate (Broze, 1992). We can thus argue
that this peptide binds to FVlla in such a way that it prevents the formation of
the FVllalTF complex and therefore FVlla does nor recognise the substrate
and does not splice the colour reagent of the chromogenic substrate. The Ki-
value is determined at 0.1232 mM. Our peptide is not a strong inhibitor of
FVlla, when the Ki-value is compared to that of other FVlla inhibitors. For
example, TFPI inhibits the activation of FXa by a Ki of 30 nM. Similarly,
73
Kunitz domain variants of TFPI inhibit the FVllalTF complex with a Ki of 1.9
nM (Dennis and Lazarus, 1994a; Dennis and Lazarus, 1994b). Another
peptide inhibitor, A-183, that binds to the exosite of the protease domain of
FVlla, inhibits FX activation with a Ki of 200 pM (De Cristofaro. 2002; Dennis
et al., 2001; Roberge et al., 2001).
One must take into account that we used Innovin (Dade Behring, Marburg,
Germany) as a source of TF in the kinetic studies. It is now know that the
Innovin contains only a small percentage of TF. This may explain the large
difference in Ki-values from other studies where they used recombinant TF.
This cyclic heptapeptide lengthens the PT dose-dependently. Comparable to
other inhibitors of TF-dependent coagulation a peptide concentration of 1.17
mM lengthened the PT 3-fold. PN7051, another cyclic inhibitor of the
FVllalTF complex that represents loop I of the second EGF-like domain of
FVII, also prolongs the clotting time dose-dependently with an ICso of 1.3 mM
(Orning et al., 200.2). The reason for the lengthening in PT may be the
binding of the peptide to FVlla and preventing the FVlla/TF complex from
forming, which prevents the activation of FXa. Our peptide also lengthens the
TT but only at concentrations in excess of 0.58 mM, indicating that it may also
inhibit FXa function. Moreover, the lengthening in TT is not as pronounced as
the lengthening in PT. At the highest concentration of 1.17 mM, the
lengthening in TT was 9.1 seconds (±0.25-fold). It may be so because
determination of the TT is not TF-dependent. This difference in the effect on
PT and TT can again be compared to that of the cyclic inhibitor, PN7051,
which inhibits the prothrombinase complex with an ICso of 0.5 mM and TF-
dependent FX activation with an ICsoof 20 ~lM using the same concentrations
of FVlla and FX (Orning et al., 2002). It is of interest to note that peptide
concentrations higher than 500 ~lM lengthened the PT and TT. In a study
where the effect of two distinct peptide exosite inhibitors of FVlla on the PT
was determined, the lengthening in PT\started at concentrations higher than
100 nM. Our peptide is therefore not as potent as exosite inhibitors of FVlla
(Roberge et al., 2001). a'.i:
74
The inhibition of our peptide on thrombin formation may be explained as
follows. This peptide prevents the formation of the FVllalTF complex and thus
prevents the activation of FX since the formation of the FVllalTF complex is
necessary to activate FX. We can therefore argue that the peptide inhibits
thrombin formation.
To summarise, one can argue that our peptide acts on three levels of the
coagulation process to inhibit thrombus formation by preventing the formation
of the FVllalTF complex and thereby preventing the activation of FX. First, at
vessel wall injury where TF within the vessel wall is exposed to FVlla in the
circulating blood (Broze, 1992). Second, on leukocytes containing circulating
TF, which is transferred to the platelet thrombus (Rauch et al., 2000). This is
the so-called blood-borne TF (Rauch and Nemerson, 2000). Third, on platelet
surfaces where the extrinsic and intrinsic tenase complexes are formed (Mann
et a/., 1990).
The existence of blood-borne TF as described by Rauch and Nemerson
(2000) provides another mechanism by which inhibitors of FVlla, or the
FVlla/TF complex, such as our peptide prevents thrombosis. They described
thrombogenic TF on leukocyte-derived mieroparticles and their incorporation
into spontaneous human thrombi. Polymorphonuclear leukocytes and
monocytes transfer the TF-positive mieroparticles to platelets, thereby making
them capable of triggering and propagating thrombosis. This is done by the
interaction of leukocytes via their CD15 receptor on the cell membrane with
CD62P (P-selectin) on the platelet membrane. P-selectin is an a-granule-
derived activation dependent adhesion molecule found on platelets. Platelets
therefore become TF-positive after exposure to the TF-positive microparticles.
The activation of the coagulation factors IX and X occurs therefore very close
to platelet surfaces and thereby favouring the formation of the intrinsic and
extrinsic tenase complexes on their highly procoagulant surfaces. Since our
",
peptide is able to prevent FX activation by preventing the formation of the
75
FVllalTF complex, one can argue that it can also act on platelets to inhibit
formation of platelet-rich thrombi.
Shortfalls
In a study of this nature where highly expensive products are used, it is
necessary to be economical. One aspect of the study that was affected is the
kinetic studies. We unfortunately did not have enough peptide or FVlla to
repeat the reactions more than 4 times. This can explain the large standard
deviation of the apparent Km-values in figure 4.13 of the kinetic studies.
Another explanation for the large Km'-values at high peptide concentrations
can be the impurities of the peptide since it maybe not 100% pure, but at least
more than 80%. Another aspect is the perfusion studies with Innovin-coated
coverslips. These studies could only be done twice at low shears and high
shears.
Future studies
The kinetic studies indicate that this peptide is a competitive inhibitor of FVlla
that prevents the binding of TF to FVlla. It would be interesting to see by X-
ray cristallography where exactly this peptide binds to FVlla. Since we could
not determine the effect of this peptide on platelet adhesion to a TF-matrix at
low shears, we would like to investigate the effect of this peptide on platelets
more intense. By using flow cytometry, we will be able to determine if this
peptide has an inhibitory effect on platelets stimulated with ionophore or
thrombin. We will measure P-selectin, CD63 (a lisosomal protein that
indicates the platelet release reaction), PAC-1 (Gpllb/llla expression on the
platelet membrane) and TF expression. We will also be able to determine
whether this peptide prevents platelet activation. It would further be
interesting to determine the in vivo effect-, of this peptide in animal models.
76
Finally, I hope that the results discussed in this thesis have convinced the
reader that the FVllalTF complex is of utmost importance not only for
maintaining normal hemostasis, but also by playing a crucial role in the
development of arterial thrombosis, still the leading cause of death in our
Western society. We also hope that the results showing inhibition of FVlla
may suggest a novel therapeutic approach to prevent thrombosis in patients
and may prove to be effective also in disorders associated with increased
blood TF. I believe that the study of FVlla and its inhibitors will remain
important research topics in years to come and that it may result in important
social benefits.
77
CHAPTER 6
G'M' wags #j,.iiïSHf .mw, ij 'éP'·!Pi rM!!iN'WMtM ik t--i**Mi*W" ktM'bbge 4 ES! "as ..?!!' iK thM a:'ijiiN· WfflW@!#'4f1MGSlAWf9il#kAi3t;!tpMaSA'"
ABSTRACT
The importance of FVlla and the FVllalTF complex for the initiation of not only
hemostasis but also thrombosis is now generally accepted. It was shown that
the blockade of coagulation at the level of FVlla provided full anti thrombotic
protection without abnormal bleeding (Harker et aI, 1996), therefore FVlla is a
suitable candidate for the development of novel antithrombotics.
We selected inhibitors to FVlla using the technique of phage display. A
repeated selection of phages from a cyclic heptapeptide and a linear 12-mere
phage display library resulted in the enrichment of phages that bind to human
FVlla. We selected twelve colonies (6 from each library) that showed the
strongest binding to FVlla. The colonies from the cyclic 7-mere library
showed a higher affinity binding for FVlla than the colonies from the linear 12-
mere library. TF also prevents the binding of one of the cyclic colonies to
FVlla. This colony as well as one colony from the linear library showed
lengthening of the prothrombin time (PT) as well as the thrombin time (TT) in
a dose-dependent manner.
A cyclic heptapeptide was synthesised with the corresponding sequence as
the sequence displayed on the cyclic 7-mere colony. The peptide showed
lengthening of the PT and TT in a dose-dependent manner with a more
pronounced effect on the PT than the TI. We also studied the effect of this
peptide on platelet adhesion on human vascular endothelial cell matrix,
collagen and TF under both venous and arterial shear stresses. The peptide
inhibits platelet adhesion to HMEC-1 under both shear stresses. The effect
on arterial shear is however more pronounced. It does not inhibit platelet
adhesion to collagen, but has a dose-dependent inhibitory effect on platelet
adhesion TF at arterial shear. Kinetic analysis of the peptide showed that this
peptide is a competitive inhibitor of FVlla by altering the Km-values but not the
78
Vmax-values. The Lineweaver-Burk plot also indicates a competitive
inhibition, because the slope of the graphs increased with increasing inhibitor
concentrations. The Ki-value was determined at 0.1232 mM.
In summary, this peptide inhibits thrombus formation by preventing FVlla from
binding to TF and therefore preventing the activation of FX by the FVllarfF
complex. This study suggests that inhibitors to FVlla provide a novel
therapeutic approach to prevent thrombosis.
79
ABSTRAK
Dit word nou algemeen aanvaar dat faktor Vila en die faktor
VllalWeefselfaktor (FVllaIWF) kompleks nie alleen 'n baie belangrike rol in
hemostase speel nie, maar ook in trombose. Deur die stollingskaskade op die
vlak van FVlla te blokkeer, lei tot volledige inhibisie van trombose sonder die
moontlikheid van 'n bloedingsneiging (Harker et al, 1996). FVlla is dus 'n
geskikte kandidaat waarteen nuwe anti-trombotiese middels ontwikkel kan
word.
Die tegnologie van peptiedblootlegging op fage is gebruik om inhibitore teen
FVlla te selekteer. Herhaalde seleksie van fage vanaf 'n sikliese
heptapeptied faag biblioteek asook 'n lineêre 12-aminosuur faag biblioteek het
gelei tot die vermeerdering van fage wat spesifiek bind aan FVIIa. Ons het
twaalf kolonies wat sterk aan FVlla bind geselekteer (6 van elke biblioteek).
Die sikliese kolonies het met hoër affiniteit aan FVlla gebind. Ons het ook
gevind dat WF di~ binding van een van die sikliese kolonies aan FVlla
verhoed. Hierdie kolonie asook een van die lineêre biblioteek het dosis-
afhanklike verlenging getoon met die protrombien tyd (PT) en die trombien tyd
(TT).
Ons het 'n sikliese hepta-peptied identies aan die peptied wat blootgelê word
op die sikliese faag kolonie, laat sintetiseer. Die peptied verleng die PT en
TT. Die effek was dosis-afhanklik en was meer uitgesproke in die PT as in die
TT. Ons het ook die effek van die peptied op plaatjie adhesie gaan
ondersoek. Ons het verskillende oppervlaktes gebruik, nl. menslike vaskulêre
endoteelsel-matriks, kollageen en WF. Daar is van beide veneuse en
arteriële skuifkragte gebruik gemaak. Die peptied inhibeer plaatjie adhesie
aan die menslike vaskulêre endoteel. Die effek was meer uitgesproke in die
geval van arteriële vloei. Die peptied het egter geen effek getoon op plaatjie-
adhesie aan kollageen nie. Daar was wel 'n dosis-afhanklike effek op plaatjie
adhesie aan WF in arteriële vloei. Ons het ook kinetiese analises van die
peptied gedoen. Die peptied is 'n kompeterende inhibeerder van FVlla. Die
80
Km-waardes varieer met verskillende peptiedkonsentrasies, maar nie die
Vmaks-waardes nie. Die Ki waarde is 0.1232 mM.
Ter opsomming kan ons sê dat hierdie peptied trombusvorming inhibeer deur
te verhoed dat FVlla aan WF bind en daardeur die aktivering van faktor X
deur die FVllalWF kompleks verhoed. Hierdie studie dui daarop dat die
ontwikkeling van inhibitore teen FVlla In nuwe benadering is om trombose te
voorkom.
81
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ACKNOWLEDGEMENTS
I wish to express my sincere gratitude to the following persons and
institutions:
.:. My promoter, Or S.M. Meiring, for her supervision, guidance and support
during the study. She became a dear friend during this study and she
gave me all her support .
•:. Prof. H.F. Kotzé and Prof. G.H.J. Pretorius for their advice and
contributions .
•:. Prof. P.N. Badenhorst, head of the Department of Haematology and Cell
Biology, for his interest in the project.
.:. Prof. J. Harsfalvi for her support, contributions and friendship during my
stay in Hungary .
•:. Dr Robert Greenfield from American Diagnostica Incorporated for his
confidence in our research work by contracting a collaboration agreement
without which this work would not be possible. .
.:. My dear friends in the laboratory, Wendy, Liezel, & Almarie, for their
interest and support.
.:. All the other people in the department who supported and encouraged me .
•:. My family for their love and encouragement and a special thanks to my
mother for her encouragement, support, patience and love.
Most important, our Father in heaven for His love and support
99