Comparison of the Inhibitory Effects of ApoB100 and Tissue
Factor Pathway Inhibitor on Tissue Factor and the
Influence of Lipoprotein Oxidation
Camille Ettelaie, Barry R. Wilbourn, Jacqueline M. Adam, Nicola J. James, K. Richard Bruckdorfer
Downloaded from http://atvb.ahajournals.org/ by guest on June 15, 2017
Abstract—The procoagulant activity of tissue factor is regulated by circulating inhibitors such as tissue factor pathway
inhibitor (TFPI) and LDL. These 2 inhibitors also readily associate making the distinction between their activities
difficult. We have examined the relative contributions of intact and C-terminal truncated TFPI and ApoB100. By
following the inhibitory potential of the preparations, over a period of 120 minutes, it was demonstrated that TFPI and
LDL-resembling particles inhibited tissue factor at different rates. TFPI was found to be a short, fast-acting inhibitor,
whereas the action of LDL-resembling particles was more prolonged but slower. The oxidation of LDL has been closely
associated with the development of cardiovascular disease, including atherosclerosis and thrombosis. Positively charged
amino acids, particularly lysine residues, are prone to alterations via the formation of adducts by lipid peroxidation
products. These residues are important in the inhibition of tissue factor activity by ApoB100. They also play an
important role in the inhibitory Kunitz domains of TFPI. We have shown that the decline in the ability of LDL to inhibit
tissue factor was as a result of modifications in LDL arising from oxidation. By examining the effects of oxidation on
full-length and C-terminal truncated TFPI bound to LDL-resembling particles, we found that TFPI is only affected when
in close association with ApoB100. C-terminal truncated TFPI was not affected significantly by oxidation. Finally,
chemical modification of lysine and arginine residues reduced the overall inhibition of tissue factor by TFPI. We
propose that TFPI and LDL act separately to inhibit tissue factor in vivo. However, the oxidation of LDL can alter both
the endogenous activity of ApoB100 and reduce that of closely associated TFPI, compromising normal hemostasis.
(Arterioscler Thromb Vasc Biol. 1999;19:1784-1790.)
Key Words: tissue factor n ApoB100 n tissue factor pathway inhibitor n LDL n oxidation n lysine n arginine n inhibition
T
shown that the inhibitory potential of TFPI may be compromised by lipoprotein oxidation.21 The first 2 Kunitz domains
within TFPI responsible for inhibiting clotting factors VIIa
and Xa contain lysine and arginine residues at the inhibition
sites.22 The third Kunitz also contains a lysine residue at this
site. Moreover, the basic C-terminal of TFPI, known to be
important in the activity of TFPI,12,13 contains a stretch of
amino acids, primarily made up of lysine and arginine
residues.22 The ability of lipid peroxides to mask the basic
amino acids by formation of adducts means that the activity
of LDL-bound TFPI may be compromised by the oxidation of
the lipoprotein. In this study, we first compared the relative
contributions of TFPI and LDL to inhibition of tissue factor.
Second, we investigated the effects of oxidation on the ability
of these proteins to inhibit tissue factor.
he procoagulant activity of tissue factor is restrained by
a number of circulating inhibitors including tissue factor
pathway inhibitor (TFPI),1–3 ApoA2 within HDL,4,5 and
ApoB100 within LDL.6 – 8 The inhibitory action of TFPI has
been well documented.9 However, it has been suggested that
the physiological concentration of TFPI is not sufficient to
control the procoagulant activity.10 Therefore, the action of
other identified tissue factor inhibitors may be physiologically significant. Moreover, 2 variants of TFPI have been
isolated11 that possess different inhibitory potentials.12,13 The
smaller form of TFPI lacks the C-terminal. The third Kunitz
domain of TFPI is thought to be involved in the interaction
with lipoproteins.14 In fact, most circulating TFPI has been
found to be associated with low-density LDL and HDL
subfractions.15–17 Oxidation of lipoproteins and LDL in particular has been closely associated with atherogenesis.18 The
inhibitory effect of LDL, arising from the ApoB100 moiety,
can be compromised by oxidation.7 The formation of lysine
adducts19 and alterations in the secondary and tertiary structure of this protein affects the residues within the domain
responsible for inhibition of tissue factor.20 It has also been
Methods
Preparation of LDL and ApoB100 and
Preparation of and Reconstituted LDL
LDLs were prepared by a modification13 of a discontinuous gradient
ultracentrifugation procedure14 from 6 individual donors. ApoB100
Received September 15, 1998; revision accepted December 23, 1998.
From the Department of Biochemistry and Molecular Biology, Royal Free and University College Medical Schools (Royal Free Campus), London, UK.
Correspondence to Camille Ettelaie, Department of Biochemistry and Molecular Biology, Royal Free Hospital School of Medicine, Rowland Hill
Street, London NW3 2PF, UK. E-mail [email protected]
© 1999 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1784
Ettelaie et al
Inhibition of Tissue Factor by ApoB100 and TFPI
was isolated as before6,7 and examined on a 5% (wt/vol) denaturing
polyacrylamide23 to ensure the homogeneity. Moreover, the nature of
the protein was examined by immunoprecipitation, using antibodies
against human ApoB100 (Immuno Ltd) and antibodies against
human TFPI (American Diagnostics) to ensure its purity. Isolated
ApoB100 was found to have a molecular mass of 530 to 540 kDa,
and was recognized by anti-human ApoB100 antibodies, but not
anti-human TFPI antibodies, indicating that the sample was free of
any contaminating TFPI (not shown). The protein was then reconstituted in lipids resembling those found in native LDL (nLDL) as
described previously.6,7 In earlier studies, it was shown that the
LDL-resembling particle (LDL-r) has a similar composition, electrophoretic mobility, and protein fold to nLDL.20
Oxidation of LDL
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LDLs (250 nmol/L protein50.125 mg of protein/mL) were prepared
from 6 different normal, healthy donors. Minimally modified LDL
were prepared under sterile conditions from nLDLs (1 g of protein/L)
by exposure to air at room temperature.24 To prepare fully oxidized
LDLs, nLDLs were exposed to oxidation with 5 mmol/L copper
sulfate for 0, 1, 3, 6, 12, 18, and 24 hours at 37°C and dialyzed
against buffer, pH 7.4, containing 1 mmol/L EDTA. Aliquots of
nLDL containing 1 mmol/L DTPA were used as controls. Lipid
peroxide products were assayed by an iodometric method.25 In
addition, cumene hydroperoxide (Sigma Chemical Company Ltd),
9-hydroperoxyoctadecadienoic acid, and 15-S-hydroperoxyeicosatetraenoic acid (Cayman Chemicals). were used to assess the effect of
peroxides on tissue factor activity.
Isolation of TFPI From Plasma and the Media of
Human Hepatocyte Carcinoma (Hep G2) Cells
Hep G2 cells were cultured in DMEM, containing 2 mmol/L
glutamine, supplemented with 10% FCS (Gibco Life Sciences) in
5% CO2 at 37°C, until the cells became confluent. The supernatant
was removed and centrifuged at 1000g for 10 minutes at 4°C to
remove any cells and was used for isolation of TFPI. Hep G-2 TFPI
was almost entirely the full-length form (43 kDa) as determined by
12% (wt/vol) denaturing polyacrylamide gel electrophoresis.26 Citrated blood was obtained from healthy volunteers and plasma
prepared by centrifugation. TFPI was isolated from human plasma27
and the media of Hep G2 cells28 by factor Xa-Sepharose affinity
chromatography. The affinity column was prepared by coupling
human factor Xa to p-nitrophenyl agarose29 (Sigma Chemical
Company Ltd). Human plasma TFPI was predominantly C-terminal
truncated (33 kDa).
Preparation of Full-Length Human
Recombinant TFPI
Full-length TFPI cDNA was prepared by reverse transcription, using
the Superscript II enzyme (Gibco Life Sciences), according to
manufacturer’s instructions and the DNA amplified by PCR, using
primers with nonidentical restriction sites. An enterokinase cleavage
site was also engineered preceding the full-length TFPI. The digested
DNA was ligated into the pinpoint Xa3 plasmid previously digested
with the same enzymes.
Competent Escherichia coli (JM109) cells were subsequently
transfected and selected by growth in Terrific broth containing
ampicillin (100 mg/mL). The cells were then grown in the presence
of 100 mg/mL isopropyl-b-thiogalactopyranoside (IPTG) and 5
mg/mL biotin, harvested, and the biotinylated protein isolated according to the manufacturer’s instructions. A sample of the isolated
TFPI was examined on a 12% (wt/vol) denaturing polyacrylamide
gel and by immunoprecipitation, using anti-human TFPI antibodies
(American Diagnostics). Recombinant full-length TFPI-fusion protein had a molecular mass of 47 kDa, which is in agreement with the
calculated value for the recombinant TFPI-biotinylated tag construct.
This TFPI preparation and those isolated from Hep G2 medium and
plasma were all recognized by the anti-TFPI antibody.
Tissue Factor Inhibition Assay
To test for any inhibitory or procoagulant effect, all samples or controls
were incubated (in triplicate) with 1-mL aliquots of human recombinant
1785
tissue factor (DADE Innovin, Sysmex UK Ltd) diluted 100 times (10
U/mL) at 37°C for up to 2 hours and the tissue factor activity measured
by means of the 1-stage prothrombin time assay or the 2-stage
chromogenic assay.30 The tissue factor stock was assumed to contain
1000 arbitrary units/mL. Tissue factor activity was calculated from
appropriate standard curves and the percentage inhibition in each sample
was calculated against an aliquot of tissue factor diluted 200 times (5
U/mL) as follows: % of inhibition51003[initial tissue factor activity
(control)2residual tissue factor activity]/initial tissue factor activity
(control).
Chemical Modification of TFPI
Lysine residues were modified selectively either to preserve the
positive charge (Lys1-modified)31 or to neutralize the charge (Lysneutmodified).31 To modify lysine residues retaining the charge, samples
of Hep G2 TFPI or BSA (2 to 10 mg) were dissolved in NaCl (0.15
mol/L), EDTA (0.01% wt/vol), BaBH4 (0.3 mol/L), pH 9.0 (Sigma
Chemical Company Ltd) at 0°C; 1 mL of formaldehyde (37% wt/vol)
was added immediately and at 3-minute intervals up to 60 minutes.
The proteins were then dialyzed against NaCl (0.15 mol/L), EDTA
(0.01% wt/vol), pH 7.0, and freeze-dried until use.
TFPI and BSA were acetylated to modify lysine residues, neutralizing the charge. The samples (10 mg) were dissolved in 3 mL of
sodium phosphate (0.005 mol/L), KCl (0.1 mol/L), pH 7.4, on ice
and 1 mL of N-acetylimidazole (0.91 mol/L) (Sigma Chemical
Company Ltd) was added and mixed. The samples were kept on ice
for 20 minutes and subsequently dialyzed and freeze-dried as above.
Arginine modification was performed as described by Stark.31 The
protein samples (1 mg) were dissolved in 2 mL of Na2B4O7 (0.25
mol/L), 1,2-cyclohexanedione (0.15 mol/L), pH 9.0 (Sigma Chemical Company Ltd). The samples were incubated at 37°C for 2 hours,
after which an equal volume of acetic acid (30% wt/vol) was added.
The samples were then consecutively dialyzed against 15%, 7.5%,
and 1% (wt/vol) acetic acid. The samples were then dialyzed and
freeze-dried as above. After chemical modification, all samples were
examined by denaturing gel electrophoresis.
Determination of Effects of Serum on Tissue
Factor Activity
Human venous blood was collected from 5 volunteers 20 to 30 years
old, allowed to clot, and the sera decanted and centrifuged to remove
any cells. The ability of sera to inhibit tissue factor (final concentration, 5 U/mL) activity was measured in the presence of either
anti-human ApoB100 antibodies (Immuno) (final concentration,
73 mmol/L) or anti-human TFPI antibodies (American Diagnostics)
(final concentration, 7.5 mmol/L), and finally in the absence of any
antibodies as before. Moreover, the total amount of LDL within the
sera was estimated by using a turbimetric precipitation method with
10% (wt/vol) dextran sulfate (T 500).32
Investigation of the Inhibitory Contributions of
ApoB100 and TFPI and the Influence of Oxidation
Samples of tissue factor (final concentration, 5 U/mL) were incubated with either reconstituted LDL (final concentration, 250 nmol/L
protein), samples of Hep G2, plasma and recombinant TFPI (final
concentration, 50 nmol/L), or combinations of LDL-r and TFPI.
Inhibitory activity of TFPI and LDL-r were assayed in the presence
of prothrombin complex (10 ng/mL) and Ca21 ions (0.5 mmol/L) and
the residual tissue factor activity. Moreover, similar samples exposed
to oxidizing conditions or chemical modifications were assessed in
the same way.
Results
Inhibitory Potential of Serum on Tissue
Factor Activity
Samples of tissue factor (5 U/mL) were incubated with 150
mL of serum at 37°C and the activity assessed at intervals up
to 90 minutes. The initial rise in inhibition plateaued after 30
minutes (Figure 1) to a maximum of 55%. This was followed
by a further increase in the inhibition, reaching a maximum
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Arterioscler Thromb Vasc Biol.
July 1999
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Figure 1. Time course study of the inhibition of tissue factor by
human serum. Tissue factor (2.85 mL; final concentration, 5
U/mL) was incubated with 150 mL of serum (F) at 37°C; 100 mL
was removed immediately and tissue factor activity measured
immediately by means of the 1-stage prothrombin time assay.
Subsequently, 100-mL samples were removed at intervals up to
90 minutes and assayed. The percentage of inhibition was calculated as the decrease in tissue factor activity, against the initial activity. In addition, similar samples of serum were incubated with either anti-human ApoB100 antibodies (f) (final
concentration, 73 mmol/L) or anti-human TFPI antibodies (Œ)
(final concentration, 7.5 mmol/L), and examined as above. The
data were obtained from 3 independent experiments
(mean6SEM values).
level of 80%, indicating the presence of at least 2 separate
tissue factor inhibitors in serum, with distinct kinetics. Preincubation of serum with anti-TFPI antibodies, before incubation with tissue factor, greatly reduced the rate of inhibition
and virtually eliminated the first inhibitory peak detected. In
comparison, preincubation with anti-ApoB100 diminished
the late wave of inhibition, without affecting the initial early
inhibitory potential of serum. The amount of LDL precipitated by dextran sulfate32 did not vary greatly between sera
used and no relation to the anticoagulant effect was evident
(not shown).
Time Course of the Inhibition of Tissue Factor by
Isolated TFPI and ApoB100
Plasma TFPI (50 nmol/L), at 5 times its mean physiological
concentration, had a transient inhibitory effect, reaching a
maximum of inhibition at '15 minutes (Figure 2). On the
other hand, both Hep G2 TFPI and recombinant TFPI
inhibited tissue factor for a longer period and to a greater
extent than plasma TFPI (Figure 2). Inhibition by LDL-r (250
nmol protein/L) took longer, ultimately causing almost total
inhibition of the tissue factor procoagulant activity (Figure 2).
Furthermore, although the inhibition by LDL-r was not
affected by the presence of anti-TFPI antibodies, the inhibition was suppressed in the presence of anti-ApoB100 antibodies (not shown). Conversely, TFPI activity was only
suppressed by the presence of anti-TFPI antibodies (not
shown).
Samples of LDL-r were combined with each of the 3
preparations of TFPI (50 nmol/L) and incubated at 37°C for
30 minutes, then incubated with recombinant human tissue
factor (final concentration, 5 U/mL) and assayed at zero time
and after 15 minutes. Preincubation of LDL-r with C-terminal
truncated plasma TFPI, before incubation with tissue factor,
had little effect on the inhibitory potential of either of the 2
proteins (Table 1). Conversely, preincubation of LDL-r with
Figure 2. Time course study of the inhibition of tissue factor by
TFPI and LDL-r. ApoB100 was reconstituted with lipids to form
the LDL-r particles. Samples of LDL-r (l; final concentration,
250 nmol/L protein), plasma TFPI (Œ; final concentration, 50
nmol/L), Hep G2 TFPI (F; final concentration, 50 nmol/L),
recombinant human TFPI (f; final concentration, 50 nmol/L),
and distilled water (control; E) were incubated with equal volumes of human recombinant tissue factor (final concentration, 5
U/mL) in the presence of prothrombin complex (10 ng/mL) and
Ca21 ions (0.5 mmol/L), at 37°C. Samples (100 mL) were
removed and assayed by means of the 1-stage prothrombin
time assay at the beginning and at intervals up to 120 minutes.
The percentage of inhibition was calculated as the decrease in
tissue factor activity, against the initial activity. The data were
obtained from 3 independent experiments (mean6SEM values).
either full-length Hep G2 TFPI or recombinant TFPI resulted
in a reduction in the activity of the sample, compared with
either LDL-r or TFPI alone (Table 1).
Time Course of Copper-Induced LDL Oxidation
and the Influence on Tissue Factor Activity
A steady rise in the concentration of lipid peroxides was
detected after incubation with CuSO4 to a maximum peroxide
concentration of 250 nmol/mg of LDL protein at 18 hours; no
further increase was apparent after 24 hours of incubation
(Figure 3). The ability of LDL to undergo oxidation varied
among the different preparations, because samples prepared
from various individuals differ in their susceptibility to
oxidation. nLDL (250 nmol/L protein) inhibited the tissue
factor activity by an average of 5368% (n56). As oxidation
progressed, the ability of LDL to inhibit tissue factor deTABLE 1. Investigation of the Combined Inhibitory Potential of
TFPI and ApoB100
% of Inhibition of
Tissue Factor Activity
Without LDL-r
With LDL-r
063
2065
4367
5566
TFPI (Hep G2)
8567
1263
TFPI (recombinant)
8067
1563
Control
TFPI (plasma)
Samples of LDL-r (final concentration, 250 nmol/L protein) were combined
with the 3 preparations of TFPI and incubated at 37°C for 30 minutes to allow
any interactions. The 3 samples were then incubated with recombinant human
tissue factor (final concentration, 5 U/ml) in the presence of prothrombin
complex (10 ng/ml) and Ca21 ions (0.5 mmol/L). Samples (100 ml) were
removed and assayed at 15 minutes by the 1-stage prothrombin time assay.
The percentage of inhibition was calculated against the tissue factor control.
The data were obtained from 3 independent experiments (mean6SEM values).
Ettelaie et al
Inhibition of Tissue Factor by ApoB100 and TFPI
1787
acid (products of LDL oxidation) or cumene hydroperoxide
(0 to 20 mmol/L) to tissue factor (10 U/mL) had little effect
on procoagulant activity at low concentrations (,4 mmol/L),
but were inhibitory in the higher range (not shown). Therefore, although peroxide formation is an index of oxidation,
they are not directly the cause of the enhancement of tissue
factor activity.
Effect of Oxidation on TFPI-Bound LDL-r
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Figure 3. Time course of LDL oxidation and its effect on tissue
factor activity. LDLs prepared from 6 different normal, healthy
donors were exposed to oxidation with 5 mmol/L copper sulfate
for 0, 1, 3, 6, 12, 18, and 24 hours. The concentration of lipid
peroxides was then measured by means of an iodometric
assay25 and interpreted against a standard curve prepared by
using H2O2. Results are expressed as an increase of peroxidation products with respect to that measured at t50 (D peroxidation product concentration). Aliquots of nLDL or oxLDL (250 mL)
were incubated with an equal volume of recombinant human
tissue factor (5 U/mL final concentration) for 1 hour and then
residual tissue factor activity was assayed by using the 1-stage
prothrombin time assay. The data were obtained from 6 independent experiments performed by using LDL prepared from 6
serum samples from different donors (mean6SEM values).
creased, eventually enhancing tissue factor activity by up to
40% (Figure 3). The change in the activity of tissue factor
was most pronounced with LDL samples more prone to
oxidation.
Effect of Low Levels of Oxidation on Tissue
Factor Activity
On exposure to air, the level of peroxides rose from 17
nmol/mg of protein in the nLDL to mean values of 45 and 70
nmol/mg of protein in the 24- and 48-hour treated LDL,
respectively. The nLDL controls retained most of their
inhibitory effect on tissue factor even after 24 and 48 hours
(Table 2). This inhibition decreased by .50% after 24 hours
of oxidation. After 48 hours of incubation, all the inhibitory
potential had been lost and tissue factor activity was enhanced slightly by 10%.
Addition of the lipid peroxides 9-hydroperoxyoctadecadienoic acid and 15-S-hydroperoxyeicosatetraenoic
TABLE 2. Effect of Minimally Modified LDL on Tissue
Factor Activity
% of Inhibition of
Tissue Factor Activity
0h
24 h
Native LDL
68610
65610
Minimally modified LDL
68610
3068
48 h
62610
21065
Minimally modified LDL (mmLDL) was prepared by exposure of native LDL
(nLDL) to air at room temperature for 0, 24, and 48 hours under sterile
conditions. As a control, an aliquot of nLDL containing 1 mmol/L DTPA as
chelator and antioxidant. Aliquots (250 ml) of nLDL or mmLDL (1 mg/ml) were
incubated with an equal volume of recombinant human tissue factor (final
concentration, 5 U/ml) for 1 hour and then residual tissue factor activity was
assayed by using the one-stage prothrombin time assay. The percentage of
inhibition was calculated against a 5 U/ml tissue factor control sample. The
data were obtained from 3 independent experiments (mean6SEM values).
Copper-mediated oxidation of TFPI-bound LDL has been
shown to suppress the inhibitory potential of the complex
toward tissue factor.21 The purpose of this part of the study
was to investigate the effects of mildly oxidized LDL on
different forms of TFPI. Aliquots of LDL-r, or lipids alone,
were incubated with recombinant TFPI, plasma TFPI, and
Hep G-2 TFPI for 30 minutes before mild oxidation by
exposure to air as described in Methods. The samples were
then incubated with recombinant human tissue factor (final
concentration, 5 U/mL) for 15 minutes in the presence of
prothrombin complex (10 ng/mL) and Ca21 ions (0.5 mmol/
L). Mild oxidation of the plasma TFPI and LDL-r mixture
slightly reduced the combined inhibitory potential toward
tissue factor, whereas the inhibitory activity of the recombinant TFPI or of Hep G2 TFPI combined with that of LDL-r
was greatly reduced by the oxidation (Table 3). In all
samples, increases in peroxidation products (40 to 45
nmol/mg of LDL protein) were detected after 24 hours,
indicating that oxidation had occurred.
Effect of Chemical Modification of Lysine and
Arginine Residues on TFPI Activity
Oxidation of lipoproteins leads to formation of adducts
between lipid peroxidation products and lysine residues. The
effects of chemical modification of basic amino acids were
therefore examined. Samples of Lys1-modified TFPI, Lysneutmodified TFPI, and Arg-modified TFPI (final concentration,
50 nmol/L) and the 3 controls Lys1-modified BSA, Lysneutmodified BSA, and Arg-modified BSA were incubated with
recombinant human tissue factor (final concentration, 5
U/mL) for 15 minutes and assayed as described in Methods.
All samples remained intact (full-length TFPI) after chemical
modification (Figure 4). The ability of TFPI to inhibit tissue
factor was reduced by modification of either lysine or
arginine residues (Table 4). However, a greater suppression
of the inhibitory potential of TFPI toward tissue factor was
achieved when the positive charges of the lysine residues
within TFPI were neutralized (Table 4). Modification of
arginine residues or lysine residues, conserving the positive
charge, were less effective in neutralizing TFPI activity,
respectively. As a control, it was shown that BSA, in its
native form or after modification of lysine or arginine
residues, had no significant effect on the overall activity of
tissue factor.
Discussion
The endogenous ability of LDL to inhibit tissue factor6 – 8 may
be diminished completely by incubation with specific antibodies against human ApoB100.6 Conversely, incubation of
preparations of LDL with anti-human TFPI antibodies does
not completely suppress the inhibitory potential of the complex.33 The inhibition pattern of tissue factor by serum
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Arterioscler Thromb Vasc Biol.
TABLE 3.
July 1999
Effect of Oxidation of LDL-r-Bound TFPI on the Inhibitory Potential of Tissue Factor
Without ApoB100 (Lipids Only)
[Lipid Peroxides]
(nmol/mg of protein)
With ApoB100
% of Inhibition of
Tissue Factor Activity
[Lipid Peroxides]
(nmol/mg of protein)
% of Inhibition of
Tissue Factor Activity
061
1065%
161
250615%
Plasma TFPI
4365
4066%
4565
3766%
Hep G2 TFPI
4065
65612%
4265
565%
Recombinant TFPI
4265
60610%
4265
665%
Control
Aliquots of LDL-r were incubated with plasma TFPI, Hep G-2 TFPI, and recombinant TFPI to allow interaction of the
2 previous mild oxidations by exposure to air as described in Table 2. In addition, samples of LDL-r lipids, devoid of
the ApoB100, were incubated with the 3 preparations of TFPI separately, and oxidized as above. The samples were
then incubated with recombinant human tissue factor (final concentration, 5 U/ml) in the presence of prothrombin
complex (10 ng/ml) and Ca21 ions (0.5 mmol/L), for 15 minutes. The residual tissue factor activity was assayed by
using the 1-stage prothrombin time assay. The percentage of inhibition because of TFPI was calculated against a 5
U/ml tissue factor control sample. The data were obtained from 3 independent experiments (mean6SEM values).
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indicated the presence of at least 2 separate inhibitors of
tissue factor, acting at differing rates. The magnitude of the
inhibition by the sera examined could not be related to the
relative amount of LDL present in this small number of
donors. It has been shown that such variation can also be
attributed to differences in the composition of the lipoproteins.7,20 However, the overall pattern of inhibition, exhibiting
2 distinct peaks (Figure 1) was the same in all the sera
examined. By using antibodies specific to ApoB100 and
TFPI, it was shown that the first inhibitory peak was the result
of TFPI activity, whereas ApoB100 had a slower, more
prolonged effect on tissue factor procoagulant activity. By
isolating TFPI and ApoB100 and examining their ability to
inhibit tissue factor, it was possible to assign the 2 observed
activities within serum to these 2 inhibitors. The peaks
exhibited by these inhibitors corresponded temporally to
those observed in the whole serum. In addition, because the
inhibitory potential of the 2 proteins was only affected by the
presence of their respective antibodies, the possibility that
LDL-bound TFPI may have a slower effect that is manifested
at a later time may be dismissed. Moreover, the combined
effect of ApoB100 and full-length TFPI was smaller than the
sum of the individual inhibitors, indicating that the 2 proteins
may, in part, mask each other. This agrees with observations
by others that TFPI activity is reduced when bound to
lipoproteins.34
The inhibitory action of nLDL against tissue factor was
abrogated by oxidation. Recently, we have demonstrated that
the inhibitory action of purified ApoB100 derives from
lysine-rich peptide within its receptor-binding domain.35,36
The residues in this region may react with the aldehydic
products of lipid peroxidation. However, mild oxidation of
LDL does not result in a significant increase in the electrophoretic mobility of LDL.37 This domain is within a region of
ApoB100 capable of local structural alterations that changes
even at low levels of LDL oxidation.20 Moreover, the oxidation of lipids alone (Table 2), or the addition of pure lipid
peroxides, did not enhance the tissue factor activity, but
rather inhibited it slightly, concurring with our previous
report that the enhancement of tissue factor activity by
oxidized LDL arises from the modification of ApoB100.7
The inactivation of TFPI as a result of oxidation of
lipoprotein has been demonstrated previously.21 In the present
study, the effect of LDL oxidation on the 2 forms of TFPI
found in vivo was examined. The full-length TFPI contains
the C-terminal domain required for direct interactions with
ApoB100.14,38 Therefore, only the full-length TFPI was
TABLE 4. Effect of Lysine and Arginine Modification on
TFPI Activity
% of Inhibition
BSA
1
Figure 4. SDS-PAGE of chemically modified TFPI. Chemical
modifications of lysines to retain the positive charge (C) and to
neutralize the charge (D) and of arginine (E) were performed on
samples of TFPI isolated from the media of Hep G2 cells. Electrophoresis of chemically modified TFPI was performed on 12%
(wt/vol) SDS–polyacrylamide gels to ensure that the full-length
TFPI samples remained intact during the procedure. Lane B
contained untreated TFPI and lanes A and F contain markers.
TFPI
Control
065
90610
Lys1-modified TFPI
065
5967
Lysneut-modified TFPI
567
4066
Arg-modified TFPI
266
7268
neut
Lys -modified TFPI, Lys -modified TFPI, and Arg-modified TFPI and BSA
(control) were prepared by chemical modification of Hep G2 TFPI. Samples of
Lys1-modified TFPI, Lysneut-modified TFPI, and Arg-modified TFPI (final concentrations, 50 nmol/L) and the 3 controls Lys1-modified BSA, Lysneut-modified
BSA, and Arg-modified BSA were incubated with recombinant human tissue
factor (final concentration, 5 U/ml) in the presence of prothrombin complex (10
ng/ml) and Ca21 ions (0.5 mmol/L), for 15 minutes, and residual tissue factor
activity was assayed by using the 1-stage prothrombin time assay. The
percentage of inhibition was calculated against a 5 U/ml tissue factor control
sample. The data were obtained from 3 independent experiments (mean6SEM
values).
Ettelaie et al
Inhibition of Tissue Factor by ApoB100 and TFPI
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influenced by LDL oxidation losing its anticoagulant function, probably because of its ability to associate with LDL.
The oxidation of lipids may also mask lysine and possibly
arginine residues within Kunitz 1 and 2 of TFPI. That
truncated (plasma) LDL is not influenced by oxidation may
be because of its inability to associate with LDL. The
aldehydes that form adducts with lysine residues on proteins
remain with the LDL and may not be able to influence the
truncated TFPI.
This study indicates that ApoB100 within LDL is capable
of inhibiting the procoagulant activity of tissue factor via a
different and independent mechanism than that of TFPI and
other tissue factor inhibitors. In addition, the rate at which the
inhibition by ApoB100 is brought about is slower, but
stronger, than that by TFPI at prevailing physiological concentrations. We propose that LDL may play an important role
in the physiological functioning of tissue factor, in vivo. Both
full-length and truncated TFPI have been detected in vivo.
Moreover, full-length TFPI may be partially degraded in
blood to the C-terminal truncated form, as observed in the
comparison between Hep G2 and plasma TFPI carried out
here. The purpose of TFPI degradation and the role in its
interaction with LDL is not understood. However, our data
are in agreement with others16,17,38 – 41 that the C-terminal of
this protein is required for the interaction that occurs with
ApoB100 as well as cell surface glycosaminoglycans. When
associated with LDL, the oxidation of TFPI–LDL complex
may result in the partial inactivation of TFPI, as well as
giving rise to a procoagulant form of LDL. Such alterations
may severely compromise correct control of hemostasis,
especially in conditions associated with the risk of tissue
factor–mediated coagulation such as atherosclerosis, and
endotoxic shock. Because neither LDL nor LDL-associated
TFPI would be capable of suppressing the procoagulant
function of tissue factor and because the procoagulant activity
of released tissue factor is augmented by the oxidized
complex, the oxidation of LDL may contribute to potentially
fatal thrombotic episodes especially those associated with
coronary heart disease. Failure to inhibit tissue factor may
also have other consequences, unrelated to thrombosis,
namely, the formation of neointima through smooth muscle
cell proliferation and angiogenesis.42
Acknowledgments
We acknowledge the support of the Wellcome Trust and the British
Heart Foundation. We also thank Dr T.W. Barrowcliffe, Dr E. Gray,
and Dr A.R. Hubbard, Division of Hematology, NIBSC, Hertfordshire, UK, for their help and support.
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Comparison of the Inhibitory Effects of ApoB100 and Tissue Factor Pathway Inhibitor on
Tissue Factor and the Influence of Lipoprotein Oxidation
Camille Ettelaie, Barry R. Wilbourn, Jacqueline M. Adam, Nicola J. James and K. Richard
Bruckdorfer
Arterioscler Thromb Vasc Biol. 1999;19:1784-1790
doi: 10.1161/01.ATV.19.7.1784
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