1. health and fitness
2. disease
3. aids and hiv

FACTOR Vila LEVELS IN
PROTHROMBOTIC CONDITIONS
By
WHALLEY K. FONG
B.Sc, Simon Fraser University, 1993
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENT FOR THE DEGREE OF
MASTER OF SCIENCE
in
THE FACULTY OF GRADUATE STUDIES
Department of Biochemistry and Molecular Biology
We accept this thesis as conforming
to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA
August 1997
© Whalley K. Fong, 1997
In, presenting
degree
freely
at
the
available
copying
of
department
publication
this
of
in
partial
fulfilment
of
the
University
of
British
Columbia,
I
agree
for
this
or
thesis
reference
thesis
by
this
for
his
thesis
and
study.
scholariy
or
her
for
I. further
purposes
financial
gain
shall
that
agree
may
representatives.
requirements
be
It
not
that
the
Library
by
understood
be
an
shall
allowed
the
of
^tocAi^'sh^
The University of British Columbia
Vancouver, Canada
Date
DE-6 (2/88)
/^OJICMJ^A
make
that
of
copying
without
^frv/c^ij
it
extensive
head
permission.
Department
advanced
permission for
granted
is
for
my
my
or
written
ABSTRACT
The coagulation cascade is continuously in a state of preparedness to rapidly
respond to vascular damage at sites of injury. However, an overactivity of the hemostasis
system resulting in a hypercoagulable state can lead to thrombotic disorders such as
coronary heart disease (CHD) and stroke. Recent prospective studies have implicated
elevated factor VII (FVII) activity as an independent risk factor in incidences of coronary
heart disease. Indeed, factor VII and its cofactor, tissue factor (TF), are generally thought
to play the principle role in the physiological initiation of clotting which may help explain
the association between FVII and the risk of CHD. Recent evidence has indicated that trace
levels of the activated form of FVII, called factor Vila (FVlIa), circulates in the blood.
This active form is unique among the clotting factors in that FVlIa has a long half life of 2
to 2.5 hours, is highly resistant to inhibitors, and can auto-activate zymogen FVII.
However, conventional assays for FVIT activity employing tliromboplastin were not able to
separate FVIIa from its zymogen precursor, FVII. This has resulted in a great deal of
controversy over whether plasma FVIIa levels per se or total FVII (zymogen and FVIIa) is
more closely associated with the risk of CHD.
In the present study, a specific assay for factor Vila was developed using a
recombinant soluble form of tissue factor (sTF), deleted of the membrane binding region,
that is deficient in promoting the conversion of factor VII to factor Vila. However, this
soluble tissue factor, in the presence of phospholipid vesicles, retains its ability to clot
plasma using the pre-activated factor Vila in the plasma sample. The sTF was engineered
by using site-directed mutagenesis to delete the transmembrane domain of the TF gene and
the truncated protein was expressed in a eucaryotic expression system employing baby
hamster kidney (BHK) cells. Using the unique properties of sTF, an automated clot-based
assay was developed and optimized to quantitatively determine the concentration of FVIIa
in plasma samples. The assay was employed to measure FVIIa levels in a normal, healthy
population and in a group of patients in the late stages of complicated pregnancies.
ii
The FVUa assay developed in the present study had a sensitivity range of 20 pg/ml
to 1000 ng/ml FVHa and required only 5 ul of plasma per assay. The within-run and
between-run coefficients of variation were 3.9% and 4.9%, respectively, indicating high
reproducibility. The assay also showed excellent correlation (r = 0.998) with a commercial
FVUa kit produced by Diagnostica Stago in a patient comparison study over a range of 0.1
ng/ml to 30 ng/ml FVUa. The present study was standardized using an international FVUa
standard and therefore FVUa levels could be measured in concentration (ng/ml) or
international activity units (mU/ml).
Results of the initial population study on FVTJa confirmed the existence of trace
amounts of FVJJa in normal healthy subjects. The normal range in the present study was
0.7 to 4.8 ng/ml (11 to 100 mU/ml) and the mean plasma FVUa was 2.08 ± 0.90 ng/ml (42
± 15 mU/ml). There was a significant correlation between FVTJa levels and increasing age
but no significant difference between FVIIa levels in men and women. Factor Vila levels
in complicated pregnancy patients were significantly higher than in an age-matched group
of healthy women. The mean FVJJa in the patients was 4.1 ± 2.2 ng/ml (85 ± 42 mU/ml)
compared to 1.86 ng/ml (37 mU/ml) in healthy women. However, more extensive clinical
studies on FVIIa and FVH in other groups at risk of CHD are required to better establish
the importance of FVUa levels in prothrombotic conditions.
iii
TABLE OF CONTENTS
ABSTRACT
ii
TABLE OF CONTENTS
iv
LIST OF TABLES
vii
LIST OF FIGURES
viii
LIST OF ABBREVIATIONS
x
ACKNOWLEDGMENTS
xiii
INTRODUCTION
1. Hemostasis
1
2. The Coagulation Cascade
3
3. Characteristics of Tissue Factor
4
4. Characteristics of Factor VH/VIIa
7
5. Thrombosis
8
6. Traditional Assays for FVTJ Activity
10
7. Soluble Tissue Factor and the Factor VTIa-specific Assay
11
8. Previous Work by Others on FVIIa-specific Assays
14
9. Clinical studies using FVIIa Assay
15
10. Objectives of the Present Study
17
MATERIALS
19
METHODS
1. Polymerase Chain Reaction of sTF
PCR amplification of sTF
21
Agarose gel electrophoresis
22
GENECLEAN DNA Purification
22
2. Cloning and DNA sequence analysis of PCR fragment
Cloning of PCR fragment into pBS
23
Transformation of E.coli cells
23
Mini-prep recovery of plasmid DNA
24
DNA Sequence Analysis
25
3. Transfection of BHK Cells and Expression of sTF
Cloning of sTF into pNUT
26
Transfection of BHK cells
27
iv
SDS-PAGE
27
Western blot analysis
28
Tissue culture of sTF-BHK cell line
29
4. Purification of sTF from Tissue Culture media
Ammonium Sulfate fractionation
30
Anion-exchange Fast Protein Liquid Chromatography
30
Gel-filtration purification
31
5. Characterization of purified sTF
32
6. Development of Factor VTJa Assay
Preparation of phospholipid vesicles
32
Optimization of sTF-Reagent
32
Assay for Factor VTIa
33
Factor Vila Standard Curve
33
Comparison of BHK vs. £.c0/i*-derived sTF
34
7. Testing of FVIIa Assay
Precision Testing
34
Accuracy Testing
34
Plasma Stability studies
35
Individual variations
35
8. Factor Vila in Normal Populations
Ethical approval for blood collection
36
Subjects
36
Phlebotomy
36
Measurement of FVIIa
36
9. Factor Vila in Complicated Pregnancies
Ethical approval
36
Subjects
37
Clinical assessment
37
RESULTS
1. Polymerase Chain Reaction of sTF
38
2. DNA sequence analysis of sTF insert
38
3. Expression of sTF in BHK cells
40
4. Ammonium sulfate fractionation
44
5. High Q FPLC purification
46
6. Sephacryl S-100 HR Gel Filtration purification
49
v
7. Characterization of sTF protein
53
8. Optimization of assay reagents
55
9. Factor VJJa Standard curve
59
10. Comparison of BHK-derived sTF and £.co//-derived sTF
62
11. Testing of FVTJa assay
62
12. Plasma FVIIa stability testing
66
13. Intra-individual variations in plasma FVTIa
69
14. Factor Vila levels in a Normal population
70
15. Factor Vila levels in Complicated Pregnancy patients
73
DISCUSSION
1. BHK-derived glycosylated sTF
2. Factor Vila assay
76
77
3. Comparison to previous FVTIa assays
4. Handling of plasma samples
79
5. Factor Vila in normal populations: effects of age and gender
6. Factor Vila in prothrombotic conditions: Complicated pregnancies
7. Future work
81
8. Possible mechanisms of FVTJa involvement in thrombotic disorders
9. Implications for treatment of thrombotic disorders
REFERENCES
vi
80
83
85
87
88
LIST O F TABLES
I.
Effects of freezing on plasma FVIIa levels
66
II.
Comparison of EDTA vs. citrate as plasma anticoagulant
68
HI.
Intra-individual variation in FVIIa levels
69
IV.
Age distribution and FVIIa levels in study sub-populations
72
V.
Clinical details of complicated pregnancy patients
73
VI.
Hemostatic variables of complicated pregnancy samples
74
Via.
Hemostatic variables for new complicated pregnancy samples
75
Vii.
FVHa levels in normal populations from several studies
81
vii
LIST OF FIGURES
1. Coagulation Cascade
2
2. Human Tissue Factor
4
3. X-ray crystal structure of extracellular domain of Tissue Factor
5
4. Dual cofactor function of Tissue Factor
6
5. Human Factor VII
7
6. Factor VII Coagulant (FVILC) Assay
10
7. Factor Vila Assay
12
8. Human TF gene and sTF gene
13
9. Oligonucleotide primers for PCR of sTFi_2i9
21
10. DNA Sequence Analysis Primers
26
11. PCR amplification of sTF gene
39
12. Not I serening of Bluescript-sTF clone
41
13. Not I and EcoRl digestion screening of pNUT-sTF clone
41
14. pNUT expression vector with cloned sTF insert
42
15. Western Blot of tissue culture media from several sTF-BHK cell lines
43
16. 55% Ammonium Sulfate fractionation
45
17. High Q FPLC purification
'
47
18. Calibration curve for Sephacryl S-100 HR Column
50
19. Sephacryl S-100 HR purification
51
20. Purification Summary gel
54
21. Clot time vs. sTF concentration
57
22. Clottimevs. Phospholipid concentration
58
23. FVUa standard curve using Sigma FVII-def plasma
59
24. FVIIa standard curve using George King FVII-def plasma
60
25. Final FVIIa Standard Curve
61
26. Comparison of BHK and E.coii-derived sTF in FVIIa Assay
63
viii
27. Patient comparison study results
64
28. Relation between Factor Vila and Age
71
29. Factor Vila Conversion Curve
79
ix
LIST O F ABBREVIATIONS
A
Adenosine
A
Angstrom
Amp
Ampicillin
APS
Ammonium persulfate
API i
Activated Partial Thromboplastin Time
BHK
Baby Hamster Kidney
Bis
N,N'-methylenebisacrylamide
BSA
Bovine serum albumin
C
Cytosine
cDNA
Complementary DNA
CHD
Coronary Heart Disease
CHO
Chinese Hamster Ovary
C-terminal
Carboxy-terminal
C. V.
Coefficient of variation
DEAE
Diethylaminoethyl
DHFR
Di-hydrofolate reductase
DMEM
Dulbecco's modified essential medium
DMF
Dimethylformamide
DNA
Deoxyribonucleic acid
dNTP
Deoxyribonucleotide triphosphate
EDTA
Ethylenediamine tetraacetic acid
EGF
Epidermal Growth Factor
ELISA
Enzyme-Linked Immunosorbent Assay
FBS
Fetal bovine serum
FTBR
Fibrinogen
FPLC
Fast Protein Liquid Chromatography
FVII
Factor VII
FVIIa
Activated Factor VII or Factor Vila
FVUa-TF
Factor VHa - Tissue Factor
FVII: Ag
Factor VII Antigen assay
FV1I:C
Factor VII Coagulant activity assay
FIX
Factor DC
FX
Factor X
FXa
Activated Factor X or Factor Xa
x
G
Guanosine
Gla
Gamma carboxylated glutamic acid
HEPES
N-2-Hydroxyethylpif^razine-N'-2-ethane sulfonic acid
HR
High Resolution
HRT
Hormone replacement therapy
IgG
Immunoglobulin G
IHD
Ischaemic Heart Disease
IPTG
Isopropyl-p^D-Thiogalactopyranoside
kb
Kilobase
kDa
KiloDaltons
LB
Luria-Bertani (media)
LUV
Large unilamellar vesicles
MI
Myocardial infarction
MTX
Methotrexate
MT-1
Zinc metallo-protein promoter
N/A
Not available
NPHS
Northwick Park Heart Study
NPP
Normal pooled plasma
N-terminal
Ammo-terminal
OD
Optical density
PAGE
Polyacrylamide gel electrophoresis
PBS
Phosphate Buffered Saline solution
pBS
Bluescript (vector)
PC
Phosphatidylcholine
PCR
Polymerase chain reaction
PE
Phosphatidylemanolamine
PL
Phospholipid
PROCAM
Prospective Cardiovascular Muenster Study
PS
Phosphatidylserine
psi
Pounds per square inch
PT
Prothrombin Time
r
Regression coefficient (linear regression analysis)
RBC
Rabbit Brain Cephalin
rpm
Revolutions per minute
SDS
Sodium dodecyl sulfate
STA
Stago
xi
sTF
Soluble Tissue Factor
T
Thymine
Taq
Thermophius aquaticus
TAE
Tris-base, acetic acid, EDTA buffer
TBS
Tris-buffered saline solution
TBE
Tris-base, borate, EDTA buffer
TE
Tris-base, EDTA buffer
TEMED
N, N, N', N'-Tetiamethylethylenediamine
TF
Tissue Factor
TFPI
Tissue Factor Pathway Inhibitor
Tris
Tri (hydroxymethyl) aminomethane
U
Units of enzyme activity
USG
Ultroser G
uv
Ultra violet
v/v
Volume-to-volume ratio
w/v
Weight-to-volume ratio
X-Gal
5-Bromo-4-chloro-3-indolyl-p-D-galactopyranoside
,
xii
ACKNOWLEDGMENTS
I was fortunate enough to work alongside an extraordinarily talented group of
researchers without whom the fruits of this work would not have emerged. I owe special
thanks to J.P. Heale and Allyson Davey who performed the preliminary stages of the
project described in this report. As well, I wish to acknowledge the contributions of Dr.
Dana Devine, Dr. Haydn Pritchard, Jeff Hewitt, Alexis Maxwell, Bea Tam, Mark Brown,
Leisa Stenberg, Gord Rintoul, Michael Murphy, and Hung Vo for their technical advice
and expertise. I wish to thank Dr. Louis Wadsworth and Dr. Cedric Carter for their help
with the clinical study, Dr. James Morrissey for the soluble tissue factor and the original
FVTIa assay, and Linda Spiller who was Ms. Everything when it came to blood. To my
supervisor, Ross MacGillivray, I cannot express my full gratitude for the support and
generosity you extended to me both as a scientist and as a friend. I also must thank my
mother and father, my brothers, Ian, Glance, and Glenn, and my sister, Karling, for their
love and support from the day I was born. Finally, I thank my wife, Naomi Uranishi.
You were the light in the darkness that showed me the way.
xiii
INTRODUCTION
1.
Hemostasis
Hemostasis, the physiological arrest of bleeding at sites of vascular injury, is one of
the most vital protective mechanisms that preserves the internal environment of vertebrates
(1). Not only does hemostasis prevent excessive bleeding but it also blocks the portal of
entry for invasive organisms (2). The complex mechanism of hemostasis involves the
interaction of three events: a transitory constriction of the damaged blood vessels at the site
of injury, the formation of a platelet plug, and the coagulation of blood (3). The first two
events, comprising the "primary hemostatic" response (4), form a soft, weak mechanical
plug that can stem the bleeding from minor injuries (1). However, hemorrhage from more
formidable wounds requires the "secondary phase" of hemostasis, blood coagulation. The
coagulation or clotting of blood results in the formation of a tough fibrin clot that provides a
scaffold for a mechanical plug to prevent blood loss while also allowing for endothelial cell
repair and the subsequent release of the fibrin clot by the fibrinolytic system (4).
The process of coagulation involves a group of blood-borne clotting proteins that
circulate as inactive zymogens. The system responds to tissue damage or other stimuli by
initiating a series of proteolytic activation reactions that lead to a cascade or waterfall of
activated clotting factors resulting in the rapid formation of a fibrin clot (Figure 1). Under
normal conditions, the hemostasis system provides an elegant and efficient system that
confers protection against vascular trauma.
However, dysfunction of the hemostatic system can lead to several different and
potentially life-threatening pathological states. A deficiency in the hemostatic system
caused by the absence or decreased activity of just one of the clotting factors can lead to a
bleeding disorder with an associated inability of the blood to clot normally. The two most
1
INTRINSIC
PATHWAY
Factor XII
Anionic Surface
Kininogen
_ Prekalllkrien
EXTRINSIC
PATHWAY
0^
-YTissue Damage I
Tissue Factor.
Factor VII
Factor X
COMMON
PATHWAY
Feedback Loop
Fibrin
Factor XHIa
Cross-linked Fibrin Clot
Figure 1. Coagulation Cascade. A simplified diagram illustrating the separation into
the two classic pathways, the intrinsic and extrinsic pathways, that converge at factor X
into the Common pathway that eventually leads to the formation of a fibrin clot.
common of these clotting disorders are hemophilia A and hemophilia B which are caused
by deficiencies in factors VIII and LX, respectively. Rare deficiencies in factors V, VII, X,
XI, XII, XIH, prothrombin, and fibrinogen as well as multiple factor deficiencies have also
been described (5).
On the other extreme is an overactivity of the hemostatic system possibly caused by
increased levels or overactivity of one or more of the clotting factors. The resulting
hypercoagulable state could lead to thrombotic disorders such as coronary heart disease,
acute myocardial infarction, and strokes. Recent studies have provided strong evidence for
2
a link between increased levels of certain clotting factors and an increased risk of
thrombotic events (6, 7, 8, 9). Thus, the hemostatic system must be tightly regulated to
maintain the very delicate balance required for the health of the individual.
2.
The Coagulation Cascade
As seen in Figure 1, the clotting cascade has been traditionally separated into two
systems, the intrinsic and extrinsic pathways, based on the location of the initiating factors
in each pathway. The mtrinsic pathway (so named because all of the components are found
in plasma) is initiated by contact of blood with anionic molecules such as kininogen or
kallikrien. The extrinsic pathway is initiated by the release of tissue factor (TF) from the
damaged tissue (located outside of the vasculature and thus extrinsic). The two series of
proteolytic activation reactions converge with the activation of factor X (FX) and then share
a common pathway leading to the formation of a cross-linked fibrin clot. Calcium ions and
phospholipid membranes are essential for the proteolytic activity of the clotting factors
(10).
In reality, there are many interactions between the two pathways and feedback
loops that regulate blood coagulation. For example, thrombin can feedback and activate
factor XI as shown in Figure 1. Other studies have shown that the extrinsic factors, factor
VII (FVII) and tissue factor (TF), play important roles in the activation of the intrinsic
system via activation of factor IX (FIX) in addition to directly activating factor X of the
common pathway (11, 12). Thus it is generally thought that the extrinsic pathway is the
primary hemostatic response to tissue damage with the factor Vila-tissue factor (FVUa-TF)
complex serving as the physiological initiator of blood coagulation (13, 14). Because of
this principal role in coagulation, the FVIIa-TF complex has become a prime target for the
investigation of thrombotic disorders.
3
3.
Characteristics of Tissue Factor
Human TF, an essential cofactor of FVIIa, is a ubiquitous membrane bound
glycoprotein of apparent molecular weight 45-47 kDa (15,16) on SDS-PAGE. A diagram
of TF is shown in Figure 2.
Figure 2.
Human Tissue Factor.
The cofactor of FVIIa consists of a large
extracellular domain followed by a single pass transmembrane domain and a short
cytoplasmic domain. There are three glycosylation sites and two disulfide bonds in the
extracellular domain.
TF is expressed as a 295-residue polypeptide which is processed to remove a 32-residue
leader peptide. The mature protein, consisting of a 219 amino acid extracellular domain
anchored to the cell membrane by a 23-residue transmembrane domain (17), is present on
subendothelial components of the vessel wall and has also been localized to^atherosclerotic
plaques (18,19). TF is glycosylated via three N-linked glycosylation sites at Asn-13, Asn126, and Asn-139 but glycosylation does not appear to be necessary for function (17).
4
Excluding post-translational modification, TF has a predicted molecular weight of 29.5 kDa
(15). TF also contains two pairs of cysteine residues on its surface domain that form
covalently stable disulfide bonds that are required for co-factor function (17).
The extracellular domain of TF (residues 1-219) has been crystallized following
partial subtilisin digestion and the X-ray structure solved to 2.4 A (20) resolution (Figure
3).
Figure 3. X-ray crystal structure of extracellular domain of Tissue Factor.
The extracellular domain consists of two fibronectin-like domains end-to-end forming an
extended structure. The normal site of membrane attachment is located at the bottom of the
figure. Courtesy of (20).
The model shows that the extracellular portion of TF adopts an extended form consisting of
two fibronectin-like domains oriented end-to-end (length = 115 A, diameter = 40-50 A)
(21). Experiments with TF-gene knockout mice have shown thattissuefactor is essential
for the development of the vasculature during embryogenesis and thus mutations in the TF-
5
gene are lethal (22). The absence of any reported homozygous mutations in the human TFgene is consistent with TF being essential for life.
Under normal conditions, TF is sequestered from the vasculature. However, upon
tissue damage, TF is exposed to the blood and forms a complex with FVII. This triggers
the activation of FVII to FVIIa and thus initiates the coagulation cascade (Figure 1). TF
actually fulfills two essential cofactor functions for FVIIa. First, TF serves as the cofactor
during activation of FVII to FVIIa and second, formation of a complex with TF
significantly increases (up to 16,000-fold) the serine protease activity of FVIIa on its
substrates (FX and FLX) (23) as shown in Figure 4.
Figure 4. Dual cofactor function of Tissue Factor. Tissue factor (TF) (a) serves
as a cofactor during activation of factor VII (FVII) zymogen to factor Vila (FVIIa) and (b)
serves as a cofactor for FVIIa serine protease activity on factor X (FX). PL =
Phospholipid surface, Ca = Calcium ions, FXa = factor Xa.
2+
6
Recently, trace amounts of plasma TF (172±135 pg/ml) have been detected by a very
sensitive ELISA method (24) and these low levels of TF may be important for the
generation and activity of trace amounts of pre-activated FVIIa in the plasma.
4.
Characteristics of Factor VLWIIa
Factor Vila belongs to a family of plasma proteins called the Vitamin K-dependent
serine proteases which also includes factors FXa, Xa, and Protein Ca. They share a
common protein structure composed of an N-terminal Gla-domain, followed by two
epidermal growth factor (EGF)-like domains, and a C-terminal serine protease catalytic
domain (Figure 5).
Figure 5. Human Factor V n . Factor VII, the zymogen precursor to the Vitamin Independent serine protease factor Vila, has several characteristic structural motifs including
an N-terminal Gla domain, two epidermal growth factor like domains, and a C-terminal
serine protease catalytic domain. Cleavage of the Argl52-Ilel53 bond activates factor VII
to factor Vila. Courtesy of (90).
7
Factor VII is secreted by the liver as a single chain zymogen of molecular weight 50 kDa
and is activated by proteolytic cleavage of the Argl52-Ilel53 bond to form FVIIa. The
resulting two polypeptide chains are joined by a disulfide bond between Cys-135 and Cys262 in activated FVIIa (25). In vitro, this activation can be catalyzed by FXa, FIXa,
FXIIa, and thrombin and requires the presence of TF as a cofactor. Recently, studies have
shown that FVIIa can also autoactivate itself (14) which is an essential property for an
initiation factor. However, it is still not well understood which of the proteases is most
responsible for FVU activation in vivo.
In addition, factor Vila has several other unique properties that make it a good
candidate for the physiological initiator of clotting. Unlike the other serine protease clotting
factors, FVIIa is highly resistant to plasma protease inhibitors. As well, once activated
most serine proteases have a half-life of a few seconds to minutes, while FVIIa has an in
vivo half-life of 2-2.5 hours (14, 26). Recent studies by several groups have confirmed
that approximately 0.5 - 1% of factor VII circulates in plasma in the pre-activated FVIIaform (26, 27) and these trace levels of FVIIa may serve a "priming" function in the
coagulation cascade. Small amounts of FVIIa-TF complexes may be involved in the
autocatalytic activation of more FVIJ-TF and/or in the generation of an initial burst of FXa
that could back-activate TF-bound FVII during coagulation. If FVIIa indeed fulfills this
role as the physiological initiator of clotting, then elevated plasma FVIIa levels may
contribute to hypercoagulable states leading to thrombotic conditions (14).
5.
Thrombosis
An overactivity of the hemostasis system can lead to a hypercoagulable state
resulting in an increase in risk of thrombotic disorders. The Northwick Park Heart Study
(NPHS) (6,7) first implicated factor VII as a potential risk factor for coronary heart disease
(CHD). This large scale, long term prospective study found that high levels of FVII
8
coagulant activity (FVII:C) were associated with an increased risk of CHD, especially for
events within 5 years of recruitment. During the 5 year period, FVII:C was a better
independent predictor of CHD than even cholesterol levels (7). The subsequent follow-up
study at Northwick Park found that FVILC was more strongly associated with fatal events
of ischemic heart disease (IHD) than non-fatal events. An increase of one standard
deviation in FVILC was found to increase the probability of dying of IHD by 44% (28).
Several other studies have corroborated this relationship between factor VII
coagulant activity and CHD risk. The PROCAM (Prospective Cardiovascular Muenster
Study) study re-affirmed FVILC as a strong independent risk factor for fatal coronary
events (8). A number of other groups have shown significant correlations between FVILC
and serum cholesterol (29, 30), triglyceride levels (31, 32), CHD risk scores (33, 34),
diabetes (35, 36), late pregnancy (37), and high fat diets (38, 39, 40, 41).
The localization of tissue factor to atherosclerotic plaques (18) provides insight into
a possible mechanism for the relationship between extrinsic activation and CHD events.
The rupture of an atherosclerotic plaque is known to be an integral event preceding
occlusive thrombosis and acute myocardial infarction (MI) (19,42, 43). The exposure of
the plaque's necrotic core containing TF to the vasculature following rupture provides the
source of thrombogenecity needed for the thrombotic event (18). Ruddock and Meade (28)
have suggested that the severity of the occluding thrombus depends on the activation state
of the extrinsic factors. Specifically, high levels of FVII activity may facilitate more
extensive initiation of clotting following exposure of TF after atherosclerotic plaque rupture
(28).
9
6.
Traditional Assays for FVII Activity
The Northwick Park Heart Study and most other studies on hemostatic variables
and CHD risk factors have measured FVII activity using the standard factor VU Coagulant
activity (FVILC) assay. This assay employs thromboplastin, a heterogeneous mixture of
the organic extracts of emulsified mammalian brain or lung tissue (44), whose active
clotting agent is tissue factor. Because of TF's ability to act as both a cofactor for the
activation of FVII to FVIIa and as a cofactor for FVIIa proteolytic activity (Figure 4),
FVILC actually measures an aggregate of both FVII zymogen and FVIIa levels in the
plasma (Figure 6).
Factor X
TF
FVIIs
TF
Factor Xa
Plasma Sample
Clotting Time
Figure 6. Factor VII Coagulant (FVIItC) Assay. Because of the dual function of
tissue factor (TF), the FVILC assay measures an aggregate of both FVU and FVUa levels.
In addition, variations in the configuration and reagents used for the FVILC assay
between laboratories are thought to affect its relative sensitivity to FVII vs. FVIIa (45).
Because of a lack of standardization of the FVILC assay, the results from various studies
may implicate different aspects of FVII activity or form. For example, the NPHS FVILC
assay was found to be considerably more sensitive to plasma FVIIa levels than the assay
used in the PROCAM study (45). This was caused by the virtual absence of protein C in
10
the substrate plasma used in the NPHS assay. As well, differences were found in the
source of tissue thromboplastin used. Human brain, rabbit brain, human placental, and
bovine brain thromboplastin are commonly used in different laboratories and it has been
shown that thromboplastin from bovine sources is most sensitive to FVIIa levels (45). A
third problem arises from the practice of measuring FVILC as a relative value with 100%
activity defined as the FVILC level for a pooled plasma sample. However, FVILC activity
of pooled plasma samples may differ from one population to another (27).
There are other assays that measure various aspects of factor VII activity and
quantity. The FVII antigen (FVILAg) assay is an ELISA method that measures the total
amount of FVII (FVII and FVIIa). The FVII "coupled" amidolytic assay first activates all
of the FVII using thromboplastin and the FVIIa, in turn, generates FXa which cleaves a
chromogenic substrate (46). A ratio of FVILC and either of these two measures of total
FVII was often used as an estimate of the "activation state" of FVII. As well, more general
clotting assays such as prothrombin time (PT), which also uses thromboplastin, can be
used to measure the activity of the extrinsic system (44). However, because most of these
assays also use thromboplastin, they suffer the same problems as the FVILC test.
7. Soluble Tissue Factor and the Factor Vila-specific Assay
Because of these innate problems with the standard FVILC assay, there has been a
great deal of controversy over the results of the NPHS and other such studies concerning
whether plasma FVIIa levels per se or total FVII (zymogen plus FVIIa) is more closely
associated with the risk of heart disease. Although providing a relative measure of the
amount of pre-activated FVIIa, the FVILC assay cannot separate previously activated FVIIa
from FVII activated by thromboplastin in the clotting assay because of the dual function of
TF (Figure 6). Thus, the FVILC assay does not provide an accurate quantitative measure
of plasma FVIIa levels due to the interference of zymogenic factor VII. An assay that is
specific for the measurement of factor Vila and insensitive to zymogenic FVH is required to
11
be able to assess the roles of FVIIa versus total FVII independently as risk factors for
thrombotic disorders.
Recently Dr. Morrissey at the Oklahoma Medical Research Foundation developed a
soluble form of tissue factor (sTF), deleted of the membrane binding region, that is
deficient in promoting the conversion of factor VII to factor Vila. However, this truncated
TF, in the presence of phospholipid vesicles and calcium ions, retains its ability to clot
plasma using the pre-activated factor Vila in the plasma sample (47). This unique partial
functionality of sTF can be exploited to develop a clot-based assay with extreme sensitivity
for factor Vila (Figure 7).
Factor X
FVIIa
sTF
Factor Xa
Plasma Sample
1
Clotting Time
Figure 7. Factor Vila Assay. Since soluble tissue factor (sTF) cannot activate FVII
to FVIIa, only pre-activated plasma FVIIa contributes to the clotting assay. Thus, the
FVIIa assay can directly measure FVIIa levels without interference from zymogenic FVII
as in the FVII coagulant assay.
Such an assay could be used to quantitatively determine the FVIIa levels in plasma without
interference from activation of zymogenic FVII.
Human TF is encoded by a 12.4 kb gene comprised of 6 exons and 5 introns
(Figure 8a) (48) located on chromosome 1 (49). Exon 1 codes for the translation initiation
12
domain and a 32-residue leader sequence that includes a signal peptide which is
subsequently cleaved. Exons 2-5 code for the 219-residue extracellular domain of the
tissue factor protein and exon 6 codes for the 23-residue transmembrane domain, the 21residue cytoplasmic domain, and the 3'-untranslated region (17). As shown in Figure 8b,
soluble tissue factor was produced by using recombinant DNA techniques to delete exon 6
of the TF gene by adding a stop codon at the end of exon 5 (50). Thus the recombinant
sTF protein consisted of only the 219-residue extracellular domain of native TF after
processing.
Tissue Factor
(295 residues)
(a)
T
Translation
Initiation
Domain
(123 bp)
Y
Extracellular
Domain
(700 bp)
Transmembrane
& Cytoplasmic
Domain
(1278 bp)
(b)
T
Translation
Initiation
J
Y
Soluble Tissue Factor
(219 residues)
Extracellular
Domain
Figure 8 (a) Human T F gene, (b) sTF gene, (a) Human TF gene is composed of
6 exons: exon 1 codes for translation initiation domain, exons 2-5 code for the extracellular
domain, and exon 6 codes for the transmembrane and cytoplasmic domains, (b) Deletion
of exon 6 results in soluble tissue factor.
Using the unique properties of sTF, Morrissey et al developed a clotting time based
assay specific for factor Vila (26,51). The method was a modification of the FVILC assay
(Figure 6) with an sTF-reagent replacing thromboplastin as the active clotting agent. Since
sTF cannot activate zymogenic FVII, the level of extrinsic pathway initiated clotting was
13
determined only by the endogenous plasma FVIIa level (Figure 7). FVIIa levels could then
be quantitated by plotting a standard curve of FVIIa concentrations versus clotting times.
Using this assay, it was possible to directly measure the levels of pre-activated FVIIa
circulating in the blood without interference from zymogen FVII.
8.
Previous Work by Others on FVIIa-specific assays
The FVIIa assay developed by Morrissey et al (26) was a clot-based assay
dependent on the limited procoagulant properties of sTF. Recently, Kario et al (27)
modified this sTF-based technique to develop a fluorogenic assay for FVIIa. Their assay
defined the reaction end point as the time elapsed to reach a relative fluorescence of 0.05
during hydrolysis of a fluorogenic thrombin substrate. The fluorogenic assay used less
reagents and exhibited high reproducibility but was less sensitive (0.2 - 1000 ng/ml) than
Morrissey's assay (0.01 - 10,000 ng/ml) (26).
Another assay specific for FVIIa was recently described by Philoppou et al (25).
Unlike Morrissey and Kario's functional assays, this new assay avoids using sTF by
employing an ELISA method. Philoppou et al were able to prepare a FVIIa-specific
antibody raised against the region of FVII exposed following its activation by cleavage at
Argl52-Ilel53. The FVIIa ELISA showed excellent correlation with the functional assay
over a range of 0.05 - 18 ng/ml FVIIa but measured plasma FVIIa levels differed
significantly from the sTF-based assays. Another problem with Philoppou's assay is that
ELISAs are innately time-consuming and labor-intensive requiring prolonged blocking and
antibody incubation times.
An important consideration in the development of any assay is the ability to
compare results between laboratories and studies. This was a major obstacle in the
interpretation of studies using the FVILC assay. An sTF-based FVIIa assay will inherently
have lower variability because the reagents are more strictly defined. It is a quantitative
assay that measures the absolute amounts of protein based on the activity of FVIIa. Thus,
14
the determined FVIIa value is completely dependent on the FVIIa standard curve and
therefore the FVIIa calibration standards. A potential problem resides in the fact that there
are several commercial suppliers of FVIIa (Novo Nordisk, Enzyme Research, etc) as well
as various in-house preparations, each of which may vary in their specific activity despite
having the same mass/volume concentration. This can be due to differences in the purity of
the FVIIa or loss of activity due to instability of the FVIIa. Thus, the National Institute of
Biological Standards (in the U.K.) has proposed the adoption of an International factor
Vila Standard based on specific activity (52).
9.
Clinical studies using FVIIa assay
Since the development of the original FVUa assay in 1993 by Dr. Morrissey, there
has been a considerable number of clinical studies taking advantage of the new assay to
improve our understanding of the role of factor Vila in prothrombotic conditions. Several
studies confirmed the presence of circulating FVIIa in plasma at concentrations of 3.6
ng/ml (26), 2.5 ng/ml (27), and 4.1 ng/ml (53) in various populations corresponding to
approximately 0.5 - 1% of the total plasma FVII. In all cases, there was considerable
variation in FVIIa levels between individuals; "normal" FVUa ranges were as wide as 0.4 14.3 ng/ml (53). The studies also showed a general trend towards increasing FVIIa levels
with age (26, 27, 53).
Studies have also examined the relationship between FVIIa levels and other CHD
risk factors. There has been mixed results with regards to an association between plasma
FVUa and elevated lipid levels. Despite the well known correlation of FVILC and FVII: Ag
with serum triglycerides (31, 32), several groups have shown that plasma FVIIa levels
were not correlated with serum triglycerides at all (40, 53, 54). However, studies have
consistently shown that FVIIa levels are transiently increased in the post-prandial
hypertriglyceridemic state following a high fat meal (38, 39,40,41,55). Studies on total
cholesterol and FVJJa have provided more conflicting results. Some groups have found no
15
significant correlations with cholesterol (27, 56) while others have shown a significant
relationship (53).
As well, different studies using various methods to assess CHD risk have come to
differing conclusions. One group found that factor Vila levels were not elevated in men
who had suffered a myocardial infarction at a young age (56). However, Kario et al (27,
54) did find that elderly patients with arterial cardiovascular diseases showed an increase in
plasma FVUa levels. Other groups have found that FVUa increased with a CHD-risk score
based on a number of CHD-risk factors in a larger general population (33, 53).
In several other potentially prothrombotic conditions, FVIIa levels have also been
shown to be elevated. Increased FVIIa levels were associated with non-insulin dependent
diabetes (35, 57), late pregnancy (26), post-menopausal women (53), cancer patients (58),
oral contraceptive-users (59), and patients suffering from juvenille arthritis (60). It is has
been postulated that some of these conditions result in endothelial tissue damage which may
activate the extrinsic pathway through release of subendothelial TF at sub-thrombotic levels
thereby increasing FVIIa levels.
On the other hand, FVIIa levels were generally not found to be associated with
smoking (26) or male gender (26, 27), factors that usually predispose CHD. As well,
FVUa levels were found to be decreased in patients treated with oral anticoagulant therapy
(26) and hormone replacement therapy (53). There also appears to be a genetic link to
FVIIa levels. Healthy subjects who are heterozygous for a common polymorphism of the
FVII gene called Mspl M2, a single G-to-A substitution changing an Arg to a Gin at
residue-353, have approximately 50% lower FVIIa levels than people homozygous for the
Mspl M l allele (61).
Some evidence suggests that the Mspl M2 allele confers a
cardioprotective effect.
Thus, there is still great potential to clarify our understanding of the role of factor
Vila in coronary heart disease and other potentially thrombotic conditions using the FVUa
16
assay. With such knowledge, more efficacious therapeutics can be developed to treat these
diseases which take the lives of nearly half a million North Americans every year.
10. Objectives of the Present Study
The purpose of the present study was to develop a fibrin clot-based FVIIa assay
using sTF in Vancouver for use in clinical studies. Recombinant soluble tissue factor was
produced by using mutagenic PCR oligonucleotide primers to delete the transmembrane
and cytoplasmic domains of the TF cDNA, cloning the truncated TF gene into a pNUT
expression vector, and expressing the sTF protein in a eucaryotic expression system
employing baby hamster kidney (BHK) cells.
The FVIIa-specific assay was developed based on the method of Morrissey (26,
51). However, the present study's FVIIa assay differs from Morrissey's assay in several
respects. The original assay employed £.co//-derived sTF and rabbit brain cephalin (RBC)
as a phospholipid source. The assay in the present study uses a BHK-derived glycosylated
form of sTF and a pure phospholipid mixture in place of RBC and studies were performed
to optimize the reagents and conditions. These changes could conceivably increase the
activity of the sTF-reagent and thus improve the efficiency of the FVIIa assay and provide
better accuracy and reproducibility. A comparison study of the two sources of sTF was
performed to determine if one source is superior. In addition, the present study's FVIIa
assay was automated using an STA coagulometer from Stago Diagnostica (Paris, France).
The robustness of the new assay was assessed using precision and accuracy
testing. Precision was judged on within-run and between-run reproducibility and accuracy
was judged by comparison of the new FVIIa assay with a commercial FVIIa kit from
Diagnostica Stago. To ensure, results from the new assay can be compared between
different laboratories, the assay was calibrated using the international FVIIa standard (52)
in addition to calibration for measurement in concentration (ng/ml).
17
An initial population study on FVIIa was performed. Factor Vila levels were
measured in a normal, healthy population (50 subjects) to determine the mean level and
normal range for plasma FVIIa and examine trends in age and gender. As well, a group of
women in the late stages of complicated pregnancies was also studied to examine changes
in FVIIa levels that accompany pregnancy and determine anomalies caused by the
complications.
18
MATERIALS
Acrylamide, N,N'-methylbisacrylamide, ammonium persulfate, bromophenol blue,
TEMED, and Econo-pac HighQ FPLC cartridges were purchased from Bio-Rad
Laboratories. Bactero-tryptone, bactero-agar, and yeast extract were purchased were Difco
grade and obtained from the Grand Island Biological Company. IPTG and X-Gal were
purchased from 5-prime —> 3-prime. Protran nitrocellulose membranes were purchased
from Schleicher & Schuell. Tissue culture flasks and roller bottles were obtained from
Corning. Centricon 30 microconcentrators, YM-30 ultrafiltration membranes, and a spiral
cartridge concentrator (CH2PRS) fitted with a S10Y10 cartridge were from Amicon.
Dialysis tubing was purchased from Spectrum Medical Industries. Ampicillin, ethidium
bromide, benzamidine, blue dextran, hemoglobin, ovalbumin, trypsin inhibitor,
cytochrome c, and bovine serum albumin were obtained from Sigma Chemical Company.
Agarose, goat anti-mouse immunoglobulin G-alkaline phosphatase conjugate, Not I and
EcoRl restriction enzymes, React 3 buffer, T4 DNA ligase, ligase buffer, HEPES
Buffered Saline, trypsin, Dulbecco's modified essential medium-Ham F12 with phenol red
dye, fetal bovine serum, and Ultroser G were from Gibco-BRL. Methotrexate was
obtained from David Bull Laboratories, antibody specific for the extracellular domain of
tissue factor was from Enzyme Research Laboratories, Coomassie blue dye was from EM
Science, and calf intestinal alkaline phosphatase and sephacryl S-100 HR chromatography
resin were purchased from Pharmacia-LKB. Deoxy-ribonucleotides were purchased from
Perkin-Elmer and Taq polymerase was prepared from a clone in our laboratory. All other
chemicals were of reagent grade or higher and were purchased from either Sigma Chemical
Company, Fisher Scientific Company, or British Drug Houses Limited.
A T7 DNA sequencing kit was obtained from Pharmacia Biotech and two
STACLOT VIIa-rTF kits were purchased from Diagnostica Stago. Recombinant factor
Vila was purchased from Enzyme Research Labs. Immunodepleted human FVII-deficient
19
plasma was purchased from Sigma Chemical Co.. Congenital human FVII-deficient
plasma was purchased from George King Biomedical. According to the suppliers, these
deficient plasmas had less than 1% residual FVJJ activity. Pure phosphatidylcholine (PC),
phosphatidylserine (PS), and phosphatidylethanolamine (PE) were purchased from
Northern Lipids Incorporated. Siliconized Vacutainer tubes containing 3.2% trisodium
citrate were obtained from Becton Dickinson.
20
METHODS
1. Polymerase Chain Reaction of sTF
PCR amplification of sTF. The polymerase chain reaction was used to
amplify the coding region for soluble tissue factor from a human cDNA library.
Oligonucleotides primers with engineered NotI restriction sites and termination codons
were designed with the aid of "Oligo" software (freeware) and synthesized on an Applied
Systems 391 DNA Synthesizer, PCR-MATE, using deoxy-ribonucleotides purchased from
Perkin-Elmer. The primers used are shown in Figure 9.
a. 5'-primer
5' - ACA GCG GCC GCT AGA CATGGA GAC CCC TGC C - 3'
Not I site
START
b. 3' - primer
5' - ACA GCfi GCC GCT CAT CAT TCT CTG AAT TCC CCT TTC TCC - 3'
Not I site
2 STOP
Figure 9.
Oligonucleotide primers for PCR of sTFi.219.
The primers were
designed with engineered Not I restriction sites for cloning. An ATG start codon was
included in the 5'-primer and two TCA stop codons were included in the 3'-primer.
PCR amplification was carried out on a Perkin Elmer Cetus DNA Thermal Cycler 480
under the following conditions: 5 pi of human brain cDNA library (100 ng), 5 pJ of 10X
PCR reaction buffer E, 1 pi of 10 mM deoxyribonucleotide triphosphates (dNTP), 1 p:l of
5'-primer (100 ng/ul), 1 ul of 3'-primer (100 ng/ul), 36.5 LLI of H 0 , and 0.5 ul of
2
Thermophius aquitus (Taq) DNA Polymerase (1 U). The human brain cDNA library was
constructed in the Escherichia coli expression vector pCDNA3 (Clonetech). Buffer E is a
10X PCR reaction buffer composed of 0.67 M Tris, pH 9.0, 0.155 M ammonium sulfate,
21
0.1 M 2-mercaptoethanol, and 25 mM magnesium chloride. The PCR cycle was begun
with a hot start at 94°C (4 minutes) and then was set to the following cycle:
Denaturation: 94°C, 30 seconds
Annealing:
50°C, 30 seconds
Extension:
72°C, 60 seconds
for a total of 20 cycles followed by a 10 minute final extension at 72°C.
Agarose gel electrophoresis. The PCR reaction mixture was checked using
agarose gel electrophoresis. The electrophoresis was performed in a Bio-Rad Mini-sub
DNA Cell apparatus. The electrode buffer used was IX TAE (diluted from a 50X T A E
stock of composition 2 M Tris base, pH 7.5, 1 M glacial acetic acid, 0.1 M EDTA); 2%
agarose gels were prepared by combining 1 g agarose with 1 ml 50X TAE in 49 ml of
distilled water, heating the mixture to boiling, adding 10 ul ethidium bromide (2.5 mg/ml)
pouring into a horizontal slab apparatus with combs, and then allowing to set at room
temperature. DNA samples were prepared for electrophoresis by adding 1/10 volume of
10X running dye (40 ul 50X TAE, 500 ul 30% Ficoll-0:5% bromophenol blue, 960 ul
water) and mixing with a pipetman. Electrophoresis was carried out at 100 V constant
voltage for 20 - 30 minutes using a Pharmacia EPCS 3000/150 power supply. Bands were
detected under UV illumination at 260 nm.
GENECLEAN
DNA Purification. The PCR-amplified DNA was purified
from the agarose gel using the GENECLEAN protocol (BIO101 Inc.): the band of interest
was cut from the agarose slab and placed into an Eppendorf tube to which 3-equivalents of
Nal was added. The agarose was melted by incubating at 55°C for 5 minutes. Five
microlitres of vortexed glass milk (BIO 101 Inc.) was added and the mixture incubated
again at 55°C for 5 minutes to bind the DNA. The solution was then centrifuged briefly
and supernatant removed. The glass milk pellet was then washed three times with 400 ul
of New Wash (20 mM Tris-HCl, pH 7.2, 0.2 M NaCl, 2 mM EDTA combined 1:1 with
100% ethanol). The washed pellet was then resuspended in 20 ul of TE-buffer (10 mM
22
Tris-HCl, pH 8.0, 1 mM EDTA) and centrifuged. The supernatant containing the clean
DNA was used for ligation reactions.
2.
Cloning and DNA sequence analysis of PCR fragment
Cloning of PCR fragment into pBS. The purified PCR fragments were
cloned into a Bluescript KS- (pBS) vector (Stratagene Cloning Systems) for DNA
sequence analysis. To produce sticky ends (6 bp overhangs) for cloning of sTF into the
pBS sequencing vector, both the amplified PCR fragment and the vector were cut with the
restriction enzyme Not I as follows:
Purified PCR fragment (sTF)
3 ul Bluescript KS-(pBS)
1 Hi Not I (10 U)
1 ul Not I (10 U)
2ul lOX-React 3 buffer
2 ul 10-X React 3 buffer
17 ul dH 0
14 ul dH 0
2
2
at 37°C for 1 hour. Following digestion, the cut pBS vector was treated with 1 ul calf
intestinal alkaline phosphatase (1 U) and incubated at 37°C for 30 minutes. Digested DNA
was purified by the Gene-Clean method.
The purified Not I-digested sTF fragment DNA and linearized pBS vector DNA
were then ligated together as follows:
2 ul Not I-digested pBS
5 ul Not I-digested sTF fragment
1 ul T4 DNA ligase (1 U)
2 ul 5X ligase buffer
at 16°C overnight (16 hours).
Transformation of E.coli cells. A colony of E.coli DH5cc was inoculated
into 50 ml of YT medium (8 g bactero-tryptone, 5 g yeast extract, 5 g NaCl, pH7 in 1 litre
of distilled water, autoclaved). The culture was shaken at 37°C until the OD600 reached 0.4
to 0.6. The culture was then placed on ice for 5 minutes before transferring to a cold sterile
23
centrifuge tube and centrifuged at 6000 rpm in an SS-34 rotor at 4°C for 5 minutes. The
supernatant was poured off, the cells gently resuspended in 12.5 ml of ice cold 50 mM
calcium chloride, and left on ice for 25 minutes to make them competent (62). The cell
suspension was re-centrifuged as before, the supernatant poured off, and the tube placed
on ice. The competent cells were resuspended very gently in 2.5 ml of ice cold 50 mM
calcium chloride.
Five microlitres of the ligation reaction mixture was transformed into 50 ul of
competent DH5a E.coli cells. The transformation was placed on ice for 30 minutes then
heat shocked at 42°C for 90 seconds. Two hundred microlitres of LB (Luria-Bertani)media (10 g bactero-tryptone, 5 g NaCl per litre H 0, pH 7.4, autoclaved) was added and
2
the mixture incubated at 37°C for 45 minutes with shaking. One hundred microlitres of
transformation mixture was plated on a selective LB-agar plate (12 g/1 agar in LB-media)
containing 25 mg/1 JPTG, 50 mg/1 X-Gal, 100 mg/ml ampicillin. The remaining 100 ul of
transformation mixture was plated on a second LB-IPTG/X-Gal/Amp plate and the plates
were incubated upside down at 37°C. Colonies were visible after 14 hours incubation.
pBS allowed for colour selection since vectors containing a cloned DNA insert produce
white bacterial colonies on the selective plates. Plasmids without inserts appeared blue.
Mini-prep recovery of plasmid DNA. White colonies were picked off the
plates using a pipette tip and grown in 4 ml of LB media in Falcon tubes for 12 hours at
37°C with shaking. Plasmid DNA (pBS) was isolated by mini-prep as follows: the
Falcon tubes were spun in a Sorvall RT6000B swinging basket centrifuge at 3000 g for 15
minutes at 4°C. The supernatant was poured off and the pellet was resuspended in 200 ul
of glucose buffer (50 mM glucose, 25 mM Tris-base, 10 mM EDTA, pH8), vortexed, and
transferred to a 1.5 ml bullet tube (Eppendorf). Four hundred microlitres of freshly
prepared alkaline-SDS solution (0.2 N NaOH, 1% SDS) was added, mixed by inversion,
and placed on ice for 5 minutes. Three hundred microlitres of 3 M sodium acetate (pH 4.8)
was added and the solution mixed by inversion. A thick precipitate formed. The solution
24
was cooled on ice for 10 minutes and then centrifuged for 5 minutes. Seven hundred and
fifty microlitres of supernatant was withdrawn and placed into a new eppendorf. Four
hundred and fifty microlitres of cold isopropanol was added, mixed well, and left for 5
minutes at 4°C to allow the DNA to precipitate. The mixture was centrifuged for 5
minutes, the supernatant decanted, the wet pellet briefly re-centrifuged, and the remainder
of the fluid removed using a pulled pasteur pipette. The pellet was allowed to air dry and
then resuspended in 200 ul of dF^O by vortexing. The pellet was then washed by adding
100 ul of 7 M ammonium acetate (pH 7.5), mixed, and then filling the tube with cold 95%
ethanol, and mixed again. The solution was centrifuged for 5 minutes and the supernatant
poured off. The pellet was re-washed using 200 ul of 95% ethanol, centrifuged for 2
minutes, and the supernatant removed using a pulled pasteur pipette as before. The washed
plasmid DNA pellet was air dried and resuspended in 50 ul of dFkO.
The recovered plasmid DNA was screened for an sTF cDNA insert by restriction
digest using Not I and electrophoresing on a 2% agarose gel. Clones containing an sTF
insert were subject to DNA sequence analysis."
DNA Sequence Analysis. Four positive clones (containing sTF insert) were
selected to be sequenced using a T7 Sequencing Kit (Pharmacia Biotech). pBS forward
and reverse primers were used in addition to two internal primers as shown in Figure 10.
Chain termination sequencing reactions (63) and S-labelling were performed using the T7
35
Sequencing kit. Electrophoresis of the DNA sequencing reactions was carried out on a
BRL vertical electrophoresis apparatus using 37.5 x 20 cm glass plates and 0.4 mm
spacers. The electrode buffer used was 0.5 X TBE (diluted from a 10 X TBE stock of
composition 0.89 M Tris, 0.89 M borate, 25 mM EDTA); the sequencing gel was
composed of 8% polyacrylamide, 8.3 M urea, and 45 mM Tris borate-1.25 mM EDTA.
Polymerization of the gel was initiated by the addition of 0.5 ml 10% (w/v) NH HS0 and
4
4
23 ul TEMED. The gel was warmed by pre-running at 50 watts constant power for 30
minutes. The DNA sequencing reaction samples were loading on the gel using Sharkstooth
25
a. pBS primers
5' - GTA AAA CGA CGG CCA GT - 3' FORWARD primer
5' - AAC AGC TAT GAC CAT G - 3'
REVERSE primer
b. Internal primers
5* - GTC CCG CGC CCC GAG ACC GC- 3' TF100
5' - TTT CTT TCC TGA ACT TGA AG -3'
TF594
Figure 10. DNA Sequence Analysis Primers. Two sets of primers, pBS primers
(a) specific for vector sequences and internal primers (b) specific for tissue factor gene
sequences, were used.
combs and electrophoresed at 32 W constant power. After electrophoresis, the gels were
dried at 80°C under vacuum on a Bio Rad gel drier for 20-30 minutes and autoradiographed
on Kodak XK-1 film overnight at room temperature.
The results of sequencing of the four clones was compared to the published
sequence of tissue factor (48). A PCR error-free clone was identified and grown in LB
medium as above. Plasmid DNA containing the error-free sTF insert was isolated by miniprep and used for sub-cloning.
3.
Transfection of BHK Cells and Expression of sTF
Cloning of sTF into pNUT. The sTF insert was cut out of the pBS vector
using a Not I restriction digest and ligated into a Not I linearized pNUT eucaryotic
expression vector (64) using the same procedure as for cloning into the pBS vector. The
ligation mixture was used to transform competent DH5cc E.coli cells as before. The
transformed cells were grown on selective LB plates, colonies were picked and cultured,
and the pNUT plasmid DNA was isolated by using the mini-prep procedure described
26
previously. Diagnostic restriction digests using Not I and EcoRl of the DNA were
performed to ensure the presence of the sTF insert in the pNUT expression vector.
Transfection of BHK cells. Baby hamster kidney (BHK) cells were grown in
Dulbecco's modified essential medium-Ham F12 (DMEM-F12) with 5% fetal bovine
serum (FBS) to approximately 10 cells per 90 mm dish. The isolated pNUT-sTF vector
7
was used to transfect the BHK cells as follows: 7 ug of mini-prepped pNUT-sTF was
resuspended in 450 ul of distilled water followed by addition of 50 ul of 2.5 M CaCk and
500 ul of 2X-HEPES buffered Saline (pH6.95). After standing at room temperature for
20 minutes, the mixture was added to 70% confluent BHK cells. After 6 hours, the
transfection media was removed and fresh 5% FBS-DMEM media added and the cells
allowed to recover for 24 hours at 37°C. The FBS-DMEM media was then replaced with
selective 5% FBS-DMEM media containing 500 uM methotrexate (MTX). The surviving
BHK colonies were trypsinized and passaged into a 75 cm tissue culture flask, still under
MTX selection. After five days, the 75 cm flasks were approaching confluency and a
Western blot was performed on thetissueculture media to detect production of sTF by the
BHK cells.
SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, based
on the method of Laemmli (65), was performed in a Hoeffer Scientific Mighty Small II
SE250 mini-protein gel apparatus using 10 x 8 cm glass plates and 1 mm spacers. The
electrode buffer used for electrophoresis was 0.015 M Tris base, pH 8.3,0.192 M glycine,
0.1% SDS (usually prepared by dilution of a 5X electrode buffer). The separating gels
used contained 0.375 M Tris-HCl, pH 8.8, 0.1% SDS, and 10% or 12% acrylamide:bis
(prepared from a 30% acrylamide (w/v) and 0.8% N,N'-methylenebisacrylamide (w/v)
stock solution). Polymerization was initiated by the addition of freshly prepared 10%
ammonium persulfate (APS) to a final concentration of 0.05% and TEMED to a final
concentration of 0.00033%. The stacking gels were prepared as 0.125 M Tris-HCl, pH
6.8,0.1% SDS, 3% acrylamiderbis (30:0.8). Polymerization was effected by adding fresh
27
10% APS to a final concentration of 0.13% and TEMED to a final concentration of
0.001%. Protein samples were prepared for electrophoresis by addition of 1/5 volume of
5X sample buffer (60 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.1% bromophenol
blue, 14.4 mM 2-mercaptoethanol) and placed in a boiling water bath for 3 minutes. After
cooling, the samples were loaded onto the stacking gel and electrophoresed at 50 mA per
gel for 30 minutes.
Protein gels were stained with Coomassie blue stain (0.25%
Coomassie blue, 10% acetic acid, 45% methanol) for 30 minutes to 1 hour and then destained using 45% methanol, 10% acetic acid de-stain solution.
Western blot analysis.
Proteins were separated by SDS-PAGE and
electrophoretically transferred (66) to a Protran nitrocellulose membrane using a Hoeffer
Scientific Transphor apparatus at 0.1 mA constant current overnight with water cooling.
The transfer buffer used was 23.4 mM Tris base, 19.3 mM glycine, and 20% methanol
(v/v). The nitrocellulose membrane was transferred to a Sarstedt culture tube and blocked
with a blotto/tween solution (5 g skim milk powder, 0.2 ml tween 20, 0.020 g sodium
azide in 100 ml phosphate buffered solution (8 g NaCl, 0.2 g KC1, 1.44 g Na HP0 , 0.24
2
4
g K H P 0 , pH 7.4, per litre PBS)) for 1 hour at room temperature with mixing. The
2
4
membrane was then incubated with a primary antibody specific for the extracellular region
of TF (1:3000 dilution in 1/10 blotto/tween) for 2 hours at room temperature with mild
agitation.
Unbound antibody was removed by washing the membrane twice with
phosphate buffer solution and twice with 150 mM NaCl, 50 mM Tris-buffer (pH 7.5).
The secondary antibody, an anti-murine IgG-alkaline phosphatase conjugate (1:5000
dilution in 1/10 blotto/tween) was then applied to the membrane and incubated for 1 hour at
room temperature with mild agitation. After washing the membrane four times using
150 mM NaCl, 50 mM Tris-buffer (pH7.5), colour detection was carried out by incubating
the membrane with 66 ul of nitro-blue tetrazolium (50 mg/ml in 70% DMF) and 33 ul
bromochloroindoyl phosphate (50 mg/ml in DMF) substrate in 10 ml alkaline phosphatase
buffer (0.1 M Tris-HCl, pH9.5,0.1 M NaCl, 5 mM MgCl ) until bands appeared.
2
28
Tissue culture of sTF-BHK cell line. Freezer stocks of the sTF-expressing
BHK cell line were prepared by trypsinizing the confluent cells and re-suspending them in
2 ml of DMEM with 50% glycerol. Aliquots were stored at -70°C. Tissue culture of the
BHK cell lines was carried out on the remaining flasks. The BHK cells were transferred to
250 ml tissue culture flasks and incubated at 37°C in 50 ml of 5% FBS-DMEM media with
500 uM methotrexate (MTX). When the cells became confluent after 3-4 days, they were
passed or split as follows: in a sterile fumehood, the old media was suctioned off and 2 ml
of trypsin was added to the flask and incubated for 50 - 60 seconds. The trypsin was
suctioned off and the treated cells were resuspended in 2 ml of fresh 5% FBS-DMEMMTX media. Ten to twelve drops of the cell suspension were added to a new 250 ml flask
containing 50 ml of fresh 5% FBS-DMEM-MTX media and the fresh flask replaced in the
37°C incubator.
After several passes, the sTF-expressing BHK cells were transferred to Roller
bottles for protein harvesting. Two 250 ml flasks containing confluent BHK cells were
trypsinized and resuspended in 5% FBS-DMEM-MTX as described above for passing of
cells. All 4 ml of the cell suspensions was added to a 2 litre roller bottle containing 150 ml
of fresh 5% FBS-DMEM-MTX and the roller bottle placed in a 37°C incubator. The cells
became confluent after 7 days at which time, the tissue culture media was switched to a
minimal 1% Ultroser G (USG)-DMEM media as follows: the 5% FBS-DMEM-MTX
media was removed by suction and 150 ml of 1% USG-DMEM media was added to the
roller bottle under sterile conditions. The roller bottle was replaced in the 37°C incubator
and the cells allowed to express sTF. After 3 days, the 1% USG-DMEM media was
reaching its limit of sustaining the cells and the media was collected into a sterile bottle
containing 0.2% sodium azide and 25 mM benzamidine. Three hundred milliltres of fresh
1% USG-DMEM was added to the roller bottle and replaced in the 37°C incubator. The
media was collected every 3.5 days and replaced with fresh media. The collected media
29
was stored at 4°C. Media was collected until the expressing BHK cells died at which time
a new roller bottle was seeded from a freezer stock and the collection cycle repeated.
4.
Purification of sTF from Tissue Culture media
Soluble tissue factor was purified from the tissue culture media by ammonium
sulfate precipitation, anion-exchange FPLC, and gel filtration. Detergent was not required
during the purification of sTF.
Ammonium Sulfate fractionation. Tissue culture media was concentrated 12fold using an Amicon CH2PRS spiral cartridge concentrator fitted with an S10Y10
cartridge at 20 psi pressure. To the concentrated crude media, solid ammonium sulfate
was slowly added to give a final concentration of 55% (36.4 g ammonium sulfate per
100 ml solution). After precipitation at 4°C overnight, the solution was transferred to
centrifuge tubes and spun at 5000 rpm for 30 minutes at 4°C using a Sorvall RC-5B
Refrigerated Super-Speed Centrifuge in an SS-34 rotor (Mandel Scientific Company). The
supernatant was separated from the precipitated proteins, concentrated 5-fold using a
Centricon 30 microconcentrator (Amicon), and then dialyzed against two changes of 4 litres
of 20 mM NaCl, 20 mM Tris-HCl, pH 7.5 (buffer A) to lower the salt concentration. The
precipitate was checked for sTF by SDS-PAGE and Western blot and discarded.
Anion-exchange
Fast Protein Liquid
Chromatography. The FPLC
apparatus consisted of Pharmacia LKB components: LCC-500 Plus Gradient Controller,
P-500 pumps, manual valve V-7, UV-MII optical unit with an HR 10 flow cell, REC-481
single channel chart recorder, and a Frac-100 fraction collector. The pumps were primed
using low salt buffer A and high salt buffer B (500 mM NaCl, 20 mM Tris-HCl, pH 7.5).
Two 5 ml Econo-Pac High Q FPLC cartridges (Bio-Rad) connected in series were used for
anion-exchange purification. The cartridges were washed with 2 volumes of buffer B and
then equilibrated with buffer A until baseline was reached prior to application of the crude
protein sample.
30
Following suction filtration through a 0.45 um filter, the dialyzed 55% ammonium
sulfate fraction was loaded onto the High Q FPLC Cartridges using a Superloop
(Pharmacia-LKB) at a flow rate of 1.0 ml/min. After washing with buffer A until a stable
baseline was re-established, the bound proteins were eluted with a continuous salt gradient
of 20 - 500 mM NaCl over 60 minutes at a flow rate of 1.0 ml/min and 1.5 ml fractions
were collected. Protein elution was detected by measuring absorbance at 280 nm and the
salt gradient was held constant during peak elution. Collected fractions were checked for
the presence of sTF using SDS-PAGE and Western blot; fractions containing sTF were
pooled and concentrated using Centricon Concentrators 30 with YM-30 ultrafiltration
membranes (Amicon) to a volume of 2.5 ml.
Gel-filtration Purification. A Sephacryl S-100 HR gel filtration column was
prepared as follows: 300 ml of 100 mM NaCl, 20 mM Tris-HCl, pH 7.5 (buffer C) was
added to 600 ml of Sephacryl S-100 HR matrix (Pharmacia LKB) and the slurry placed on
ice and de-gassed for 10 minutes. At 4°C, the de-gassed slurry was poured slowly into a
Bio-Rad 55 x 2.4 cm column to a height of 52 cm giving a bed volume of 300 ml. The gel
filtration column was equilibrated with 3 bed volumes of buffer C passaged at 1 ml/min
using a Gilson Miniplus HP4 peristaltic pump. Void volume was determined using blue
dextran and the column calibrated using 3 ml of a protein standard mix containing 2.5 mg
each of hemoglobin, ovalbumin, trypsin inhibitor, and cytochrome c.
The concentrated High Q fraction (2.5 ml) was loaded onto the Sephacryl S-100
HR gel filtration column. Proteins were eluted with buffer C at a flow rate of 0.60 ml/min
using a peristaltic pump (Gilson Miniplus HP4) and 3.0 ml fractions were collected using a
2112 Redirac Fraction collector (LKB Bromma) Protein elution was determined by
measuring absorbance at 280 nm on a Perkin Elmer Lambda 3A UV/Visible
Spectrophotometer. Fractions containing protein were checked for sTF using SDS-PAGE
and Western blot; fractions containing pure sTF were pooled and concentrated using
Centricon Concentrators 30.
The sTF concentration was determined spectrophoto31
metrically using a 1% extinction coefficient at 280 nm of 14.8 (67) and a molecular weight
of 39,000. Aliquots of the pure sTF were stored frozen at -70°C.
5.
Characterization of purified sTF
The purified sTF was characterized using SDS-PAGE on a 12% polyacrylamide gel
and Western blotted as described above. Amino-terminal sequence analysis of sTF was
performed using an Applied Biosystems 476A Sequencer. The presence of glycosylated
residues was also determined using the 476A Sequencer.
6.
Development of Factor V i l a Assay
Preparation of phospholipid vesicles. Phospolipid vesicle mixtures of
composition POPS = 75:25 and PC:PS:PE = 64:6:30 were prepared as follows: the
phospholipids in the desired molar composition were frozen in liquid nitrogen, lyophilized,
and then resuspended in distilled water by vortexing. The phospholipid mixture was then
transferred to a cryovial (Simport Plastics) and subjected to 5 freeze-thaw cycles alternating
between a liquid nitrogen bath and a 35°C water bath. Large unilamellar vesicles (LUVs)
were prepared by extruding the freeze-thawed mixture through a 100 nm pore size
polycarbonate filter (Poretics Corporation) using a Lipex Extruder (Lipex Biomembranes
Inc.) under 300 psi Nitrogen gas pressure for 10 cycles. Vesicle size was determined
using a Nicomp Model 270 Submicron Particle Sizer (Pacific Scientific) and concentration
of the phospholipid mixtures was determined using a standard phosphate assay (68).
Optimization of sTF-Reagent. The sTF-reagent was composed of purified
BHK-derived soluble tissue factor and a phospholipid mixture dissolved in TBS/BSA
(50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% bovine serum albumin, 0.02% NaN ).
3
To determine the optimal concentration of sTF, a series of sTF-reagents with sTFconcentrations ranging from 0.01 umol/1 to 1.0 umol/1 was prepared in a 0.27 mM
PC:PS:PE (64:6:30) phospholipid, TBS/BSA mixture. As well, an sTF-negative reagent
32
containing no sTF was also prepared. To determine the optimal composition and
concentration of the phospholipid mixture, a series of sTF-reagents with varying
concentrations of PC:PS:PE (64:6:30) or PC:PS (75:25) phospholipid were prepared in a
0.5 uM sTF, TBS/BSA mixture. All sTF-reagents were stored frozen at -70°C and
underwent exactly one freeze-thaw cycle before use.
Assay for Factor Vila. The factor Vila assay was performed on an automated
STA Coagulometer (Diagnostica Stago) at Vancouver Hospital. The assay was performed
ontitratedplasma samples thawed at 37°C and held at room temperature until placed in the
coagulometer.
Sample volumes per assay (plasma, sTF-reagent, and 25 mM CaCl
2
solution) were 50 ul each. Factor VH-deficient plasma, test plasma, and the sTF-reagent
were held in a 16°C temperature block and the 25 mM CaCl solution was held in a 37°C
2
temperature block prior to use.
Assays were carried out in cuvettes containing
electromagnetic ball-bearings for mixing. Five microlitres of titrated test plasma was
diluted 10-fold in FVJJ-deficient plasma to give a final volume of 50 ul in a cuvette; 50 ul
of sTF-reagent was added, the solution mixed, and incubated at 37°C for 120 seconds.
Fifty microlitres of pre-warmed CaCl was added and the solution electro-magnetically
2
mixed. Clotting times were measured from the addition of calcium to the freezing of the
ball-bearing to electromagnetic mixing indicating clot formation. All assays were
performed in duplicate and a third assay was run if the duplicates did not agree within 7%.
If the third assay did not agree with either of the duplicates, an error was reported.
Factor Vila Standard curve. A stock factor Vila concentrate was prepared by
diluting pure FVIIa to a concentration of 300 ng/ml in titrated normal pooled plasma
(obtained from the Hematology Department, UBC Hospital). Aliquots of this FVIIa stock
solution were stored at -70°C. Factor Vila standards of 30, 3.0, 0.3, and 0.03 ng/ml
FVUa were prepared by serial dilution of the factor Vila 300 ng/ml stock in FVII-deficient
plasma. The clotting times of the four FVIIa standards were measured using the FVIIa
assay as described above. Using the calibration mode of the STA Coagulometer, the
33
clotting times were plotted versus the FVIIa concentrations on a log-log scale and a
polynomial curve was fit to the line using linear regression analysis. Using this calibration
curve, factor Vila concentrations in ng/ml could be generated automatically from the
measured clotting times of the test plasma samples.
Comparison of BHK vs. E.coli-derived sTF. Soluble tissue factor derived
from BHK cells was compared to Zs.co/i'-derived sTF (a generous gift from Dr. James H.
Morrissey, Oklahoma Medical Research Foundation) by preparing two sTF-reagents of
identical composition 0.5 uM sTF, 1.0 mM PC:PS:PE (64:6:30) in TBS/BSA, except
using BHK-derived sTF in one preparation and Rco/i-derived sTF in the other preparation.
The four FVIIa standards (30, 3, 0.3, 0.03 ng/ml) and fivetitratedplasma samples were
measured using both sTF-reagents in the FVIIa assay described above. Linear regression
analysis of the results was performed to compare the two sources of sTF.
7.
Testing of FVIIa Assay
Precision Testing. To test the reproducibility of the FVIIa assay, precision
testing for within-run and between-run reproducibility was performed. A citrated plasma
sample was collected from a single donor and 15 aliquots were frozen at -70°C. One
aliquot was measured 10 times (in duplicate) on a single day to establish within-run
precision. To establish between-run precision, an aliquot from the donor was measured 4
times per day on 5 additional days . Mean values, standard deviations, and coefficients of
variation for within-day and between-day precision were calculated.
Accuracy Testing. To test the accuracy of the FVIIa assay, a patient comparison
study was performed between the present study's FVIIa assay and a commercial FVIIa kit
available from Diagnostica Stago. The STACLOT VHa-rTF kit was set up on the STA
Coagulometer according to the manufacturer's instructions. A standard curve using FVIIa
international standards (mU/ml), included with the kit, was established for the STACLOT
VIIa-rTF assay. Twenty citrated plasma samples ranging from 0.1 ng/ml to 30 ng/ml
34
FVIIa concentration were measured using both the present study's FVIIa assay and the
STACLOT VIIa-rTF kit. Linear regression analysis was performed to assess the accuracy
of the FVIIa assay in relation to the commercial FVUa kit. A FVIIa conversion curve was
plotted to convert FVUa concentrations in ng/ml to international units (mU/ml).
Plasma Stability studies. To determine the stability of plasma FVIIa, a series
of plasma stability studies was performed. To determine the effects of freezing and frozen
storage time on a plasma sample, a freshly collectedtitratedplasma sample was measured
for FVIIa and then aliquots were frozen at -70°C for 15 minutes, 2 days, and 7 days and
re-measured. To test the effects of repeated freeze-thaw cycles on plasma FVIIa, three
plasma samples were subjected to three freeze-thaw cycles and measured for FVIIa after
each cycle. To compare the effects of different anti-coagulants, one titrated and one
EDTA-anti-coagulated plasma sample was drawn from a single donor from the same
venepuncture. Precision testing, as described above, was performed on both the EDTA
andtitratedplasma samples and the results compared. To determine the effects of dilution
during the FVIIa assay, 10titratedplasma samples were measured neat and after 10-fold
dilution in FVII-deficient plasma and the measured FVIIa compared after correction for
dilution.
Individual variations. To determine the intra-individual variations in plasma
FVIIa levels, 6titratedplasma samples were drawn from a single donor over a 3 month
period (December, 1996 to March, 1997). FVIIa levels were measured for each sample to
determine individual fluctuations in plasma FVUa.
35
8.
Factor V i l a in Normal Populations
Ethical approval for blood collection. Ethical approval for the collection of
blood from volunteer donors, who gave informed written consent, was obtained from the
University of British Columbia ethical review board.
Subjects. Fifty normal healthy volunteers (29 female, 21 male) aged 20-52 years
with no history of CHD provided blood samples. No effort was made to control for ethnic
origin and samples were collected in the fasting state or within 2 hours after a light
breakfast.
Phlebotomy. Venous blood (9 volumes) was collected between 9 and 10:30 am
sequentially into 2 siliconized Vacutainer tubes (Becton Dickinson) containing 1 volume of
3.2% trisodium citrate. The first tube of blood collected after venepuncture was discarded.
Only the second tube was used for analysis. Platelet poor plasma was obtained by
centrifugation at 3000 rpm for 20 minutes and aliquots in plastic tubes were frozen at -70°C
within one hour of collection. At the time of assay, citrated plasma samples were thawed
rapidly at 37°C, mixed gendy, and stored at room temperature until tested. Plasma samples
were never subjected to more than one freeze-thaw cycle.
Measurement of FVIIa. FVIIa was determined with the automated FVIIa assay
employing sTF as described above. The composition of the sTF-reagent used was 0.5 uM
sTF, 1.0 mM PC/PS/PE (64:6:30) in TBS/BSA. Clotting times were converted to a FVIIa
concentration in ng/ml using the FVIIa standard curve and into FVIIa international activity
units (mU/ml) using the FVIIa conversion curve. FVIIa levels in the normal population
were examined by age and gender.
9.
Factor V i l a in Complicated Pregnancies
Ethical approval. Ethical approval for collection of blood samples from hospital
patients was obtained through Dr. Cedric Carter of Vancouver Hospital and Dr. Louis
Wadsworth of British Columbia Children's Hospital.
36
Subjects. Nineteen pregnant women aged 22-40 years who experienced
complications during their pregnancy provided blood samples. Plasma collection was
performed as described above.
Clinical assessment. Details on the complications and stage of pregnancy of
each subject were noted. In addition to the measurement of plasma FVIIa as described
above, plasma samples were variously measured for prothrombin time (PT), activated
partial thromboplastin time (APTT), plasma fibrinogen (FD3R), and platelet counts at the
physicians' discretion.
37
RESULTS
1.
Polymerase Chain Reaction of sTF
The initial step in the present study was the isolation of a truncated form of tissue
factor. Since sTF does not occur naturally, the mutant protein had to be produced using
recombinant DNA techniques. Previous studies have generated sTF by isolating restriction
fragments from cDNA libraries and then ligating synthetic linker oligonucleotides to the
restriction fragments' 3'- and 5'-ends to generate termination codons and cloning sites
(50). The present study simplified the process by using polymerase chain reaction with
mutagenic primers incorporating engineered stop codons and cloning sites to selectively
amplify the coding region for soluble tissue factor.
A 785-bp DNA fragment consisting of 752 nucleotides of the TF gene encoding
amino acids -32 to 219 as described in (15) flanked by a synthetic Not I restriction site on
the 5'-end and two synthetic stop codons followed by a synthetic Not I site on the 3'-end
was amplified using PCR. Successful amplification of the sTF fragment by PCR was
confirmed using agarose gel electrophoresis.
Figure 11a shows the results of PCR
amplification of sTF using a human brain cDNA library as the template. The 785 bp band
in lane 2 was cut out and used as a template for a second round of PCR. The results
shown in Figure lib indicate a stronger 785 bp band confirming specific amplification of
the desired fragment.
2.
DNA Sequence analysis of sTF insert
The PCR fragment with its engineered NotI restriction sites was subsequently
cloned into a Bluescript sequencing (pBS) vector and transformed into an E.coli host. pBS
allowed for colour selection since vectors containing a cloned DNA insert produced white
bacterial colonies on the selective LB-IPTG/X-Gal/Amp plates while plasmids without an
38
Figure 11. PCR amplification of sTF gene. 2% agarose gel electrophoresis,
visualized at 260 nm UVillumination of (a) PCR amplification reaction from
human cDNA library of sTF gene. A faint band of 785 bp was visible in lane 2.
This band was cut out and used for a second round of PCR amplification,
(b) Results of PCR. amplification using the excised band as template show two very
strong bands of 785 bp in lanes 2 and 3. These bands were excised, punfied by
GENECLEAN, and used for D N A sequencing. The large, smudged bands on the
bottom of the gels areRNA. Lane 1 shows a standard 1 kb ladder.
39
insert appeared blue. Figure 12 shows the results of the Not! digestion screening to
confirm the presence of the sTF insert in the isolated pBS from a white colony.
Since the Taq polymerase employed in the PCR reactions lacks a 3'-exonuclease for
proof-reading (69), errors in the amplification product often occur. To ensure that an errorfree clone was selected for sTF protein expression, four of the positive clones were
analyzed for their DNA sequence. Of the four clones sequenced, only one was found to be
free of PCR errors when compared to the known sequence for TF (48).
3.
Expression of sTF in B H K cells
The sTF insert from the error-free pBS clone was sub-cloned into a pNUT
expression vector again via its Not! restriction sites. The presence of the sTF insert in the
pNUT vector was confirmed by Not! and EcoRl digestion screenings. Figure 13 shows
the 782 bp Not I band and a 600 bp EcoRl band verifying the presence of the sTF insert.
The 600 bp band is a result of two EcoRl restriction sites, one at the 3'-end of the sTF
insert and one in the pNUT poly linker as shown in Figure 14.
Expression of sTF has previously been carried out in Chinese hamster ovary
(CHO) cells (50), S.cerevisiae (70), and E.coli (67). The present study used baby hamster
kidney (BHK) cells for expression. The BHK cells provided a eucaryotic expression
system without the disadvantages of hyperglycosylation conferred by yeast systems
(S.cerevisiae) (70) and the low sTF expression levels of CHO cells (0.5-1.0 mg/1) (50).
The pNUT vector serves as a eucaryotic expression vector, with a zinc metalloprotein promoter driving transcription of the cloned sTF insert (Figure 14). The isolated
pNUT-sTF vector was used to transfect competent BHK cells and the cells were grown in
selective media containing the cytotoxic agent methotrexate (MTX). Since the pNUT
vector contains a genetic cassette for di-hydrofolate reductase which breaks down
40
p B S - 3000 bp
sTF —782 bp
Figure 12. Not I screening of Bluescript-sTF clone. 2% agarose gel
electrophoresis, visualized at 260 nrn U V illumination of isolated bluescript sTF plasmids following digestion with Not I. Lanes 1 to 9 show the results
from 9 white colonies. Lane 11 shows a standard 1 kb ladder. The upper
(3000 bp) band corresponds to the bluescript vector D N A and the lower
(782 bp) band corresponds to the cloned sTF -insert. Thus all clones were
positive.
MW 1 2 3 4 5 6 7 8 9 10
(<=)
- 782 bp
MW1 2 3 4 56 7 8 910
•600 bp
Figure 13. Not I and EcoRl screening of pNUT-sTF clone. 2% agarose gel
electrophoresis, visualized at 260 nm TJV illumination of isolated pBS-sTF
plasmid following digestion with (a) Not I or (b) EcoRl. The results indicate a
782 bp band presentin all lanes except 3 and 6 of the Not I screen (a) and a 600 bp
band in lanes 2, 4, and 8 of the EcoRl screen (b) confirming the presence of the
sTF insert in the pNUT expression vector. A 1 kb ladder is shown in lane MW.
41
• T F cDNA
DHFR em—Urn
Figure 14.
pNUT expression vector with cloned sTF insert.
A schematic
representation of the pNUT eucaryotic expression vector with the sTF cDNA cloned into its
polylinker region. EcoRl and Not I restriction sites are indicated as well as the zinc
metallo-protein promoter (MT-1) and the di-hydrofolate reductase (DHFR) cassette that
confers resistance to methotrexate.
methotrexate, only B H K cells successfully transfected with the pNUT-sTF plasmid
survived drug selection.
Successful transfection was apparent by day nine of methotrexate selection when
discreet surviving colonies were visible after dead cells were removed and fresh selective
media re-applied. After transferring to tissue culture flasks, a Western Blot was performed
on thetissueculture media to detect production of sTF by the BHK cells. The results of
the immunoblot are shown in Figure 15.
All of the BHK clones were found to be successfully expressing and secreting
soluble tissue factor. It should be noted that the expressed sTF protein contains an
endogenous export signal peptide in its 32-residue leader peptide (17) which is
42
1
2
3
4
5
6
7
8
Full-length s T F - *
>M
Positive Control
*%M| •»* M l ^-BHK-expressed sTF
(E.coii-derived sTF)
Figure 15. Western Blot of tissue culture media from several sTF-BHK cell lines.
Samples of tissue culture media were run on a 10% SDS-PAGE and Western blot analysis
was performed using an antibody raised against the extracellular portion of tissue factor.
A l l 7 clones (lanes 2-8) were positive for expression of sTF protein as indicated by the
results of Western blot. Western blot analysis of non-transfectedBHK cells (results not
shown) showed no positive clones. Lane 1 shows a positive control containing sTF and
full-length TF expressed in E.coli.
43
subsequently cleaved by a signal peptidase. It was via this peptide that the sTF protein was
secreted from the BHK cells into the media. (Had such an endogenous signal peptide not
existed in the sTF protein structure, a synthetic oligonucleotide coding for such a peptide
would have had to be ligated to the sTF fragment or the sTF would have had to be purified
from within the BHK cells). Lane 1 shows a positive control of sTF provided by Dr.
Morrissey.
Since all the BHK clones were positive for sTF expression, they were
combined.
Tissue culture of the sTF-expressing BHK cell line was carried out in roller bottles
at 37°C. Culture media was collected (and replaced with fresh media) when the cells
appeared to be stressed. A pH-sensitive phenol red indicator dye in the DMEM-F12 media
changed from red to orange-yellow when the media had reached its limit to support the
BHK cells. After 5 to 6 changes of media, the sTF-BHK cell line eventually died and new
roller bottles were seeded. BHK cells generally express at their highest level when
stressed. Approximately 7 litres of tissue culture media from the sTF-expressing BHK
cells was collected.
4.
Ammonium sulfate fractionation
The first step in the purification of soluble tissue factor from the tissue culture
media was an ammonium sulfate fractionation. This served two purposes: to concentrate
the crude sample to a workable volume and to remove some protein impurities. Prior to
addition of ammonium sulfate, the tissue culture media was concentrated 12-fold using an
Amicon concentrator to reduce the volume. To determine the optimal ammonium sulfate
concentration for fractionation, ammonium sulfate cuts of 30%, 40%, 50%, 60%, 70%,
and 80% were performed on samples of the concentrated media. Western blots indicated
that sTF began precipitating at 60% ammonium sulfate (results not shown).' Thus, it was
decided that a 55% ammonium sulfate fractionation would produce a good initial
purification with negligible loss of sTF protein (Figure 16).
44
(a)
1
2 3
4
3 minor <
impurities
(b)
5
1
2 3 4 5
130 kDa
bo kDa
M kDa
130 kDa
90 kDa
68 kDa
\43
43 kDa
kDa
29 kDa
29 kDa
18 kDa
18 kDa
Major 65 kDa Impurity
sTF
Figure 16. 55% Ammonium Sulfate Fractionation. Supernatant and precipitate
following 55% ammonium sulfate precipitation were run on a 12% SDS-PAGE. The
protein gel was visualized with Coomassie blue (a) and Western blot analysis (b) was
performed using an antibody raised against the extracellular portion of tissue factor.
Lanes 1 and 4 are crude protein samples, lane 2 is the precipitate fraction, lane 3 is the
supernatant fraction, and lane 5 contains the M W markers. Western blot (b) analysis
indicated that no sTF precipitated at 55% ammonium sulfate (lane 2) but the supernatant fraction (lane 3) contained a large amount of sTF of molecular weight 39 kDa.
The Coomassie stained gel (a) shows three minor impunty proteins were removed in
the 55% precipitate (lane 2) but a major 65 kDa impunty remained in the supernatant
fraction with the sTF (lane 3).
45
A photograph of the SDS-PAGE and Western blot of the supernatant and precipitate
after 55% ammonium sulfate fractionation of the concentrated tissue culture media is shown
in Figure 16. Comparison to low MW standards show that the BHK-derived sTF runs
with an apparent MW of 39 kDa indicating a glycosylated form (70) as expected with a
eucaryotic expression system. Figure 16a shows only a small amount of purification
occurred with the 55% ammonium sulfate cut. Three minor protein impurities (65 kDa,
60 kDa, 25 kDa) were removed in the precipitate but a very large impurity of approximately
65 kDa remained in the supernatant fraction containing sTF. However, the immunoblot in
Figure 16b indicated that no sTF precipitated at 55% ammonium sulfate.
5.
High Q F P L C Purification
In order to remove the major 65 kDa impurity (likely bovine serum albumin (BSA)
from the media), FPLC purification was employed.
Using the PC-Gene computer
application, the isoelectric point of sTF was determined to be pH 4.88. Thus an anionexchange column was chosen for purification. A MonoQ FPLC column using a phosphate
buffer (pH 6.0) and continuous NaCl gradient failed to separate the sTF from the major
impurity in initial attempts with anion-exchange FPLC.
However, changing to a High Q FPLC cartridge using a Tris-buffer (pH 7.5) and
continuous NaCl gradient resulted in excellent resolution between sTF and the major
impurity. The purification profile of the High Q FPLC run is shown in Figure 17 along
with SDS-PAGE and Western blot analysis of the collected fractions. The salt gradient
was held constant during elution of each peak. As can be seen from the results of the SDSPAGE and Western blot, sTF eluted in the first peak at approximately 100 mM NaCl along
with some other minor (but significant) impurities. The major 65 kDa impurity eluted in
the very large second peak at 160 mM NaCl; the resolution between the peaks was
excellent Fractions containing the semi-purified sTF were pooled.
46
Figure 17.
High Q F P L C purification. The ammonium sulfate fraction was
purified using a High Q FPLC cartridge and a linear salt gradient of 20 - 500 mM NaCl in
20 mM Tris-HCl, pH 7.5 buffer. The salt gradient was held constant during peak elution;
four peaks eluted at 100, 160, 210, and 285 mM NaCl as shown in the elution profile (a).
Fractions from peak #1 and #2 were run on a 12% SDS-PAGE and stained with
Coomassie blue (b) or Western blotted using an antibody specific for sTF (c). The results
indicate that sTF eluted in Peak #1 along with a high MW doublet impurity. However, the
major 65 kDa impurity (BSA) was separated and eluted in Peak #2. Lane 1 is the crude
ammonium sulfate fraction, lane 2 is the FPLC flow-through, and lane 11 is MW markers.
47
Peak £2
1
H8&1.1M,.
/
I
,
•
a
II /•'
160 mM..
I
Peats #3
Poak J»4
1 « II
I
|<
Peaktfl
>|
Pk#2
1 2 3 4 5 6 7 8 9 10 11 12 13
|<
Peak#l
>|
Pk#2
1 2 3 4 5 6 7 8 9 10 11 12 13
()
-130kDa
-90kDa
-68 kDa
c
- 43 kDa
- 29 kDa
/
Major 65 kDa Impurity
High M W Doublet
48
sTF
One disadvantage of the High Q FPLC cartridge was that it had a limited capacity
for total protein loaded. If overloaded, sTF would elute with the flow-through during
loading of the crude sample because of the relatively low affinity of sTF for the column.
Thus, during preparative purification of sTF, two High Q cartridges were connected in
series and a maximum of 100 mg of total protein was loaded onto the column. Several
runs had to be performed to purify all of the protein. A second problem was caused by the
pH-indicator dye in the tissue culture media. The phenol red dye bound very strongly to
the anion-exchanger during loading. This was one of the main reasons the column was so
easily overloaded. As well, since the dye bound so strongly, washing the column with
concentrated salt (2 M NaCl) solutions for long periods was necessary to re-generate the
anion-exchanger.
To avoid the problems caused by the dye, DMEM-F12 media without indicator dye
can be purchased from Gibco-BRL and used for tissue culture. Another choice is to
perform an initial batch purification of the ammonium sulfate fractionated sample on a
DEAE-Sepharose equilibrated in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl (16) to remove
the dye prior to loading onto the FPLC column. In this case, the dye would bind to the gel
and the sTF would flow through.
6.
Sephacryl S-100 HR Gel Filtration Purification
Following FPLC, the High Q-purified sample still contained some impurities in the
form of a high MW doublet of approximately 70 kDa as seen in Figure 17b (lanes 4, 5, 6).
Thus for the final stage of purification, a size exclusion column was used to separate the
relatively small (39 kDa) sTF protein from the high MW doublet impurity. A high
resolution Sephacryl S-100 HR gel filtration column with a height-to-diameter ratio of 20:1
and bed volume of 300 ml was prepared. Void volume for the column was determined to
be 105 ml using blue dextran and calibration using a mixture of protein standards indicated
49
excellent resolution between proteins of 64.5 kDa (hemoglobin), 45 kDa (ovalbumin), and
20 kDa (trypsin inhibitor) molecular weight as shown in Figure 18.
0.4
E
c
o
-Trypsin
Hemoglobin
Inhibitor
0.3 H
CO
CM
d
> 0.2 H
o
a
o
w
.a
o.H
<
0.0
0
10
20
Fraction
Figure 18.
30
40
number
Calibration curve for Sephacryl S-100 HR Column.
Elution
profile of protein standards run on Sephacryl S-100 HR size exclusion column using
100 mM NaCl, 20 mM Tris-HCl, pH 7.5. The protein standards were hemoglobin
(64.5 kDa), ovalbumin (45 kDa), trypsin inhibitor (20 kDa), and cytochrome c (13 kDa).
Flow rate was 0.6 ml/min. Void volume for the column was 105 ml.
To ensure the highest resolution, the High Q-purified sample was concentrated to
2.5 ml or 1% of the bed volume prior to loading onto the Sephacryl S-100 HR column and
separation was carried out at slow flow rate of 0.6 ml/min.
The A
2 8 0
profile of the gel filtration purification run is shown in Figure 19a.
Surprisingly, there was some overlap between the elution peaks of the high MW doublet
(70 kDa) and sTF (39 kDa). Arrows indicate elution volumes of the protein standards used
for calibration. Fortunately, SDS-PAGE and Western blot (Figure 19b and 19c) indicated
50
Figure 19. Sephacryl S-100 HR Purification. The High Q fraction (2.5 ml) was
loaded onto the Sephacryl S-100 HR size exclusion column and eluted with 100 mM NaCl,
20 mM Tris-HCl, pH 7.5 at a flow rate of 0.6 ml/min. The elution profile (a) shows that
the two peaks were not completely resolved. However fractions from peak #1 and #2 were
run on a 12% SDS-PAGE and stained with Coomassie blue (b) or Western blotted using an
antibody specific for sTF (c). The results of the electrophoresis indicate that fractions
containing pure sTF (lanes 8 - 14) were isolated. The reason for the poor resolution
despite the large apparent size difference was due to the extended structure of sTF.
51
(a)
2
Fraction
(b)
,
4
5
6
number
(0
9
10 11 12 13 14 15
1
2 3 4
J
6
7 8 9 10 11 12
13 14 15
-90 kDa
v J68 kDa
- 43 kD a
29
Pure sTF
52
kDa
Pure sTF
Low M W sTF
that sTF had successfully been separated from the high MW doublet in the collected
fractions with nearly no overlap. Thus, fractions containing the pure sTF were pooled.
The poor resolution between the sTF and the high MW doublet was surprising
since the size exclusion column had successfully resolved hemoglobin and ovalbumin, two
proteins with an apparent molecular weight difference smaller than sTF and the high MW
doublet. However, consideration of the recently published X-ray crystal structure of sTF
(20, 21) revealed the reason for the apparent discrepancy. Soluble tissue factor has an
elongated structure, approximately 115 A long and 40-50 A wide (Figure 3). Thus, the
apparent Stokes radius of the sTF protein is much larger than a globular protein of similar
molecular weight. Since size exclusion chromatography actually separates proteins by
Stokes radius (71) and not molecular weight, sTF behaves like a protein much larger (ie.
higher MW) than its apparent molecular weight of 39 kDa on SDS-PAGE. This resulted in
the poor resolution between sTF and the high MW doublet impurity.
A longer, narrower column would improve resolution for future size separations
using the same gel filtration matrix. Sephacryl S-100 HR is an excellent size exclusion
material that offers high resolution due to its small bead size (25-75 um) and mechanical
strength due to its extensive cross-linking. The fractionation range of 1,000 to 100,000
suits the purification requirements well.
7.
Characterization of sTF Protein
Figure 20 shows an SDS-PAGE at different stages of purification of sTF from the
tissue culture media. The crude tissue culture media in lane 1 was dominated by the major
BSA impurity (65 kDa) which was removed after the High Q FPLC purification (lane 4).
Gel filtration on a Sephacryl S-100 HR column removed the remainder of the impurities
(lanes 5-8).
53
12
3 4 5 6 7 8 9
. 130 kDa
- 90 kD a
- 68 kD a
- 43 kDa
9 kDa
/ «
/
\
Major 65 kDa Impurity
H i g h M W doublet
sTF
LowMW
sTF
Figure20. Purification Summary gel. Samples from different stages of purification
of sTF were run on a 12% SDS-PAGE and stained with Coomassie blue. Lane 1 shows
the crude tissue culture media, lane 2 shows the supernatant of the 55% ammonium
sulfate fractionation; lane 3 shows a (trial) batch purification, lane 4 shows the results
after HighQ FPLC purifi cation, lanes 5-8 show several fractions of the purified sTF
after Sephacryl S-100 HR size exclusion chromatography. Lane 9 shows the molecular
weight markers In lane 8, a high M W (39 kDa) and a low M W (35 kDa) form of sTF
are clearly visible.
54
The purified sTF consists of a major band of 39 kDa. However, Western blot
analysis also detects at least one slightly smaller form of sTF below the major band (Figure
19c, lanes 12-14 and Figure 20, lane 8). This minor band of apparent molecular weight
35 kDa most likely represents a partially glycosylated (or unglycosylated) form of sTF
(70). Treatment with endoglycosidase F could confirm this characteristic of the minor
band. The minor band behaved identically to the major band when used in the sTF-reagent
for the factor Vila assay.
Amino-terminal sequence analysis of the purified sTF determined two peptide
sequences: Ser-Gly-Thr-Thr-Asn-Thr-Val and Thr-Thr-Asn-Thr-Val-Ala-Ala in equal
proportions. The second sequence is identical to the first sequence from the third residue
(Thr) on. The two sequences represent two alternate processing sites as described for
natural full length tissue factor (15). Thus the N-terminal sequence of the purified protein
is in agreement with the expected composition of sTF. The N-terminal sequence analysis
also confirmed the purity of the sample to be greater than 98% and the presence of
glycosylation in the form of fucose.
The concentration of sTF was determined spectrophotometrically using a 1%
extinction coefficient at 280 nm of 14.8 for the pure protein (67). Purification of 2.4 litres
of tissue culture media yielded 7.36 mg of pure sTF protein. Thus, the overall expression
level of the sTF-BHK cell line was approximately 3 mg/litre. This amount represented
about 1% of the total protein in thetissueculture media and was a significant improvement
over expression levels of CHO cells for sTF (50). Fifty microlitre aliquots of the purified
sTF were stored frozen at -70°C prior to use.
8.
Optimization of Assay Reagents
The first step in the development of the FVIIa assay was to optimize the assay
reagents. The assay uses three reagents to measure the FVIIa-level in a plasma sample: the
55
sTF-reagent, CaCl solution, and FVII-deficient plasma. The calcium chloride solution is a
2
standard 25 mM aqueous solution and the FVII-deficient plasma is available commercially
from several manufacturers.
The sTF-reagent is analogous to thromboplastin in a
prothrombin time (PT) or FVII coagulant (FVILC) assay . The sTF-reagent contains the
soluble tissue factor protein and phospholipid diluted in a TBS/BSA solution. One of the
keys to a robust FVIIa assay is the optimization of the sTF-reagent.
According to Morrissey's original paper (26), the sensitivity of the FVIIa assay
could be manipulated by adjusting the sTF concentration in the sTF-reagent. A "high
sensitivity" reagent containing nearly saturating concentrations of sTF was determined to be
optimal. Thus to determine the optimal concentration of sTF, a set of sTF-reagents with
varying concentrations of sTF was prepared and the clotting time for a normal pooled
plasma (NPP) sample was measured using each. A plot of clotting time versus sTF
concentration is shown in Figure 21.
Clotting times began to plateau at around 0.1 uM sTF concentration and reached a
minimum at 0.5 uM sTF. This was lower than the 1.0 uM sTF required in the original
assay to achieve saturating levels (26). An sTF-negative reagent failed to produce a clot in
400 seconds confirming the BHK-derived sTF as the active clotting agent in the sTFreagent.
The original assay used rabbit brain cephalin (RBC) as a phospholipid source in the
sTF-reagent. Since Sigma no longer sells RBC, a phospholipid replacement was required.
Initially a PC/PS (75:25) pure phospholipid mixture was used. However, it was found that
this composition was not particularly stable to freezing resulting in variable clotting times
after a single freeze-thaw cycle. According to a recent paper by Neuenshwander et al (72),
the addition of phosphatidylethanolamine (PE) to a standard PC/PS phospholipid mixture
enhanced the activity of the FVIIa-TF complex in its activation of factor X and factor IX.
An sTF-reagent containing a PC/PS/PE (64:6:30) mixture was prepared and found to be
extremely stable to freeze-thaw. To determine the optimal concentration of PC:PS:PE
56
300
0H
0.0
•
1
0.2
•
1
0.4
•
1
0.6
sTF-conc
Figure 21.
•
1
0.8
•
1 • 1
1.0
1.2
(uM)
Clot time vs. sTF concentration.
Clotting times of a neat citrated
pooled plasma sample were measured using an sTF-reagent composed of 0.27 mM
PC/PS/PE (64:6:30), TBS/BSA with varying concentrations of sTF (0.01 - 1.0 uM).
Clotting times began plateauing at 0.1 uM and reached a minimum of aprox. 0.5 uM sTF.
(64:6:30) phospholipid mixture, a set of sTF-reagents with varying concentrations of
PC/PS/PE was prepared and the clotting time for a normal pooled plasma (NPP) sample
was measured using each. A plot of clotting time versus phospholipid concentration is
shown in Figure 22. Clotting times reached a plateau at approximately 1.0 mM PC/PS/PE
(64:6:30).
An updated protocol for the FVIIa assay provided by Dr. Morrissey describes the
use of a 0.2 mM PC/PS/PE (40:40:20) mixture as the phospholipid source (51).
Comparison of the two PC/PS/PE compositions revealed that the 0.2 mM PC/PS/PE
(40:40:20) mixture yielded 30% shorter clotting times than the, 1.0 mM PC/PS/PE
57
(64:6:30) using the same sTF concentration. The shorter clotting times obviously were a
result of the higher composition (40%) of phosphatidylserine (PS), a negatively charged
phospholipid. The danger of using such a high concentration of negatively charged
phospholipids (ie. 60%) is that the negative phospholipids could initiate contact activation
of coagulation which could in turn activate FVU to FVIIa via factor XUa (37). This would
result in a misleading increase in the measured plasma FVIIa levels.
The main objective of optimizing the sTF-reagent composition was to achieve a
reproducible assay with convenient clotting times while conserving valuable sTF. An sTFreagent of composition 0.5 uM sTF, 1.0 mM PC/PS/PE (64:6:30) in TBS/BSA gave
convenient clotting times of approximately 60 seconds for neat NPP samples and 120
seconds for 10-fold diluted NPP samples using the automated assay and thus was chosen
as the sTF-reagent for the FVIIa assay.
140 T
1
u
CD
40 H
0
1
1
1
•
2
1
PC/PS/PE-concentration
Figure 22.
•
1
3
(mM)
Clot time vs. Phospholipid concentration. Clotting times of a neat
citrated pooled plasma sample were measured using an sTF-reagent composed of 0.5 uM
sTF, TBS/BSA with varying concentrations of PC/PS/PE (64:6:30) phospholipid (0.05 2.0 mM). Clotting times reached a minimum at approx. 1.0 mM PC/PS/PE.
58
9. Factor V i l a Standard Curve
In order to quantitate FVIIa levels, a calibration curve to convert clotting times to
factor Vila concentration was prepared. Initially, FVIIa standards of 30, 3.0, 0.30, and
0.03 ng/ml were prepared by serial dilution in Sigma immuno-depleted FVII-deficient
plasma. However, as shown in Figure 23, the calibration curve began to plateau at FVIIa
concentrations below 0.10 ng/ml.
2.2 T
1.4
H
•
• 2
1
1
23.
1
- 1
FVIIa
r
0
Log
Figure
1
:
FVIIa
standard
1
2
coiic
curve
using
Sigma
FVII-def
plasma.
Measurement of clotting times of FVIIa calibration standards of 30, 3, 0.3, and 0.03 ng/ml
FVIIa prepared in Sigma brand immuno-depleted FVII-deficient plasma using the FVIIa
assay. A plateau in clotting times is apparent at FVIIa concentrations below 0.10 ng/ml due
to the high endogenous levels of FVIIa in the Sigma FVII-deficient plasma.
Measurement of the FVIIa level in the FVII-deficient plasma revealed that there was 0.06 0.07 ng/ml residual FVIIa in the Sigma product. When the FVIIa standard curve was
corrected for this endogenous FVIIa, a linear curve resulted.
59
However, the endogenous FVIIa would cause significant deviations in measured
plasma FVIIa levels due to the fact that plasma samples would be diluted 10-fold in the
Sigma FVII-deficient plasma. For example, a plasma sample containing 2.0 ng/ml FVIIa
diluted 10-fold would be expected to give 0.2 ng/ml. However, the endogenous FVIIa in
the FVII-deficient plasma used for dilution would increase the apparent FVIIa concentration
to 0.27 ng/ml. After correction for dilution, the measured FVIIa would be 2.7 ng/ml,
significantly different from the actual FVIIa concentration.
Thus, congenital FVII-deficient plasma was purchased from George King
Biomedical as an alternative source. A FVIIa calibration curve prepared using FVIIa
standards serially diluted in the new FVII-deficient plasma is shown in Figure 24.
2.4 -
S
2.2-
**
fit)
.5
"•3
2
-°"
©
1.8-
5
O
J
1.61.4 -2
-1
Log
Figure 24.
0
FVIIa
1
2
cone
FVIIa standard curve using George King FVII-def plasma.
Measurement of clotting times of FVIIa calibration standards of 30, 3, 0.3, and 0.03 ng/ml
FVIIa prepared in George King Biomedical brand congenital FVII-deficient plasma using
the FVIIa assay. Only a small plateau in clotting times was apparent at low concentrations
of FVIIa due to the very low levels of endogenous FVIIa in the George King plasma.
60
There was still a slight plateau at low FVIIa levels but the George King Biomedical FVTJdeficient plasma provided a significant improvement over the Sigma product Measurement
of the endogenous FVIIa revealed that there was only 0.02 ng/ml FVTJa in the new FVIIdeficient plasma. Although not completely negligible, this was judged to be acceptable for
the dilution of normal plasma samples. The FVIIa standard curve was corrected for the
endogenous FVIIa by plotting the FVIIa standards as 30.02, 3.02, 0.32, and 0.05 ng/ml
FVTJa. The final FVUa standard curve used for the assay is shown in Figure 25.
CALIBRATION
Factor UII Activate!
Date
61/27/1937 18:65:26
Control 1
Control 2
Calibrators Rom Data
ng/al
sec.
39.82
26.9
36.62
27.3
3.62
53.3
3.62
53.2
6.32
111.1
6.32
111.3
6.65
263.6
6.65
261.1
Interpol.
ng/itl
28.82
27.51
3.31
3.33
6.32
6.32
6.65
6.65
1
0.05
r-
0.32
3.02
oancfMrxtion n o ^ l
LogCO - <- 3.1(7 • UgCt» • 5.988
r » - 1.000
Figure 25. Final F V U a Standard Curve. Factor Vila standard curve prepared by
STA
coagulometer calibration program using corrected FVIIa standard concentrations of
30.02, 3.02, 0.32, and 0.05 ng/ml diluted in George King Biomedical FVII-deficient
plasma. This curve was used by the STA coagulometer to convert clottingtimesto FVUa
concentrations in ng/ml.
The linear regression coefficient for the standard curve of -1.000 shows a ^-magnitude
linear range for the FVTJa assay. Clot times ranged from 26 seconds to 200 seconds for the
61
measurable range with normal 10-fold diluted plasma samples falling in the middle of the
range. Plasma FVIIa concentrations as low as 20 pg/ml could be measured indicating an
extremely sensitive assay. The FVIIa standard curve shown in Figure 25 was used by the
STA Coagulometer to automatically convert clotting times of plasma samples into FVIIa
concentrations in ng/ml.
10.
Comparison of BHK-derived sTF and E.co/i-derived sTF
As previously mentioned, the original FVIIa assay employed a recombinant sTF
protein expressed in E.coli. (26). To study the effects of glycosylation on sTF-function,
sTF-reagents containing BHK-derived sTF and E.co/i-derived sTF (a gift from Dr.
Morrissey) were prepared and several FVIIa standards and plasma samples were measured
using each sTF-reagent. Figure 26 shows the results of linear regression analysis of the
FVIIa concentrations measured using each sTF-reagent.
Measurement of the clotting times for the FVIIa standards (Figure 26a) using each
sTF-reagent gave excellent agreement with a linear regression coefficient of r = 1.00.
Figure 26b shows the results of measurement of the 5 normal plasma samples. Linear
regression analysis shows the correlation between the FVIIa values measured using either
sTF-reagent was also extremely good, r = 0.976.
The results indicate that BHK-derived sTF behaved identically to the E.co/i-derived
form and glycosylation did not appear to affect the co-factor activity of sTF nor its activity
in the FVIIa assay. This was in agreement with the findings of Dr. Morrissey (26) and
others (50, 70).
11.
Testing of FVIIa Assay
The next stage of development of the FVIIa assay was to establish the precision and
accuracy of the assay. Reproducibility studies established within-run and between-run
coefficients of variation of 3.9% and 4.9%, respectively, for the present study's FVIIa
62
Clot
Figure 26.
times
w/
E.coli-sTF
(sec)
FVIIa
w/
E.coli-sTF
(ng/ml)
Comparison of B H K and E.co/i-derived sTF in FVIIa Assay.
Results of linear regression analysis of (a) measurement of clotting times of 4 FVIIa
calibrators (30, 3, 0.32, 0.05 ng/ml FVIIa) and (b) measurement of FVIIa concentrations
of 5 normal citrated plasma samples using sTF-reagents composed of 1.0 mM PC/PS/PE
(64:6:30), TBS/BSA, and either 0.5 uM BHK-derived sTF or 0.5 uM £.co//-derived sTF.
assay. This compared very well with Morrissey's results of 2.2% and 8.1% (26), Kario's
results of 1.3% and 4.2% (27), and Phillippou's results of 4.5% and 9.8% (25),
respectively.
A patient comparison study was performed to establish the accuracy of the FVIIa
assay. Since no absolute FVIIa standards were available as a comparison, a commercial
STACLOT VIIa-rTF kit from Diagnostica Stago was used to define the FVIIa levels of a
series of citrated plasma samples. The Stago kit measured FVIIa in international activity
units (mU/ml) and included a sample of the FVIIa international standard (52). Since it is
the only commercial kit available on the market for measuring FVIIa, the Stago kit was
regarded as the standard by which a new FVIIa assay should be tested. Although the kit
also used a clot-based method of detection, the assay procedure (which was also automated
63
on the STA Coagulometer) was slightly different. Plasma samples were diluted 10-fold in
a physiological buffer and then diluted 1:1 with FVII-deficient plasma prior to
measurement. The present study's FVIIa assay diluted plasma samples 10-fold directly in
FVII-deficient plasma. However, since the FVIIa standards and test plasma samples were
treated identically in each assay, the difference in dilution protocols should not have an
effect on the measured FVIIa levels.
The results of linear regression analysis of the patient comparison study are shown
in Figure 27.
0
200
STAGO
Figure 27.
400
FVIIa
600
800
(mU/ml)
0
20
STAGO
Patient comparison study results.
40
60
FVIIa
80
100
(mU/ml)
Results of linear regression
analysis of measurements of 19 citrated plasma samples containing 0.1 - 30 ng/ml FVIIa
using present FVIIa assay and STACLOT VIIa-rTF kit (a). Two spiked high FVIIa
samples (15 and 30 ng/ml) were included. The normal human plasma samples gave FVTIa
values of 5.0 ng/ml (100 mU/ml) or less. Linear regression analysis of only these 17
normal plasma samples are shown in (b).
The plasma samples were chosen to achieve a broad range of FVIIa levels. To assess
accuracy at extremely low FVIIa levels, plasma samples were diluted 10-fold in FVII-
64
deficient plasma before loading for measurement. To determine accuracy at extremely high
FVIIa levels, two plasma samples were spiked with FVIIa to give concentrations of 15 and
30 ng/ml FVIIa. A total of 19 samples were measured using both assays. As shown in
Figure 27, over the full range of FVIIa (0.1 - 30 ng/ml), correlation between the two
assays was excellent, r = 0.998. When considering only the normal human plasma
samples, the results were still extremely good, r = 0.995.
It should be noted that the FVIIa calibration curve of the Stago kit had a smaller
range than the present study's FVIIa assay. The upper limit of the kit's standard curve was
250 mU/ml which corresponded to approximately 10 ng/ml FVIIa. Thus, the 15 ng/ml
FVIIa sample was off-scale and its corresponding international activity level of 400 mU/ml
had to be determined manually. The limited FVIIa ceiling was partly due to the faster
clotting times of the STACLOT kit. As FVIIa levels increase, the clotting times decrease.
For example, the 30 ng/ml FVIIa sample clotted in 26.8 seconds with the STACLOT kit
compared to 54.4 seconds using the present FVIIa assay. At extreme FVIIa levels, clot
times would reach a lower limit using the STACLOT kit more quickly than with the present
study's FVIIa assay. This would limit its use in measurement of extremely high FVIIa
levels.
The inclusion of the FVIIa international standard in the Stago kit gave the
opportunity to establish a conversion curve for FVIIa concentrations. Since the results of
the patient comparison study were so accurate, it was concluded that both assays were
indeed measuring the same relative level of FVIIa but expressing them in different units.
Thus, the linear regression curve of Figure 27 could be used as a FVIIa conversion curve
to convert FVIIa concentrations measured in ng/ml to international activity units in mU/ml
to allow for better inter-laboratory comparison.
65
12.
Plasma FVIIa Stability Testing
It was important to clearly establish the method of handling plasma samples to
ensure the stability of plasma FVIIa. To determine whether samples that were measured
after being freshly drawn (as often is the case in a hospital) could be compared to plasma
samples placed in frozen storage, samples were measured immediately after being drawn
and again after varying lengths of storage at -70°C. Table la below shows the results of the
experiment.
Table I.
a.
Effects of freezing on plasma' FVIIa levels ,
8
Results of fresh vs freeze-thawed
Sample
Fresh FVIIa
Time Frozen
Freeze-thawed FVIIa
Norm lC:pm
3.1 ng/ml
15 minutes
3.1 ng/ml (100%)
Norm lE:pm
3.2 ng/ml
2 days
3.0 ng/ml (94%)
Norm lE:pm
3.2 ng/ml
7 days
3.1 ng/ml (97%)
b.
Results of repeated freeze-thaw cycles.
Number of Freeze-Thaw Cycles
Sample
Norm lC:am
Norm lE:pm
Norm lE:pm
a
One
Two
Three
2.2 ng/ml
2.0 ng/ml
1.0 ng/ml
(91%)
(45%)
2.8 ng/ml
3.2 ng/ml
(93%)
(107%)
3.4 ng/ml
3.1 ng/ml
(110%)
(100%)
3.0 ng/ml
3.1 ng/ml
Plasma FVIIa levels measured using present study FVIIa assay after indicated time of
frozen storage and/or number of freeze-thaw cycles. Values in brackets refer to relative%
FVIIa measured compared to fresh sample (a) or single-freeze-thaw sample (b).
66
The results indicate that plasma FVIIa levels are not affected by a single freeze-thaw cycle
nor by the length of frozen storage; the differences between fresh and freeze-thawed FVIIa
levels are within the inter-assay variations of 4.9%.
To test whether further freeze-thaw cycles would affect plasma FVIIa levels, the
samples were subjected to two more freeze-thaw cycles. The results in Table lb show that
FVIIa levels were reasonably stable after even two freeze-thaw cycles but the change in
FVIIa could not be explained solely by inter-assay variation. However, after the third
freeze-thaw cycle, gross changes in FVIIa levels were observed in at least one sample
(Norm lC:am). In most assays for hemostatic variables (PT, APTT, FVII:C, etc.), plasma
samples are measured fresh or after.at most a single freeze-thaw cycle (73). Thus, plasma
samples for the clinical study in this investigation were restricted to a maximum of one
freeze-thaw cycle.
Another important consideration for the stability of the plasma samples is the
anticoagulant used during blood collection. Typically;- plasma used for measurement of
hemostatic variables is collected in citrate. To determine whether plasma samples collected
in EDTA tubes could be used for measurement of FVIIa levels, a plasma sample from the
same, donor was collected in a citrate tube and a second collected in an EDTA tube and both
were measured using the FVIIa assay. The results of precision testing on both samples are
shown in Table II.
The results show very poor agreement between the EDTA and citrated sample in
measured FVIIa level. In addition, inter-assay reproducibility of the EDTA sample was
very poor with a coefficient of variation of 21.4% versus 4.9% for the citrated sample.
EDTA is a much stronger chelator than citrate and thus requires much higher concentrations
of calcium to overcome its anti-coagulant effects (74). As well, EDTA can cause clumping
of platelets in plasma that may also affect the measured FVUa levels. It was concluded that
67
Table II. Comparison of EDTA vs. Citrate as plasma anticoagulant.
b
Avg FVIIa-conc
Intra-assay C V . Inter-assay C V .
Citrated plasma
1.65 ± 0.08 ng/ml
3.9%
4.9%
EDTA plasma
2.19 ± 0.47 ng/ml
7.1%
21.4%
b
Measurement of a citrated and EDTA plasma sample collected from a single donor at the
same collection using the FVIIa assay. Mean FVIIa with standard deviation and results of
intra-assay and inter-assay precision testing for reproducibility are shown.
EDTA was a poor choice as an anti-coagulant for FVIIa measurement. Thus, plasma
samples were collected only in citrated tubes for the clinical study.
Plasma samples were routinely diluted 10-fold in FVII-deficient plasma for
measurement of FVIIa and then the measured FVIIa concentration was multiplied by 10 to
correct for the dilution. However, the expense of FVII-deficient plasma suggested
measurement of plasma samples neat. The results of a comparison between plasma
samples measured undiluted versus 10-fold diluted (data not shown) indicated mixed
results. In some cases the measured FVIIa levels agreed between undiluted and diluted
measurements whereas in other cases undiluted samples gave higher or lower readings for
FVIIa when compared to the 10-fold diluted samples (after correction for dilution).
i
Consideration of the mechanism of coagulation suggested the difference in FVIIa
between undiluted and diluted samples was caused by differences in the amounts of clotting
factors other than FVIIa (such as fibrinogen, FX, etc.). If the test plasma had different
amounts of the other clotting factors than in the FVII-deficient plasma in which the
standards were diluted, then these would also influence the clotting time. Samples that
gave similar FVIIa levels whether diluted or not probably had levels of the other clotting
factors (other than FVII) that were similar to those in the FVII-deficient plasma. Thus, the
clotting times of samples that are measured undiluted are determined not only by their
68
endogenous level of FVIIa but also by the levels of other clotting factors. Ten-fold dilution
ensures that 90% of the other clotting factors come from the same source, the FVIIdeficient plasma. Thus, the differences caused by the other clotting factors (which also
vary from individual-to-individual) are diluted out and only differences between FVIIa
levels will affect the clotting times and thus the measured FVIIa concentration (26). For
this reason all test plasma samples were diluted 10-fold in FVII-deficient plasma.
Although formal studies were not carried out, the effects of cold activation and
post-prandial hyperlipidemia on FVIIa levels were examined briefly. The results indicated
that prolonged storage of plasma samples at 4°C caused an increase in FVIIa levels. As
well, plasma samples drawn 3 hours or more after a high fat meal had elevated FVIIa levels
compared to the fasting state. However such an increase in FVIIa levels was not observed
after light (low fat) meals up to 2 hours before collection. These results are in agreement
with several other studies which found that cold activation (26, 27) and post-prandial
hyperlipidemia (38, 39, 40, 41, 55) are associated with an increase in FVIIa.
13.
Intra-individual variations in plasma FVIIa
To study the effects of individual variations in FVIIa, six citrated plasma samples
were collected over a 3 month period from the same donor. The results are shown in Table
III.
Table III.
FVIIa
c
Intra-individual variation in FVIIa levels.
c
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
2.2 ng/ml
2.1 ng/ml
2.0 ng/ml
2.3 ng/ml
2.2 ng/ml
2.0 ng/ml
Measurement of FVIIa levels in six citrated plasma samples collected from a single donor
over a period of 3 months using the FVIIa assay. Values are from duplicate measurements
agreeing within 7%. Average FVIIa level is 2.13 ± 0.12 ng/ml (CV. = 5.6%).
69
The results indicate that FVTIa levels are quite stable within the same donor. The variations
are not within inter-assay variability and thus there is a small amount of real fluctuation in
FVUa levels within an individual donor. However, generally it appeared that FVIIa levels
were very characteristic of an individual. This was also in agreement with previous studies
(26, 27).
14. Factor Vila Levels in a Normal Population
The first stage of the clinical study on FVIIa was to establish the normal range for
FVIIa and to determine the effects of age and gender on FVIIa levels. A population study
was undertaken in which plasma samples were collected from 50 healthy donors aged 20 52 years. The overall mean FVIIa level was 2.08 ng/ml (42 mU/ml) with a standard
deviation of 0.90 ng/ml (15 mU/ml). The range of FVIIa in the study group was 0.7 ng/ml
(11 mU/ml) to 4.8 ng/ml (100 mU/ml) corresponding to 33.7% to 231% of the mean
value. Previous studies by others have determined mean FVIIa levels to be 3.48 ng/ml
(26), 2.5 ng/ml (27), and 4.1 ng/ml (53). The lower mean FVIIa level in the present study
could be a result of the smaller population size surveyed or simply characteristic of a
different population than in the other studies. However as Figure 28 shows, FVIIa levels
increased with age.
The average age of the donors in the present study was 35.4 ± 8.9 years which was
significantly lower than the studies by Kario (57.9 years) and Scarabin (45.5 years) but
similar to Morrissey's group (36.2 years). However, the present study did not include any
elderly patients (over 55) who would be expected to have higher FVIIa levels as indicated
by the trend in Figure 28 and previous studies (26, 27, 53). Thus the absence of elderly
subjects could have resulted in the lower mean FVIIa value recorded in the present study.
As Figure 28 shows, a strong correlation between increasing age and FVIIa levels existed
(r = 0.443 for males, r = 0.390 for females).
70
•
Males (n=21)
•
Females (n=29)
R = 0.443 for Males
R = 0.390 for Females
10
20
30
40
50
60
Age (years)
Figure 28. Relationship between Factor Vila and Age. Plasma FVIIa levels in
a normal population of 50 healthy subjects (21 male, 29 female, average age = 35.4 year,
age range = 20 - 52 years) as a function of age. Linear regression coefficients (R) are
indicated separately for males and females. Mean factor Vila of the normal population was
2.08 ± 0.90 ng/ml.
The association between increasing age and FVIIa had very similar patterns in both
males and females as shown by the two lines in Figure 28. In addition, there was no
significant differences between the FVIIa levels of men and women in terms of mean
FVIIa.
Statistics on sub-populations (by age and gender) of the study subjects are
summarized in Table IV.
71
Table IV. Age Distribution and FVIIa levels in Study Sub-populations'
1
Sub-population
Mean FVIIa
Average Age
Group size
All Normals
2.08 ± 0.90 ng/ml
35.4 ± 8.9 years
n = 50
Normals, <40 yrs
1.82 ± 0 . 7 6 ng/ml
29.8 ± 5.2 years
n = 32
Normals, >40 yrs
2.55 ± 0.97 ng/ml
45.3 ± 3.9 years
n = 18
All Females
2.10 ± 0 . 9 0 ng/ml
34.7 ± 8.4 years
n = 29
All Males
2.07 ± 0.94 ng/ml
36.3 ± 9.6 years
n = 21
All Females, <40 yrs
1.86 ± 0.75 ng/ml
30.1± 5.2 years
n = 20
All Females, >40 yrs
2.62 ± 1.00 ng/ml
45.0 ± 3.3 years
n == 9
All Males, <40 yrs
1.76 ± 0.80 ng/ml
29.3 ± 5.3 years
n = 12
All Males, >40 yrs
2.48 ± 0 . 9 9 ng/ml
45.7 ± 4.6 years
n ==9
d
Mean ± Standard deviation of plasma FVIIa levels for various sub-populations of the
study group. Mean ± SD for age and number of subjects per sub-population are also
indicated.
No significant difference between males and females was observed in any of the subpopulations. In both genders, FVIIa levels increased with age at approximately the same
rate. Corresponding male and female sub-populations in Table IV were closely agematched. Thus, gender did not seem to affect FVIIa levels in the study population.
72
15.
Factor V i l a Levels in Complicated Pregnancy Patients
Clinical details on nine patients who experienced complications during pregnancy
are summarized in Table V.
Table V. Clinical details of Complicated pregnancy patients
Sample
Comments
Age
Clinical Stage
Platelet count
H-26
On Heparin
40 years
Ante-partum
N/A
H-77
Had anti-cardiolipin
32 years
Ante-partum
311
26 years
Ante-partum
N/A
H-16
35 years
Post-partum
N/A
H-106
22 years
Post-partum
450
32 years
Post-partum
170
36 years
Post-partum
126
36 years
Post-partum
191
28 years
Post-partum
267
antibody
H-39
H-61
22+ weeks
Post-partum
Hemorrhage
H-134
H^tl
New-bom had
hemolytic disease
H-179
The patients were either full-term and plasma samples were drawn following a complicated
delivery (post-partum) or mid- to late-term and experienced complications during their
pregnancy (ante-partum) when the plasma samples were taken. Normal hemostatic
variables measured may have included platelet counts, prothrombin time (PT), activated
partial thromboplastin time (APTT), and plasma fibrinogen (FIBR) at the physician's
discretion. FVUa levels were measured in all subjects. The results are shown in Table VI.
73
Table VI. Hemostatic variables of complicated pregnancy samples
Sample
PT
(10.4-12.2)
APTT
(23.3-32.8)
FIBR
(3.75-6.3)
FVIIa
(0.7-4.8)
H-26
12.1 sec
31.5 sec
N/A
0.9 ng/ml
H-77
10.9 sec
27.4 sec
N/A
3.9 ng/ml
H-39
10.6 sec
N/A
N/A
1.8 ng/ml
H-16
10.7 sec
24.9 sec .
6.7
6.2 ng/ml
H-106
10.5 sec
31.9 sec
N/A
4.8 ng/ml
H-61
11.1 sec
22.0 sec
N/A
2.2 ng/ml
N/A
27.0 sec
4.16
5.2 ng/ml
H^U
9.5 sec
27.5 sec
7.5
6.0 ng/ml
H-179
10.6 sec
31.2 sec
N/A
6.0 ng/ml
H-134
Overall, for the 9 pregnancy samples the mean FVIIa was 4.11+2.0 ng/ml (85±40 mU/ml)
which was significantly higher than the mean FVIIa for normal subjects. This agrees with
previous studies by Morrissey on FVIIa in pregnancy (26) and supports the general
increase in hemostatic activity accompanying pregnancy; factor VII levels normally increase
to 150 - 250% of normal levels during pregnancy (75) as a protective mechanism.
However, there appeared to be a few obvious outliers among the pregnancy
samples. Specifically, samples H-26 (0.9 ng/ml), H-61 (2.2 ng/ml), and H-39 (1.8 ng/ml)
exhibited significantly lower FVIIa levels than the other pregnancy samples. Closer
inspection of the patient details in Table V provided explanations for the outliers. Patient
H-26 was on heparin therapy, an anticoagulant. Factor Vila levels have been previously
shown to be lowered in patients on anticoagulants (26). The PT and APTT times were also
extended indicating the anti-clotting effects of heparin. Patient H-61 suffered from postpartum hemorrhage indicating failure of the protective hemostatic mechanisms that normally
stops excessive bleeding after delivery. The normal level of FVIIa in the patients blood
was an indication of the unprepared state of the hemostatic system to the massive blood
loss accompanying parturition. Patient H-39 was only 22 weeks or 5 months into her
pregnancy and thus activation of the hemostatic system had not reached its peak.
74
However, other undocumented complications in the patient may also have influenced the
low plasma FVIIa concentration.
After removing the 3 outliers, the mean FVIIa concentration for the remaining
pregnancy samples was 5.35 ± 0.89 ng/ml (115 ± 15 mU/ml) which was the same mean
FVIIa level measured by Morrissey (26) in pregnant subjects. This was a very significant
increase over the mean FVIIa of 1.86 ± 0.75 ng/ml (37 ± 12 mU/ml) in an age-matched
group of non-pregnant female subjects.
There did not appear to be any significant
correlation between FVIIa levels and any of the other hemostatic variables measured in
Table VI.
An additional 10 plasma samples from complicated pregnancy patients were
received several weeks after the initial study. Unfortunately, complete clinical details (as in
Table V) wereriotavailable for the new patients at the time of publication. However,
FVIIa concentration, PT, and APTT were measured for each sample. The results are
presented in Table Via.
Table V i a . Hemostatic variables for new complicated pregnancy samples
Sample
Age
PT
APTT
FVIIa
10
22
10.8 s
N/A
2.6 ng/ml
12
30
11.1 s
26.2 s
3.0 ng/ml
13
33
11.0 s
30.6 s
5.1 ng/ml
14
31
10.7 s
25.5 s
4.6 ng/ml
15
33
N/A
44.4 s
0.8 ng/ml
16
36
10.5 s
35.3 s
4.2 ng/ml
17
30
10.1 s
28.8 s
1.9 ng/ml
18
29
11.5 s
30.2 s
1.7 ng/ml
19
34
11.5 s
27.0 s
8.7 ng/ml
20
29
11.7 s
32.5 s
7.7 ng/ml
The overall mean FVIIa level for all nineteen complicated pregnancy samples was 4.07 ±
2.2 ng/ml (84 ± 45 mU/ml), similar to the mean FVIIa for the first nine samples.
75
DISCUSSION
Recent studies implicating factor VII as an independent risk factor for coronary
heart disease (7, 8, 28, 33) have necessitated improved techniques to characterize plasma
levels of the serine protease. Conventional assays for the measurement of FVII have
typically relied on thromboplastin to target the extrinsic factors. However, recent studies
exposing the heterogeneity of such assays (23, 45) as well as the postulation of the
existence of trace amounts of pre-activated factor Vila in plasma (76), which may be
important in the initiation of clotting, have suggested the need for an assay specific for
factor Vila. Until recendy, there was no way to separate the activated form of FVIIa from
its zymogen precursor, FVII, using a functional assay. The development of a soluble
mutant form of tissue factor (47, 50) has provided an avenue for the design of a clot-based
assay specific for factor Vila.
The FVIIa assay in the present study was developed based on the original FVIIa
assay employing sTF reported by Morrissey et al (26). The present study's assay modified
the original procedure in several important ways. First, a glycosylated form of sTF was
uniquely expressed in BHK cells rather than E.coli as in the original assay. Second, a new
phospholipid source had to be found to replace rabbit brain cephalin (RBC) which was
used in the original paper but has since been discontinued by the manufacturer (Sigma).
Third, the present study's assay was run on an automated STA Coagulometer. Finally, the
present assay was calibrated to determine plasma FVIIa activity in international standard
units (mU/ml) as well as in concentration (ng/ml).
1.
BHK-derived glycosylated sTF
Soluble tissue factor protein was successfully expressed in baby hamster kidney
cells at a level of 3 mg per litre and purified using anion-exchange FPLC and gel filtration
chromatography. Previous studies have used E.coli, yeast, and Chinese hamster ovary
76
cells for expression. The advantage of using a mammalian eucaryotic expression system
such as BHK or CHO is that they confer post-translational modifications to the
recombinant proteins similar to native human forms of the protein. The BHK-expressed
form of sTF was glycosylated with a molecular weight of 39 kDa which was similar to the
protein produced in CHO cells (50) and the minor form of sTF produced in yeast (70). A
minor lower molecular weight form of the sTF protein was also detected by immunoblot in
BHK expression. Its apparent molecular weight of 35 kDa on SDS-PAGE matched the
CHO- and yeast expressed forms of sTF following treatment with endoglycosidase F (50,
70) suggesting that the minor species was a less glycosylated form of sTF. Glycosylation
of sTF occurs at three N-linked sites: Asn-11, Asn-124, and Asn-137 (70). Analysis of
the BHK-derived protein confirmed the presence of fucosyl residues in the sTF.
Comparison with a 25 kDa unglycosylated form of sTF expressed in E.coli,
however, revealed that the BHK-derived sTF behaved identically to the £.co//-derived form
in the clotting assay. The 39 kDa and 35 kDa forms of the BHK-derived proteins were
also functionally identical. Therefore, despite the increased molecular mass, glycosylation
had no effect on the cofactor activity of sTF. This was consistent with previous studies
(26, 70). However, there were other advantages of producing sTF in BHK cells over
bacteria. The signal peptide included in the cloned sTF cDNA sequence ensured that the
protein was secreted by the BHK cells into the growth medium. This allowed for milder
purification conditions since harsh methods of homogenization were not required.
Previous studies had used affinity chromatography to purify sTF (16, 67, 77). The present
study showed that the high expense of affinity columns could be avoided by purifying sTF
using general commercially available columns and matrices.
2.
Factor V i l a Assay
The FVIIa assay developed for the present study was optimized to run on an STA
coagulometer using an sTF-reagent composition of 0.5 uM sTF, 1.0 mM PC/PS/PE
77
(64:6:30) in TBS/BSA. The method required only 5 ul of test plasma and 45 ul of FVIIdeficient plasma per assay and as many as 100 samples could be measured simultaneously
using the STA coagulometer. In addition, only 50 ul of sTF-reagent, which contains just
0.8 ug sTF, was used per assay; thus 1.0 mg of sTF would be good for over one thousand
assays. The assay was sensitive to as little as 20 pg/ml FVIIa and samples up to 300 ng/ml
FVIIa could be measured, giving the assay a tremendous range. Furthermore, the upper
limit could easily be extended to 1000 ng/ml with no modifications. However, most
plasma samples would be expected to be well within the established range.
Precision testing of the assay revealed high reproducibility; within-run and
between-run coefficients of variation were 3.9% and 4.9%, respectively. The FVIIa assay
also proved to be extremely accurate when compared to a commercial FVIIa kit produced
by Diagnostica Stago. The two assays agreed over a range of 0.1 ng/ml to 30 ng/mPFVTIa
with a correlation coefficient of r = 0.998. All plasma samples measured fell within this
range of FVIIa concentrations. The inclusion of an international FVIIa standard in the
Stago kit allowed for the standardization of the present study's FVIIa assay. Thus samples
measured for FVIIa in ng/ml could be converted to international activity units (mU/ml)
using a FVUa conversion curve (Figure 29).
Use of standardized units of FVIIa activity would facilitate comparison of results
between different laboratories. This was a major shortcoming of previous assays for FVII
coagulant activity using thromboplastin. The use of FVIIa standards from different sources
for calibration of the FVIIa assay would produce problems since different methods of
assigning mass concentration (eg. optical density or ELISA antigen) are often used in
different labs (52). Results from studies using the present investigation's FVIIa assay can
be compared directly with other FVIIa assays that have been standardized using the
international FVIIa assay (including the STACLOT VUa-rTF kit).
78
40
FVIIa (mU/ml)
Figure 29. Factor Vila Conversion Curve. Factor Vila values measured in ng/ml
using the present study's FVIIa assay can be converted to international units (mU/ml) using
this conversion curve to improve between-laboratory comparison.
3.
Comparison to previous FVIIa assays
Previous assays described by Morrissey (26), Kario (27), and Phillopou (25) have
not made use of the international FVIIa standard. Generally, the FVIIa assay in the present
study is on par with or improves upon these previous assays. Testing revealed that the
present study's FVIIa assay (referred to simply as the "present assay" for this comparison)
was at least as robust as the other assays. The inter-assay reproducibility of 4.9% for the
present assay was significantly better than both Morrissey's and Phillopou's assays (8.1%
and 9.8%, respectively). Use of the STA coagulometer ensured reproducibility because of
its excellent automated duplicate run program which repeated an assay if the duplicate
results did not agree within 7%. Another advantage the present assay had over the
Morrissey assay was that the present assay used an sTF-concentration of 0.5 uM compared
to 1.0 uM sTF recommended in the Morrissey assay. Thus, using the present assay, twice
as many samples can be measured using the same amount of soluble tissue factor.
79
The present assay's sensitivity down to 20 pg/ml FVIIa is another strong point that
sets it apart, especially from Kario's assay which only measures to 200 pg/ml. Another
disadvantage of Kario's assay is that the fluorescent endpoint typically take 200 - 500
seconds (3-8 minutes) to be reached after addition of the initiating reagent. This results in
a slow assay. The present assay's reagents were optimized to achieve convenient clotting
times for the FVIIa standards (26 - 200 s) and the normal plasma FVIIa range (90 - 180 s).
Typically, a single measurement required about 4 minutes total assay time (including a 2
minute incubation period).
One problem of the extreme sensitivity of the present assay is that it is easily
influenced by contamination or carry-over of thromboplastin from other assays. Since
thromboplastin is a very sticky substance, it tends to be easily carried over on syringes or
other pipetting devices despite rinsing. A small amount of carryover can activate zymogen
FVII in a plasma sample and thus cause an increased FVIIa reading when measured using
the present assay. Since FVIIa values are multiplied by 10 to correct for dilution, the error
in FVIIa due to thromboplastin carryover is also compounded. Error of this type was
apparent when plasma samples were measured for FVIIa and FVII:C or PT,
simultaneously, using the STA coagulometer. To avoid the problem, samples had to be
measured separately when both FVIIa and a thromboplastin-based assay were used and
generally, it was better to measure FVIIa levels first to avoid thromboplastin carryover.
4.
Handling of plasma samples
The results of plasma FVIIa stability testing emphasized some guidelines for the
collection and handling of plasma samples for the FVIIa assay. Samples should be
collected only in siliconized tubes using trisodium citrate as the anti-coagulant. Two tubes
of blood were sequentially collected from the same venepuncture. The first tube of blood
was collected to remove the tissue plug that might result in in vivo tissue factor mediated
activation of FVII. Only the second tube was used for FVIIa measurement. However,
80
studies by other groups have suggested this precaution is unnecessary (26, 27).
Consumption of a light (uncooked, low fat) breakfast within 2 hours of blood collection
did not appear to affect FVIIa levels. However, due to the difficulty of controlling meal
content intake, fasting (12 hours) samples are highly recommended. Once collected, the
plasma samples may be measured immediately or after at most a single freeze-thaw cycle.
Frozen samples should be thawed at 37°C and held at room temperature until assay.
Samples should never be stored at 4°C for extended periods to avoid cold activation nor
should they have any contact with untreated glass to avoid contact activation.
5.
Factor V i l a in a normal population: effects of age and gender
The present study confirmed the existence of trace amounts of FVIIa in normal
healthy subjects as previously reported (26, 27). The normal range in the present study
was 0.7 to 4.8 ng/ml and the mean plasma FVIIa was 2.08 ± 0.90 ng/ml. Previous studies
have determined mean FVIIa levels to be 3.48 ng/ml by Morrissey et al (26), 2.5 ng/ml by
Kario et al (27), and 4.1 ng/ml by Scarabin et al (53) with ranges of 0.5 - 8.4 ng/ml, 1.2 5.1 ng/ml, and 0.4 - 14.3 ng/ml, respectively (Table VII), using larger populations.
Table VII. FVIIa Levels in Normal Populations from Several Studies
Study
Mean FVIIa
FVIIa Range
Study size
Age of Group
Morrissey
3.48 ng/ml
0.5 - 8.4 ng/ml
n= 188
36.2 yrs (18-72)
Kario
2.5 ng/ml
1.2 - 5.1 ng/ml
n= 110
57.9 yrs (21-84)
Scarabin
4.1 ng/ml
0.4 - 14.3 ng/ml
n = 684
41.1 yrs (26-64)
Present
2.08 ng/ml
0.7 - 4.8 ng/ml
n = 50
35.4 yrs (20-52)
As in previous studies, FVIIa levels varied substantially between individuals in the normal
population. In fact, the 7-fold variation of FVIIa levels in the present study was small
81
compared to other studies which found FVIIa levels to vary up to 36-fold (53). Normal
FVII antigen levels typically vary from 130 to 1000 ng/ml, a 7.7-fold range (78). Thus,
differences in FVII activity (ie. FVII:C) associated with CHD risk could be due to these
large variations in FVIIa levels observed in other studies. Assuming an average FVII
antigen concentration of 470 ng/ml, the mean FVIIa would represent 0.5% to 0.9% of the
total factor W protein in plasma.
The reason for the smaller range of FVIIa in the present study was partially due to
the smaller population surveyed (n=50) and partly due to the limited age-range of the
individuals. The oldest subject in the present study was 52 years old and the mean age of
the population was 35.4 years which was significantly lower than the studies by Kario
(57.9 years) and Scarabin (45.5 years) but similar to Morrissey's group (36.2 years).
However, as Table VII indicates the present study did not include any elderly patients (over
55) who would be expected to have higher FVIIa levels as indicated by the trends observed
in Figure 28 and the previous studies (26, 27, 53). Thus the mean FVIIa value and the
overall FVIIa range in the present study was skewed lower due to the absence of elderly
subjects.
The significant correlation between FVIIa levels and increasing age in both men and
women was also consistent with previous studies (26, 27). The results of the present
study indicated that there was no apparent difference in FVIIa levels between men and
women in any age group. However, some studies have shown that FVIIa levels increase
significantly in post-menopausal women (53). Menopausal status was not considered in
the present study.
Interestingly, the general trend of increasing FVIIa levels with advancing age and
menopause parallels the increase in CHD risk with age and onset of menopause. Of
course, many other changes in hemostatic status and other CHD risk factors such as lipid
levels also accompany age and menopause. However, since FVII coagulant activity has
been implicated in fatal incidences of CHD (28) and FVIIa plays an essential role in the
82
initiation of coagulation (13), it is conceivable that FVIIa is an important factor in
thrombotic conditions.
6.
Factor V i l a in prothrombotic conditions:
Complicated pregnancies
A comprehensive prospective clinical study is the only way to confirm an
association between risk of coronary heart disease and FVIIa levels. Although such a
study is clearly beyond the scope of the present investigation, an initial study to examine
potential correlations between FVIIa levels and certain prothrombotic conditions was
undertaken. A group of 9 patients at B.C. Children's Hospital were included in a study of
FVIIa levels in women experiencing complicated pregnancies. A significant increase in
FVIIa levels in pregnant women was observed; the corrected mean FVIIa concentration
during pregnancy was 5.35 ± 0.89 ng/ml. This was substantially higher than the mean
FVIIa in an age-matched group of non-pregnant females (1.86 ± 0.75 ng/ml) as well as the
population average of 2.08 ng/ml.
The overall effect of pregnancy is to increase the activity of the coagulation system
as a protective mechanism against post-partum hemorrhage. However, these changes can
put the patient at increased risk of venous thrombosis. Total factor VII levels reach 150 to
250% of normal levels during pregnancy (75) and this has been shown to be reflected in a
substantially higher FVII coagulant activity (79). The present study found FVIIa levels
during pregnancy to be on average 287% of normal levels. Although this high level of
FVIIa activity may have been in part due to the complications experienced by the patients
during their pregnancy, it does seem to indicate that FVIIa increases proportionally as much
or more than the total FVII mass during pregnancy. To better understand the interaction
between FVIIa and total FVII levels, FVII antigen levels must be measured (by ELISA)
and the ratio of FVIIa-to-FVII: Ag examined.
Although a complete study was not performed to determine the effects of anticoagulants on FVIIa, the results of one of the complicated pregnancy subjects provided
83
some insight into the cardio-protective effects conferred by heparin. The patient, a 40 year
old woman, was placed on heparin therapy because she was considered to be a high-risk
pregnancy. Because of her age, the risk of thrombotic complications during pregnancy
was greater than average. Previous results would suggest that her age and her pregnancy
would combine to produce extremely elevated FVIIa levels. However, her plasma FVIIa
was 0.9 ng/ml, below average even for a non-pregnant subject. This was likely a direct
result of the heparin therapy. Thus, heparin's cardio-protective effect is in part mediated
through its ability to directly decrease FVIIa levels or FVIIa activity in the plasma.
Although this may not be surprising since heparin is known to target serine proteases (80),
the 500% decrease in FVIIa activity/concentration is very large.
The only complicated pregnancy patient with lower than expected FVIIa levels
(2.2 ng/ml) after delivery suffered from post-partum hemorrhaging. Interestingly, the.
patient's PT and APTT times were normal. Hence the patient's risk of post-partum
bleeding could not have been predicted using the standard tests. However, the FVIIa assay
was able to detect a problem that would have alerted medical personnel to the potential
danger. Unfortunately, plasma fibrinogen was not measured for this subject as a
comparison to determine if other clotting factors were also depressed; however, the fact that
FVIIa was critically low (for a full term pregnancy) but both PT, a measure of extrinsic
activity, and APTT, a measure of the intrinsic pathway (44), were normal could be a clue
that depressed FVIIa levels are sufficient to cause bleeding problems post-partum.
The remaining 10 complicated pregnancy samples (Table Via) were not included in
the above discussion because clinical details about the patients' conditions were not
available at the time of publication. However, as with the first nine patients, the overall
mean FVIIa level was significantly elevated compared to normal, healthy donors. This
group included patients with extremely high FVIIa levels (samples 19, 20) as well as a few
patients with lower than expected FVIIa levels (samples 15, 17, 18). However, more
information about the patients is required for a full discussion.
84
7.
Future Work
Clearly more work must be done to establish FVIIa as an independent risk factor in
prothrombotic states. The clinical investigation undertaken in the present study had many
deficiencies. The healthy population used to establish the normal range of FVIIa was small
(n=50) and comprised a narrow age range (20-52 years). As well, even though subjects
were healthy with no known history of CHD, it would have been useful to better
characterize them by measuring BMI, blood pressure, total cholesterol, total triglycerides,
and hemostatic variables such as FVILC, FVILAg, and fibrinogen. Thus, correlations
between these different variables and FVIIa levels could have been determined.
However, the most indispensable test not included in the present study was for
factor VH antigen. Measurement of FVH:Ag would allow the determination of the ratio of
FVIIa-to-FVII: Ag, a more useful value representing the actual proportion of FVII in the
activated form. For example, an increase in FVIIa could represent either an overall increase
in the total amount of FVII protein with the same proportion of activated FVIIa or it could
represent an actual increase in the proportion of total FVII in the activated FVIIa form.
Without data on FVILAg, it was impossible to differentiate specific increases in activated
FVIIa from general increases in total FVII mass. Thus, the significance of the observed
increase in FVHa levels during pregnancy was not completely clear.
The study of FVIIa in prothrombotic states is presently incomplete. To this point,
only 19 samples from complicated pregnancies have been tested. More samples from
various clinically defined groups are absolutely required before a comprehensive study can
be achieved. It would be interesting to study FVIIa levels in patients with dyslipidemia to
determine if there are associations between FVIIa levels and various lipid components.
Studies by other groups have shown that a common polymorphism of the FVII gene is
associated with lower FVIIa levels. It would be interesting to follow such individuals to
determine if they suffered from a lower incidence of CHD. Recent studies have found a
85
close link between factor VII activity and fatal incidences of CHD (28); this suggests a
study that would separate subjects by the outcome of an MI event and measure each
group's mean FVIIa levels at the time of the event
The most meaningful study would be a long term, large scale prospective study
such as the Northwick Park Heart Study (7): The best way to accomplish such an
extensive study would be to examine several hemostatic variables together in a well defined
group of subjects who could be recruited and followed according to a well defined study
plan over at least a 10 year period. The best groups to study would be those that fit into
traditionally high risk categories but with no prior history of heart disease such as middleaged to elderly men or post-menopausal women.
Besides research on CHD risk, the FVIIa assay developed in the present study
would be useful for routine clinical work such as monitoring of patients receiving
recombinant FVIIa therapy or anti-coagulant therapy in which FVE levels must be carefully
controlled. As well, the assay could help in biochemical studies of cold and contact
activation of clotting factors, the mechanism of the tissue factor pathway, and determination
of the importance of pre-activated FVHa in initiation of normal clotting. Since FVII can be
activated by several different factors (thrombin, FXa, FXIIa, FVIIa) in vitro (81, 82), the
new assay could be used to elucidate which of these factors is most important in FVII
activation in vivo by performing studies in patients with various clotting factor
deficiencies.
Another use of the FVIIa assay could be in studying individuals with FVIIdeficiencies.
Since individuals deficient for FVII exhibit a broad range of clinical
symptoms (some suffer from severe bleeding disorders while others have no apparent
problems (83)), measurement of such subjects' FVIIa levels may provide some insight into
the apparent discrepancies. For example, a study could be done on two patients who each
have only 20% of normal FVII antigen levels; however one suffers a severe bleeding
disorder while the other does not. Measurement of their FVIIa levels could reveal that the
86
patient who does not bleed maintains normal levels of FVIIa despite the decreased total
FVII mass.
8.
Possible mechanisms of FVIIa involvement in thrombotic disorders
Many recent studies have improved the understanding of the significance of FVIIa
levels in various pathological conditions. The results of many of the clinical studies on
FVIIa were described in the introduction. Other studies have provided insight into the
possible mechanism of FVIIa and tissue factor in thrombotic states Here, speculation into
possible mechanisms of FVIIa's role as a prothrombotic risk factor in atherosclerosis,
diabetes, and menopause are discussed.
Histological evidence has confirmed the presence of TF in atherosclerotic plaques
(18). The rupture of a plaque is often the critical event in acute MI (19,42). The exposure
of TF from the plaque core to the vasculature initiates an explosive thrombus formation
near the site of plaque rupture. The size of the thrombus formed and thus the outcome of
the MI event may be determined by the plasma FVIIa level. High levels of circulating
FVIIa would produce a stronger and faster activation of extrinsic coagulation initiated by
the released TF and thus a more severe occlusion at the site of injury resulting in a greater
chance of the event ending fatally. Thus, FVIIa levels may not be a direct indicator of the
risk of CHD but may be important in determining the severity of the event after it has
occurred. This is consistent with the studies of Northwick Park where they found FVII:C
levels were better predictors of fatal incidences of CHD than non-fatal events (28).
Several studies have also determined that plasma FVIIa levels are elevated in
patients with non-insulin dependent diabetes mellitus (35, 57). This is a pathological state
that has been associated with endothelial damage. Studies have show that the greater the
extent of endothelial damage, the higher the associated FVIIa levels (57). Endothelial
damage results in the release or exposure of TF, a component of the subendothelium.
Thus, increased plasma TF would result in activation of FVII. However, the increased
87
extrinsic activity would remain sub-thrombotic and be characterized only by elevated FVIIa
levels. One of diabetes' most devastating side effects is an increased risk of CHD and
sudden cardiac death (84). Thus, the elevated FVIIa level mediated by diabetes-related
endothelial damage could be the cause of the CHD risk associated with diabetes.
Hormone replacement therapy (HRT) in post-menopausal women has been shown
in several studies to offer cardio-protective benefits (85). HRTs seems to confer these
cardio-protective benefits through their effect on the vascular wall and specifically the
endothelial layer. Thus, HRT may in fact be reversing the endothelial damage that releases
TF and activates FVII resulting in lower risk of CHD. It has been shown by Scarabin et al
(53) that HRT decreases FVIIa levels in post-menopausal women and such women suffer
from fewer incidences of CHD than post-menopausal women not on such therapy (85).
Since factor W activity and possibly FVHa may be closely linked to the outcome of
a coronary event, measures of FVII activity (FVILC, FVIIa, FVII: Ag) could be useful in
determining the short-term risk of CHD. If FVII(a) levels were found to increase quickly
over a short period of time, it could be an indication of plaque rupture or excessive
endothelial damage which might lead to an acute thrombotic event. Thus, careful
monitoring of FVIIa levels could be important for the survival of patients who are already
at known risk of CHD such as patients suffering from atherosclerosis, diabetes, or postmenopausal women.
9.
Implications for treatment of thrombotic disorders.
Because of the growing amount of epidemiological evidence implicating factor VII
and more recently factor Vila to the risk of CHD and cardiovascular death, the TF-FVHa
complex has become a target for the development of new anticoagulants for the treatment of
thrombotic disorders. Several groups have begun work on therapeutics which specifically
focus on the inhibition of factor Vila and/or the extrinsic pathway.
88
Some groups are exploring the use of recombinant forms of natural inhibitors of
factor VII. Yamanobe et al (86) have studied the use of recombinant tissue factor Pathway
Inhibitor (TFPI) as a therapeutic anticoagulant.
They found that TFPI injected
intravenously into rats initially binds to endothelial cells and hepatocytes removing it from
circulation. However, upon injection of heparin, the TFPI was released and improved the
anti-coagulant effects of the heparin. Another group, Sorensen et al (87) are working on
the use of an active site-inhibited FVIIa as a potential antithrombotic agent. The group
treated FVIIa with D-Phe-L-Phe-L-Arg-chloromethyl-ketone to inhibit its active site. Early
work has shown that, the inhibitor alters FVIIa's affinity for tissue factor thus inhibiting
TF-initiated coagulation.
Other groups are working on recombinant proteins and synthetic polypeptides as
FVfla-targeting anticoagulants. Kelley et al (88) have developed a mutant form of soluble
tissue factor which binds FVIIa with the same affinity as the native form but shows a 34fold reduction in catalytic efficiency for FX activation. The sTF mutant was shown to
successfully prolong prothrombin times. Finally, Ronning et al (89) have studied the
possibility of using synthetic peptide analogs of TF and FVII to inhibit FXa formation by
the TF-FVIIa complex. They prepared several synthetic polypeptides corresponding to
regions of TF and FVII which have been suggested to be important in binding between TF
and FVUa or the TF-FVIIa complex and FX to determine the potential inhibitory effects on
coagulation. Early results were encouraging.
. Thus, there is much excitement over this new potential area for the development of
anti-thrombotic agents targeting factor Vila and the tissue factor pathway. Further
understanding of the specific role and mechanism that FVIIa plays in thrombotic disorders
will allow better targeting of the potential therapeutics. Therefore, continued studies using
the FVIIa assay developed in the present investigation are essential to the development of
more efficacious anti-thrombotic drugs targeting the extrinsic pathway of coagulation.
89
REFERENCES
1. Ratnoff, O.D. Hemostasis and coagulation. In: Physiology, edited by Beme, R.M. and
Levy, M.N. St. Louis: The C V . Mosby Company, 1988, p. 371-391.
2. Kay, L.A. Whole blood Coagulation in Haemostasis. In: Essentials of Haemostasis and
Thrombosis, edited by Kay, L.A. Edinburgh: Churchill Living Stone, 1988, p. 40-53.
3. Jones, P. Bleeding and Clotting. In: Living with Haemophilia, edited by Jones, P.
Oxford: Oxford University Press, 1995, p. 39-58.
4. Saito, H. Normal Hemostatic Mechanisms. In: Disorders of Hemostasis, edited by
Ratnoff, O.D. and Forbes, C D . Orlando: Grune and Stratton, Inc., 1984, p. 23-42.
5. Bowie, E.J.W. and Owen, C A . The Clinical and Laboratory Diagnosis of
Hemorrhagic Disorders. In: Disorders of Hemostasis, edited by Ratnoff, O.D. and Forbes,
C D . Orlando: Grune and Stratton, Inc., 1984, p. 43-72.
6. Meade, T.W. Factor VII and Ischaemic Heart Disease: Epidemiological Evidence.
Haemostas 13:178-185, 1983.
7. Meade, T.W., Brozovic, M . , Chakrabarti, R.R., Haines, A.P., Imeson, J.D.,
Mellows, S., Miller, G.J., North, W.R.S., Stirling, Y. and Thompson, S.G. Hemostatic
Function and Ischaemic Heart Disease: Principal Results of the Northwick Park Heart
Study. Lancet 533-537, 1986.
8. Heinrich, J., Balleisen, L., Schulte, H., Assman, G. and van de Loo, J. Fibrinogen
and Factor VII in the Prediction of Coronary Risk. Results from the PROCAM Study in
Healthy Men. Arterioscler Thrombos. 14:54-59, 1994.
9. Hultin, M.B. Fibrinogen and Factor VII as Risk Factors in Vascular Disease. Prog
Hemostas Thrombos 10:215-241, 1991.
10. Ratnoff, O.D. The Evolution of Knowledge about Hemostasis. In: Disorders of
Hemostasis, edited by Ratnoff, O.D. and Forbes, C D . Orlando: Grune and Stratton,
1984, p. 1-22.
11. Osteoid, B. and Rapaport, S.I. Activation of factor LX by the reaction product of tissue
factor and factor VII: additional pathway for initiating blood coagulation. Proc Natl Acad
Sci USA 74:5260-5264, 1977.
12. Zur, M., Shastri, K. and Nemerson, Y. Kinetics of the tissue factor pathway of blood
coagulation: activation of factor DC. Blood 52:198,1978.
13. Nemerson, Y. Regulation of the Initiation of Coagulation of Factor VII. Haemostas
13:150-155, 1983.
14. Morrissey, J.H. Tissue Factor Modulation of Factor Vila Activity: Use in Measuring
Trace Levels of Factor Vila in Plasma. Thrombos Haemostas 74:185-188,1995.
15. Morrissey, J.H., Fakhrai, H. and Edgington, T.S. Molecular Cloning of the cDNA for
Tissue Factor, the Cellular Receptor for the Initiation of the Coagulation Protease Cascade.
Cell 50:129-135, 1987.
90
16. Paborsky, L.P., Tate, K.M., Harris, R.J., Yansura, D.G., Band, L., McCray, G.,
Gorman, C.M., O'Brien, D.P., Chang, J.Y., Swartz, J.R., Fung, V.P., Thomas, J.N.
and Vehar, G.A. Purification of Recombinant Human Tissue Factor. Biochemistry
28:8072-8077, 1989.
17. Edgington, T.S., Mackman, N., Brand, K. and Ruf, W. The Structural Biology of
Expression and Function of Tissue Factor. Thrombos Haemostas 66:67-79,1991.
18. Wilcox, J.N., Smith, K.M., Schwartz, S.M. and Gordon, D. Localization of tissue
factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci USA
86:2839-2843, 1989.
19. Falk, E. Plaque rupture with severe pre-existing stenosis precipitating coronary
thrombosis. Characteristics of coronary atherosclerotic plaques underlying fatal occlusive
thrombi. Br Heart J 50:127-134,1983.
20. Muller, Y.A., Ultsch, M.H., Kelley, R.F. and de Vos, A.M. Structure of the
Extracellular Domain of Human Tissue Factor: Location of the Factor Vila Binding Site.
Biochemistry 33:10864-10870, 1994.
21. Banner, D.W., D'Arcy, A., Chene, C , Winkler, F.K., Guha, A., Konigsberg,
W.H., Nemerson, Y. and Kirchhofer, D. The crystal structure of the complex of blood
coagulation factor Vila with soluble tissue factor. Nature 380:41-46,1996.
22. Carmeliet, P., Mackman, N., Moons, L., Luther, T., Gressens, P., Van Vlaenderen,
I., Demunck, H., Kasper, M., Breier, G., Evrard, P., Muller, M., Risau, W., Edgington,
T.S. and Collen, D. Role of tissue factor in embryonic blood vessel development. Nature
383:73-75, 1996.
23. Hayes, E.Y., Pike, J. and Tracy, R.P. Factor VII Assays. Arch Pathol Lab Med
117:52-57, 1993.
24. Albrecht, S., Kotzsch, M., Siegert, G., Luther, T., Grobmann, H., Grober, M . and
Muller, M. Detection of Circulating Tissue Factor and Factor VII in a Normal Population.
Thrombos Haemostas 75:772-777', 1996.
25. Philoppou, H., Adami, A., Amersey, R.A., Stubbs, PJ. and Lane, D.A. A Novel
Specific Immunoassay for Plasma Two-Chain Factor Vila: Investigation of FVUa Levels
in Normal Individuals and in Patients with Acute Coronary Syndromes. Blood 89:767775, 1997.
26. Morrissey, J.H., Macik, B.G., Neuenschwander, P.F. and Comp, P.C. Quantitation
of Activated Factor VII Levels in Plasma Using a Tissue Factor Mutant Selectively
Deficient in Promoting Factor VII Activation. Blood 81:734-744,1993.
27. Kario, K., Miyata, T., Sakata, T., Matsuo, T. and Kato, H. Fluorogenic Assay of
Activated Factor VII. Plasma Factor Vila Levels in Relation to Arterial Cardiovascular
Diseases in Japanese. Arterioscler Thrombos. 14:265-274,1994.
28. Ruddock, V. and Meade, T.W. Factor VII activity and ischaemic heart disease: fatal
and non-fatal events. QJMed 87:403-406,1994.
91
29. Hoffman, C.J., Miller, R.H. and Hultin, M.B. Correlation of Factor VII Activity and
Antigen with Cholesterol and Triglycerides in Healthy Young Adults. Arteriol Thrombos
12:267-270, 1992.
30. Miller, G.J., Walter, S.J., Stirling, Y., Thompson, S.G., Esnouf, M.P. and Meade,
T.W. Assay of factor VII activity by two techniques: evidence for increased conversion of
VII to Vila in hyperlipidaemia with possible implications for ischaemic heart disease. Br J
Haemostas 59:249-258, 1985.
31. Mukherjee, M., Dawson, G., Sembhi, K. and Kakkar, V.V. Triglyceride Dependence
of Factor VII Coagulant Activity in Deep Venous Thrombosis. Thrombos Haemostas
76:500-501, 1996.
32. Folsom, A.R., Ma, J., Eckfeldt, J.H., Shahar, E. and Wu, K.K. Plasma phospholipid
fatty acid composition and factor VII coagulant activity. Atheroscler 111:199-207,1994.
33. Miller, G.J., Bauer, K.A., Barzegar, S., Cooper, J.A. and Rosenberg, R.D.
Increased Activation of the Haemostatic System in Men at High Risk of Fatal Coronary
Heart Disease. Thrombos Haemostas 75:767-771, 1996.
34. Pankow, J.S., Folsom, A.R., Province, M.A., Rao, D.C., Eckfeldt, J.H., Heiss, G.,
Shahar, E. and Wu, K.K. Family History of Coronary Heart Disease and Hemostatic
Variables in Middle-aged Adults. Thrombos Haemostas 77:87-93, 1997.
35. Kario, K., Sakata, T., Matsuo, T. and Miyata, T. Factor VII in non-insulin-dependent
diabetic patients with microalbuminuria. Lancet 342:1552,1993.
36. Fuller, J.H., Keen, H., Jarret, R.J. and et al., Haemostatic variables asociated with
diabetes mellitus and its complications. Br Med J 2:964-966,1979.
37. Mitropoulos, K.A., Martin, J.C., Reeves, B.E.A. and Esnouf, M.P. The Activation of
the Contact Phase of Coagulation by Phhysiologic Surfaces in Plasma: The Effect of Large
Negatively Charged Liposomal Vesicles. Blood 73:1525-1533,1989.
38. Mennen, L.I., Schouten, E.G., Grobbee, D.E. and Kluft, C. Coagulation Factor VII,
Dietary Fat and Blood Lipids: A Review. Thrombos Haemostas 76:492-499, 1996.
39. Sanders, T.A.B., Miller, G.J., de Grassi, T. and Yahia, N. Postprandial Activation of
Coagulant Factor VII by Long-chain Dietary Fatty Acids. Thrombos Haemostas 76:369371, 1996.
40. Green, D., Chamberlain, M.A., Ruth, K.J., Folsom, A.R. and Liu, K. Factor VII,
Cholesterol, and Triglycerides: The CARDIA Study. Arterioscler Thrombos Vase Biol.
17:51-55, 1997.
41. Miller, G.J., Martin, J.C., Mitropoulos, K.A., Esnouf, M.P., Cooper, J.A.,
Morrissey, J.H., Howarth, D.J. and Tuddenham, E.G.D. Activation of Factor VII during
Alimentary Lipemia occurs in Healthy Adults and Patients with Congenital Factor XII or
Factor XI Deficiency, But not in Patients with Factor IX Deficiency. Blood 87:4187-4196,
1996.
42. Ridolfi, R.L. and Hutchins, G.M. The relationship between coronary artery lesions
and myocardial infarct: Ulceration of atherosclerotic plaques precipitating coronary
thrombosis. Am Heart J 93:468-486, 1977.
92
43. Levin, D.C. and Gardiner, G.A. Coronary Atheoslerotic Plaque Morphology and its
Consequences. In: Heart Disease: A textbook of Cardiovascular Medicine, edited by
Braunwald, E. Philadelphia: W.B. Saunders Company, 1988, p. 295-348.
44. Kay, L . A . The Coagulation Laboratory. In: Essentials of Haemostasis and
Thrombosis, edited by Kay, L.A. Edinburgh: Churchill Livingstone, 1988, p. 228-276.
45. Miller, G.J., Stirling, Y., Esnouf, M.P., Heinrich, J., van de Loo, J., Kienast, J.,
Wu, K.K., Morrissey, J.H., Meade, T.W., Martin, J.C., Imeson, J.D., Cooper, J.A. and
Finch, A. Factor VD-Deficient Substrate Plasma Depleted of Protein C Raise the Sensitivity
of the Factor VII Bio-Assay to Activated Factor VII: an Internation Study. Thrombos
Haemostas 71:38-48, 1994.
46. Seligsohn, U., Osterud, B. and Rapaport, S.I. Coupled Amidolytic Assay for Factor
VII: ITs use with a Clotting Assay fo Determine the Activity State of Factor VII. Blood
52:978-988, 1978.
47. Neuenschwander, P.F. and Morrissey, J.H. Deletion of the Membrane Anchoring
Region of Tissue Factor Abolishes Autoactivation of Factor VII but not Cofactor Function.
JBiol Chem 267:14477-14482, 1992.
48. Mackman, N., Morrissey, J.H., Fowler, B. and Edgington, T.S. Complete Sequence
of the Human Tissue Factor Gene, a Highly Regulated Cellular Receptor That Initiates the
Coagulation Protease Cascade. Biochemistry 28:1755-1762, 1989.
49. Carson, S.D., Henry, W.M. and Shows, T.B. Tissue Factor Gene localized to
Chromosome 1. Science 229:991-993, 1985.
50. Ruf, W., Rehemtulla, A. and Edgington, T.S. Phospholipid-independent and
-depandent Interactions Required for Tissue Factor Receptor and Cofactor Function. J Biol
Chem 266:2158-2166, 1991.
51. Morrissey, J.H. Clotting Assay for Measuring Plasma Factor Vila Based on Soluble
Mutant Tissue Factor (sTF). Personal Communication , 1996.
52. Hubbard, A.R. and Barrowcliffe, T.W. Measurement of Activated Factor VII Using
Soluble Mutant Tissue Factor - Proposal for Standardization. Thrombos Haemostas
72:649-650, 1994.
53. Scarabin, P.Y., Vissac, A.M., Kirzin, J.M., Bourgeat, P., Amiral, J., Agher, R. and
Guize, L. Population Correlates of Coagulation Factor VII: Importance of Age, Sex, and
Menopausal Status as Determinants of Activated Factor VII. Arterioscler Thrombos Vase
Biol. 16:1170-1176, 1996.
54. Kario, K., Matsuo, T., Sakata, T. and Miyata, T. Factor VII hyperactivity and
ischaemic heart disease. Lancet 343:233,1994.
55. Silveira, A., Karpe, F., Blomback, M., Steiner, G., Walldius, G. and Hamsten, A.
Activation of Coagulation Factor VII During Alimentary Lipemia. Arterioscler Thrombos.
14:60-69, 1994.
93
56. Moor, E., Silveira, A., van't Hooft, F., Suontaka, A.M., Eriksson, P., Blomback, M .
and Hamsten, A. Coagulation Factor VII Mass and Activity in Young Men With
Myocardial Infarcation at a Young Age. Role of Plasma Lipoproteins and Factor VII
Genotype. Arterioscler Thrombos Vase Biol. 15:655-664, 1995.
57. Kario, K., Matsuo, T., Kobayashi, FL, Sakata, T. and Miyata, T. Activation of Tissue
Factor-Induced Coagulation and Endothelial Cell Dysfunction in Non-Insulin-Dependent
Diabetic Patients With Microalbuminuria. Arterioscler Thrombos VascBiol. 15:1114-1120,
1995.
58. Kakkar, A.K., DeRuvo, N., Chinswangwatanakui, V., Tebbut, S. and Williamson,
R.C.N. Extrinsic-Pathway activation in cancer with high factor Vila and tissue factor.
Lancet 346:1004-1005, 1995.
59. Quehenberger, P., Loner, U., Kapiotis, S., Handler, S., Schneider, B., Huber, J. and
Speiser, W. Increased Levels of Activated Factor VII and Decreased Plasma Protein S
Activity and Circulating Thrombomodulin during Use of Oral Contraceptives. Thrombos
Haemostas 76:729-734, 1996.
60. Inamo, Y., Pemberton, S., Tuddenham, E.G.D. and Woo, P. Increase of Activated
Factor Vila and Haemostatic Molecular Markers in Juvenile Chronic Arthritis. Br J Rheum
34:466-469, 1995.
61. Silveira, A., Green, F., Karpe, F., Blomback, M., Humphries, S. and Hamsten, A.
Elevated Levels of Factor VII Activity in the Postprandial State: Effect of the Factor VII
Arg-Gln Polymorphism. Thrombos Haemostas 72:734-739, 1994.
62. Dagert, M. and Ehrlich, S.D. Prolonged Incubation in Calcium Chloride Improves the
Competence of Escherichia coli Cells. Gene 6:23-28,1979.
63. Sanger, F., Nicklen, S. and Coulsen, A.R. DNA Sequencing with Chain Terminating
Inhibitors. Proc Natl Acad Sci USA 74:5463-5467, 1977.
64. Palmiter, R.D., Behringer, R.R., Quaife, C.J., Maxwell, F., Maxwell, I.H. and
Brinster, R.L. Cell lineage ablation in transgenic mice by cell-specific expressed toxic
gene. Cell 50:435-443, 1987.
65. Laemmli, U.K. Cleavage of Structural Proteins during the assembly of the head of
Bacteriophage T4. Nature 227:680-685, 1970.
66. Towbin, H., Staehelin, T. and Gordon, J. Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl
Acad Sci USA 76:4350-4354, 1979.
67. Reziae, A.R., Fiore, M.M., Neuenschwander, P.F., Esmon, C.T. and Morrissey,
J.H. Expression and purification of a soluble tissue factor fusion protein with an epitope
for an unusual calcium dependent antibody. Prot Express Purif3:453,1992.
68. Fiske, C H . and Subbarow, Y. The colorimetric determination of phosphorus. J Biol
Chem 66:375-379, 1925.
69. Saiki, R.K., Glefand, D.H., Ttoffel, S.J., Schart, S.J., Higuchi, R., Horn, G.T.,
Mullis, K.B. and Erlich, H.A. Primer-directed Enzymatic Amplification of DNA with a
thermostable DNA Polymerase. Science 239:487-491,1988.
94
70. Shigematsu, Y., Miyata, T., Higashi, S., Miki, T., Sadler, J.E. and Iwanaga, S.
Expression of Human Soluble Tissue Factor in Yeast and Enzymatic Properties of Its
Complex with Factor Vila. J Biol Chem 267:21329-21337, 1992.
71. Porath, J. and Flodin, P. Gel Filtration: a method for desalting and group separation.
Nature 183:1657-1659,1959.
72. Neuenschwander, P.F., Bianco-Fisher, E., Rezaie, A.R. and Morrissey, J.H.
Phosphatidylethanolamine (PE) augments Factor Vila-Tissue Factor Activity:
Enhancement of Sensitivity to Phosphatidylserine. Biochemistry 34:13988-13993,1995.
73. Williams, C.E. Critical Factors in Haemostasis, ChichestenEllis Horwood Ltd, 1988.
pp. 1-11.
74. Williams, C.E. Global screening test in coagulation. In: Critical Factors in
Haemostasis, edited by Williams, C.E., Short, P.E., George, A.J. and Entwistle, M.B.P.
Cambridge: Ellis Horwood, 1988, p. 1-13.
75. Bonnar, J. Haemostasis and coagulation disorders in pregnancy. In: Haemostasis and
Thrombosis, edited by Bloom, A.L. and Thomas, D.P. Edinburgh: Churchill Livingston,
1981, p. 454-469.
76. Miller, B.C., Hultin, M.B. and Jesty, J. Altered factor VII activity in hemophilia.
Blood 65:845, 1985.
77. Morrissey, J.H., Revak, D., Tejada, D., Fair, D.S. and Edgington, T.S. Resolution
of Monomeric and Heterodimeric Forms of Tissue Factor, The High-Affinity Cellular
Receptor for Factor VII. Thrombos Res 50:481-493, 1988.
78. Zur, M . and Nemerson, Y. Tissue factor pathways of blood coagulation. In:
Haemostasis and Thrombosis, edited by Bloom, A.L. and Thomas, D.P. Edinburgh:
Churchill Livingstone, 1981, p. 124-139.
79. Hubbard, A.R., Parr, L.J. and Baines, M.G. Pregnancy and Factor VII. Br J
Haemostas 80:265, 1992.
80. Barrowcliffe, T.W. and Thomas, D.P. Antithrombin III and heparin. In: Haemostasis
and Thrombosis, edited by Bloom, A.L. and Thomas, D.P. Edinburgh: Churchill
Livingstone, 1981, p. 712-724.
81. Prydz, H. The Role of Factor VII in the Intrinsic Pathway: A Brief Review.
Haemostas 13:156-160, 1983.
82. Nakagaki, T., Foster, D C , Berkner, K.L. and Kisiel, W. Initiation of the Extrinsic
Pathway of Blood Coagulation: Evidence for the Tissue Factor Dependent Autoactivation
of Human Coagulation Factor VII. Biochemistry 30:10819-10824, 1991.
83. Roberts, H.R. and Lefkowitz, J.B. Inherited Disorders of Prothrombotic Conversion.
In: Haemostasis and Thrombosis: Basic Principles and Clinical Practice, edited by
Colman, R.W., Hirsh, J., Mardy, V.J. and Salzman, E.W. Philadelphia: J.B. Lippincott
Company, 1994,
95
84. Winocour, P.D. and Richardson, M. Thrombosis and Atherogenesis in Diabetes. In:
Diabetes and Atherosclerosis: Molecular basis and Clinical aspects, edited by Draznin, B.
and Eckel, R.H. New York: Elsevier Science Publishing Company, 1993, p. 213-254.
85. Sitruk-Ware, R. Benefits and risks of hormonal replacement therapy in
postmenopausal women. In: Medical-Economic Aspects of Hormone Replacement
Therapy, edited by Cossery, J.M. Basel, Switzerland: 1993, p. 1-16.
86. Yamanobe, F., Mochida, S., Ohno, A., Ishikawa, K. and Fujiwara, K. Recombinant
Human Tissue Factor Pathway Inhibitor as a possible Anticoagulant targeting hepatic
sinusoidal walls. Thrombos Res 85:493-501, 1997.
87. Sorensen, B.B., Persson, E., Freskgard, P.O., Kjalke, M., Ezban, M., Williams, T.
and Rao, L.V.M. Incorporation of an active site inhibitor in Factor Vila alters the affinity
for Tissue Factor. J Biol Chem 272:11863-11868, 1997.
88. Kelley, R.F., Refino, C.J., O'Connel, M.P., Modi, N., Sehl, P., Lowe, D., Pater,
C. and Bunting, S. A Soluble Tissue Factor Mutant is a Selective Anticoagulant and
Antithrombotic agent Blood 89:3219-3227, 1997.
89. Ronning, H.F., Risoen, U.C., Orning, L., letten, K. and Sakariassen, K.S. Synthetic
peptide analogs of Tissue Factor and Factor VII which inhibit Factor Xa formation by the
Tissue Factor/Factor Vila complex. Thrombos Res 84:73-81,1997.
90. Hagen, F.S., Gray, C.L., O'Hara, P., Grant, F.J., Saari, G.C., Woodbury, R.G.,
Hart, C.E., Insley, M., Kisiel, W., Kurachi, K. and Davie, E.W. Characterization of a
cDNA coding for human factor VII. Proc Natl Acad Sci USA 83:2412-2416, 1986.
96