© 1999 American Heart Association, Inc.
Thrombosis |
Comparison of the Inhibitory Effects of ApoB100 and Tissue Factor Pathway Inhibitor on Tissue Factor and the Influence of Lipoprotein Oxidation
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 Camille{at}RFHSM.AC.UK
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Abstract |
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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.
Key Words: tissue factor • ApoB100 • tissue factor pathway inhibitor • LDL • oxidation • lysine • arginine • inhibition
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Introduction |
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The procoagulant activity of tissue factor is restrained by a number of circulating inhibitors including tissue factor pathway inhibitor (TFPI),1 2 3 ApoA2 within HDL,4 5 and ApoB100 within LDL.6 7 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 16 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 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.
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Methods |
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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 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
LDLs (250 nmol/L protein=0.125 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 µmol/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 µmol/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-hydroperoxyeicosatetra-enoic 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 µg/mL). The cells were then grown in the presence of 100 µg/mL isopropyl-ß-thiogalactopyranoside (IPTG) and 5 µg/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 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
inhibition=100x[initial tissue factor activity (control)-residual
tissue factor activity]/initial tissue factor activity (control).
Chemical Modification of TFPI
Lysine residues were modified selectively either to preserve the
positive charge
(Lys+-modified)31 or to
neutralize the charge
(Lysneut-modified).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 µL 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 µmol/L) or anti-human TFPI antibodies
(American Diagnostics) (final concentration, 7.5
µmol/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
Ca2+ 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.
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Results |
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Inhibitory Potential of Serum on Tissue Factor Activity
Samples of tissue factor (5 U/mL) were incubated with 150 µL 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
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).
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) (final concentration, 73 µmol/L) or anti-human
TFPI antibodies (
) (final concentration, 7.5 µmol/L), and
examined as above. The data were obtained from 3 independent
experiments (mean±SEM values).
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).
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; final concentration, 250
nmol/L protein), plasma TFPI (
) 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 Ca2+ ions (0.5 mmol/L), at
37°C. Samples (100 µL) 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 (mean±SEM
values).
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 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).
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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 53±8% (n=6). As oxidation progressed, the
ability of LDL to inhibit tissue factor decreased, 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.
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peroxidation product concentration).
Aliquots of nLDL or oxLDL (250 µL) 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 (mean±SEM values).
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%.
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Addition of the lipid peroxides 9-hydroperoxyoctadecadienoic acid and 15-S-hydroperoxyeicosatetraenoic acid (products of LDL oxidation) or cumene hydroperoxide (0 to 20 µmol/L) to tissue factor (10 U/mL) had little effect on procoagulant activity at low concentrations (<4 µmol/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
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 Ca2+ 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.
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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 Lys+-modified TFPI,
Lysneut-modified TFPI, and Arg-modified TFPI
(final concentration, 50 nmol/L) and the 3 controls
Lys+-modified BSA,
Lysneut-modified 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.
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Discussion |
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The endogenous ability of LDL to inhibit tissue factor6 7 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 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 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 39 40 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
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Acknowledgments |
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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.
Received September 15, 1998; accepted December 23, 1998.
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