Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:639-647
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:639-647.)
© 1996 American Heart Association, Inc.
The Mechanism of Inhibition of Factor III (Thromboplastin) Activity by Apolipoprotein B-100
Protein-Protein Interactions
Camille Ettelaie;
Nicola J. James;
Barry Wilbourn;
Jacqueline M. Adam;
Khalid M. Naseem;
K. Richard Bruckdorfer
From the Department of Biochemistry and Molecular Biology, Royal Free
Hospital School of Medicine, London, UK.
Correspondence to Camille Ettelaie, Department of Biochemistry and Molecular Biology, Royal Free Hospital School of Medicine, Rowland Hill St, London, UK, NW3 2PF. E-mail camille@rfhsm.ac.uk.
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Abstract
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Abstract Factor III (thromboplastin) activity is inhibited by
apoB-100,
but the mechanism of inhibition is unknown. By examining the
effect
of purified apoB-100 on factor III activity, we showed that
apoB-100
can inhibit factor III via a different mechanism from that
caused
by the tissue-factor pathway–inhibitor,
which is mainly
carried on the surface of lipoproteins. Although the
presence
of calcium ions and factors X and VII may enhance the rate of
inhibition,
they are not a prerequisite for the inhibition of factor
III
by apoB-100. In addition, by investigating the changes in the
UV
spectra of apoB-100 on interaction with factor III and factors
X and
VII and by assigning the shifts in absorption spectra
to particular
amino acids, we showed that these interactions
involve negative and
positive residues within these proteins.
By following the rates of
interactions between apoB-100 and
either factors III, X, or VII, a
two-step mechanism for the
inhibition process involving factors X
and VII was postulated.
In this mechanism, the primary interaction of
apoB-100 with
factor III is followed by a rate-limiting step that
can be accelerated
by the presence of either factor X or VII and leads
to the inhibition
of factor III. Furthermore, a computer-based
analysis of the
sequences of factor III revealed a possible
binding site for
apoB-100.
Key Words: apolipoprotein B-100 • factor III (thromboplastin) • factor VII • factor X • UV spectroscopy
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Introduction
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The exposure of FIII
upon injury to endothelium initiates the
extrinsic
pathway of coagulation and leads to formation of a
blood clot on the
injured surface.
1 2 3 Following clot formation
the
procoagulant effect of FIII is restrained by a number of
circulating
inhibitors within the serum. Previously, the
inhibitory
action of LDL was thought to be derived solely
from an associated
protein known as TFPI (also known as
lipoprotein-associated
coagulation inhibitor), which
requires the presence of activated
clotting factors X and VII
and calcium ions for its inhibitory
effect.
4 5 6
Recently, it was demonstrated that LDL7 8 9 10 and
specifically the protein component of LDL (apoB-100) was capable of
inhibiting the ability of FIII to initiate the extrinsic coagulation
pathway.11 Studies with other FIII inhibitors,
such as TFPI,4 5 6 apoA-II,12 and placental
anticoagulant protein,13 have shown that certain
conditions must be met for FIII-inhibitor complexes to
form. These conditions include the presence of activated
clotting factors X and VII and calcium ions as well as thermodynamic
criteria.5 14 In addition, the inhibition of FIII activity
by TFPI seems to occur at a faster rate than that by
apoB-100.7 8 9 10 The aim of this work was to elucidate the
mechanism of FIII inhibition by apoB-100 in the absence of TFPI.
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Methods
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Isolation and Reconstitution of ApoB-100 and FIII
ApoB-100 was isolated to homogeneity
10 11 and
reconstituted
(1.9 µmol/L) in a microemulsion of soybean
phosphatidylcholine
(1 g/L) (Sigma Chemical Co) and dispersed in sodium
deoxycholate
(6 mmol/L) (Calbiochem Novabiochem).
10 15 16
Commercial rabbit
brain FIII (Sigma) and recombinant rabbit FIII
(Gamidor Ltd)
were used throughout this work so that any procoagulant
activity
measured would be standardized, as the activity of FIII varies
among
differing preparations. On the other hand, in some experiments,
the
presence of impurities made the use of crude FIII questionable.
In
these experiments, the assays were performed with rabbit
brain FIII and
recombinant rabbit FIII and then repeated with
affinity-purified
rabbit brain FIII. The purification of FIII
from the crude rabbit brain
extract was performed by using monoclonal
antibodies to FIII
(Calbiochem) coupled to
p-nitrophenyl
chloroformate–activated
agarose (Sigma).
17
The purified FIII exhibited a single band
(47 kD) on 12% denaturing
polyacrylamide gel electrophoresis.
However, the yield of the
purified protein was not sufficient
for measurement of FIII activity
and hence was used for spectroscopic
measurements. The rFIII had the
advantage of purity at measurable
quantities but the disadvantage of
incomplete glycosylation
and posttranslational modifications, which may
be necessary
for ideal activity. The activity of FIII and the
percentage
inhibition were calculated according to an arbitrary
scale.
10 11
Measurement of Inhibition of FIII by ApoB-100
Assays to measure inhibition of FIII activity were performed as
described below, and the results were interpreted.10 11
Samples (1 mL) of purified apoB-100 (0.95 µmol/L), reconstituted in
soybean phosphatidylcholine (final concentration, 0.5 g/L) and
dispersed in sodium deoxycholate (6 mmol/L), were incubated with equal
volumes of rabbit brain FIII or rFIII (final concentration,
50x103 U/L) and CaCl2 solution (final
concentration, 0.25 mmol/L) at 37°C either in the absence of
additional factors or the presence of FX and FVII (final concentrations
for both, 10 U/L) (Sigma). At these concentrations, FX and FVII had no
significant effect on the assay method used. The study was repeated in
the presence of FVII (10 U/L) only, FX (10 U/L) only, and finally at
4°C in the absence of other factors. Samples were removed from the
incubation at intervals over a period of 180 minutes, and
residual FIII activity was measured by means of the one-stage
prothrombin time assay. Deoxycholate had no effect on FIII activity at
this concentration.18 The percent inhibition in each case
was calculated as the reduction in activity divided by the initial
activity multiplied by 100.
Samples (1 mL) of purified apoB-100 (final concentration, 0.95
µmol/L), reconstituted in soybean phosphatidylcholine (final
concentration, 0.5 g/L) and dispersed in sodium deoxycholate (6
mmol/L), were incubated with FX or FVII (final concentration, 10 U/L)
together with the CaCl2 solution (final concentration, 0.25
mmol/L) at 37°C for 60 minutes before the addition of rabbit brain
FIII or rFIII (final concentration, 50x103 U/L). The
incubation was continued and samples were removed at intervals over a
period of 180 minutes and assayed as described above. In addition,
samples of FIII (1 mL) were preincubated at 37°C for 60 minutes with
the same amounts of either FX or FVII before the addition of apoB-100
and monitored for any inhibition over 180 minutes.
Agarose Gel Electrophoresis of the FIII–ApoB-100
Complex
Samples of reconstituted apoB-100 (0.95 µmol/L) were incubated
with rFIII at 37°C for 0, 10, 30, and 60 minutes. Samples of
reconstituted apoB-100 and FIII were used as a reference. All samples
and references were prestained with the Chromatophor protein
stain (Promega Corp). The samples were then examined on a 50x50-mm
glass-plate agarose gel (10 g/L) in 0.12 mol/L barbitone buffer, pH
8.6. Electrophoresis was performed at 1 V/mm until the bands were
clearly separated.
UV Spectroscopy of the Interaction Between FIII and
ApoB-100
The interaction of equal volumes of apoB-100 (0.95 µmol/L
reconstituted in 0.5 g/L soybean phosphatidylcholine) and FIII
(50x103 U/L) in the absence of other factors was monitored
at a wavelength range of 230 to 400 nm. Experiments were performed with
commercial rabbit brain FIII and repeated with affinity-purified
rabbit brain FIII and recombinant rabbit FIII. Difference spectra at 20
and 40 minutes were obtained by zeroing the reading for the measured
range immediately after the addition of the reactants. When using crude
and purified FIII, the samples were constantly mixed to prevent
sedimentation of the FIII. The difference spectra were then used to
calculate the resultant second derivative spectra. Similar spectra of
the interaction between apoB-100 (0.95 µmol/L reconstituted as
before) and FVII (10 U/L) recorded at 15-, 90-, and 180-minute
intervals and FX (10 U/L) recorded at 15-, 60-, and 120-minute
intervals were obtained.
The spectra of rFIII and apoB-100 were recorded individually over a
range of pH values (5.5 to 9.0). Aliquots of FIII were reconstituted
(final concentration, 50x103 U/L) in 0.5 mL Tris-HCl (0.01
mol/L) adjusted at pH 5.5 to 9.0. The spectra (300 to 400 nm) were
obtained on a Beckman DU-70 spectrophotometer, and the spectra were
superimposed. Similarly, the spectra of apoB-100 (0.95 µmol/L),
reconstituted as before in 0.5 mL Tris-HCl (0.01 mol/L) adjusted to pH
5.5 to 9.0, were obtained. These were compared with the spectra of
monomeric and polymeric amino acids, histidine, arginine, lysine,
tyrosine, and glutamic and aspartic acids to assign the various shifts
in the spectra of particular residues.
The interactions of 1-mL samples of apoB-100 (0.95 µmol/L,
reconstituted as before) with 1 mL of either rFIII or purified FIII
(50x103 U/L) were followed individually by measuring the
absorption at 270 nm until no further rise in absorption was detected.
In addition, the interaction of apoB-100 and FIII was followed at 383
nm as described above. The assays were then repeated with 1 mL of
100x103 and then 200x103 U/L FIII. Similar
experiments were performed with three concentrations of FVII (5, 10,
and 20 U/L) or FX (5, 10, and 20 U/L) and followed individually by
measuring the absorption at 270 nm until no further rise in absorption
was detected.
Sequence Analysis
Finally, any homology between the primary structure of FIII and
the LDL receptor protein (obtained from GenBank) was investigated by
using the Bestfit and Gap programs (Genetic Computing Group, University
of Wisconsin).
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Results
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Inhibition of FIII by ApoB-100
The isolation of the apoB-100 to purity ensured the absence
of any
clotting factors or other FIII inhibitors.
11
On incubation
with FIII, apoB-100 was capable of inhibiting the
procoagulant
activity of FIII at 37°C but not 4°C (Fig 1
). The inhibition
of FIII by apoB-100 did not require
the presence of clotting
factors X or VII in either the
activated or zymogen form, nor
was it calcium dependent.
However, the presence of either the
activated or zymogen form
of these factors, together with calcium
ions, enhanced the rate of
inhibition (Fig 1
). In the absence
of calcium ions (not shown) all the
inhibition assays had similar
rates of increase to that in which no
additional factors were
added, indicating that the accelerating effect
of either FVII
or FX was dependent on calcium ions.
In the following experiments, apoB-100 was preincubated with either FX
or FVII for 60 minutes before the addition of FIII and measuring. The
inhibition was completely blocked for 30 minutes on preincubation of
apoB-100 with FVII prior to the addition of FIII and monitoring. The
delay was increased to 90 minutes when apoB-100 was preincubated with
FX before the addition of FIII (Fig 2). Next, FIII was
preincubated with either FX or FVII for 60 minutes before the addition
of apoB-100. Preincubation of FIII and FVII together, before adding
apoB-100, partially protected the FIII activity, as indicated by a
slower rate of inhibition. The preincubation of FIII with FX had no
effect on the rate of inhibition following the addition of
apoB-100.
Agarose Gel Electrophoresis of the FIII–ApoB-100
Complex
The samples containing reconstituted apoB-100, rFIII, and
apoB-100–FIII that were incubated for 0, 10, 30, and 60 minutes were
examined on an agarose gel. The reconstituted apoB-100 showed a
fast-running band, whereas the FIII band exhibited a slower, more
diffuse band, probably due to heterogenous association
with lipids. Agarose gel electrophoresis of FIII–apoB-100, following a
minimum of 30 minutes' preincubation, indicated the formation of a
complex with a mobility between that of FIII and apoB-100 (Fig 3). With incubations of less than 30 minutes both the
FIII and apoB-100 bands were visible, whereas after longer periods of
incubation the FIII band disappeared and there was a retardation of the
apoB-100 band, suggesting the formation of a complex between the two
proteins.
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Figure 3. Samples of reconstituted apoB-100 were incubated
with rFIII at 37°C for 0, 10, 30, and 60 minutes. Samples of
reconstituted apoB-100 and FIII were included as a reference. All
samples and references were prestained with the Chromatophor protein
stain. The samples were examined on a 50x50-mm glass-plate agarose
gel (10 g/L) in 0.12 mol/L barbitone buffer, pH 8.6. Electrophoresis
was performed at 1 V/mm until the bands were clearly separated. The
lanes contained (left to right) (A) FIII, (B) apoB-100, and
FIII+apoB-100 incubated at (C) 0, (D) 10, (E) 30, and (F) 60 minutes.
The figure is one of four separate experiments.
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UV Spectroscopy of the Interaction Between FIII and
ApoB-100
The incubation of apoB-100 with FIII produced various shifts in
the UV spectrum of these proteins. The difference spectra of the
interaction of apoB-100 and the three preparations of FIII were used to
calculate the resultant second derivative spectra after incubation for
20 or 40 minutes. The spectra from the interaction of apoB-100 with
either crude, purified, or recombinant FIII produced essentially the
same difference spectra: the spectra for interaction with rFIII are
shown in Fig 4. In the first 20 minutes, the shift in
the wavelengths corresponding to the aromatic amino acids (241, 246,
257, and 285 nm) and cystine/cysteine (235, 250, and 323 nm) to longer
wavelengths indicated the formation of a hydrophobic
environment.19 20 After incubation for a further 20
minutes, the shifts in wavelengths arising from tryptophan residues
(278 and 285 nm), alterations in aspartic and glutamic residues (>390
nm), and the polarization of 
-amino groups (383 and 318 nm) of
lysine and arginine residues were detected.20 Measurements
at wavelengths >400 nm were too ambiguous, possibly due to masking by
the absorptions arising from the presence of lipids.
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Figure 4. Spectroscopic analysis of the interaction of
rFIII and reconstituted apoB-100. The interaction of equal volumes of
reconstituted apoB-100 and rFIII in the absence of other factors was
monitored at a wavelength range of 230-400 nm, and spectra were
recorded at various times. Difference spectra at 20 (A) and 40 (C)
minutes were obtained by subtracting the initial spectra of rFIII and
apoB-100 from those of recorded spectra at these intervals. These
were then used to calculate the resultant second derivative spectra at
20 (B) and 40 (D) minutes. The spectra of interaction of apoB-100 with
crude and purified FIII produced essentially the same shifts in
wavelengths. Results are typical of those from three separate
experiments.
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The interaction between apoB-100 and FVII (Fig 5)
produced bathochromic shifts in the wavelengths corresponding to the
aromatic amino acids (240 to 290 nm). Furthermore, some shifts in the
wavelengths corresponding to delocalization of free amino side chains
of positively charged amino acids were observed (383
nm).20 However, all the shifts occurred much more slowly
and simultaneously. The interaction between apoB-100 and FX
(Fig 6) produced bathochromic shifts in wavelengths
corresponding to aromatic amino acids similar to those with FVII.
Moreover, the shifts occurred at rates that were faster than those for
FVII and were completed within 90 minutes.
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Figure 5. Spectroscopic analysis of the interaction of
FVII and reconstituted apoB-100. The interaction of equal volumes of
reconstituted apoB-100 and FVII in the absence of other factors was
monitored at a wavelength range 230-400 nm, and spectra were
recorded at various intervals. Difference spectra at 15 (A), 90
(C), and 180 (E) minutes were obtained by subtracting the spectra of
FVII and apoB-100 from those of recorded spectra at the three
intervals. These were then used to calculate the resultant second
derivative spectra at 15 (B), 90 (D), and 180 (F) minutes. Results are
typical of three separate experiments.
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Figure 6. Spectroscopic analysis of the interaction of
FX and reconstituted apoB-100. The interaction of equal volumes of
reconstituted apoB-100 and FX in the absence of other factors was
monitored at a wavelength range 230-400 nm, and spectra were
recorded at various intervals. Difference spectra at 15 (A), 60
(C), and 120 (E) minutes were obtained by subtracting the spectra of FX
and apoB-100 from those of recorded spectra at the three intervals.
These were then used to calculate the resultant second derivative
spectra at 15 (B), 60 (D), and 120 (F) minutes. Results are typical of
three separate experiments.
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The interaction of apoB-100 with either FIII, FX, or FVII produced
shifts in the wavelengths corresponding to those of tyrosine residues
(223 to 240 and 274.5 to 293.5 nm), which may have been due to
ionization/deionization of the hydroxyl groups of these
residues.20
Assignment of Absorption Wavelengths
With increasing pH, the spectrum of FIII exhibited wavelength
shifts (>390 nm). This indicated the polarization of the carboxyl
groups of glutamic and aspartic acid residues (Fig 7),
since spectra of aspartate and glutamate monomers and polymers
exhibited similar shifts (not shown) as the pH was increased. Moreover,
this corresponded to the shift observed at these wavelengths on
incubation with apoB-100. Therefore, the shift in absorption at >390
nm was assigned to the involvement of the negatively charged residues
within FIII. The increase in pH produced specific shifts in the
absorption of apoB-100 at 318 and 383 nm (Fig 8). The
spectra of histidine, arginine, and lysine, either in monomer or
polymer form, exhibited similar shifts (not shown) at lower pH that
were assigned to a delocalization of the side-chain nitrogen at
neutral pH values. The shifts were greatest in histidine and lowest in
lysine residues, as expected. These shifts in absorption were also
detected on interaction of apoB-100 with FIII.
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Figure 7. Spectroscopic analysis of rFIII at various
pH values. Aliquots of FIII were reconstituted in 0.5 mL Tris-HCl
buffer, pH 5.5-9.0. Spectra were obtained on a Beckman DU-70
spectrophotometer and superimposed. The shift in wavelengths <300 was
not significant, but the shift in wavelengths >390 indicate the
polarization of the negative residues within FIII. Results are typical
of two separate experiments.
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Figure 8. Spectroscopic analysis of the apoB-100 at
various pH values. Aliquots of apoB-100 were reconstituted in 0.5 mL
Tris-HCl buffer, pH 5.5-9.0. Spectra were obtained on a Beckman DU-70
spectrophotometer and superimposed. The shift in wavelengths <300 was
insignificant, but the shift in wavelengths 318 and 383 nm indicate the
delocalization of the positive residues within apoB-100. Spectra were
divided into 300-370 nm (A) and 370-400 nm (B) for clarity. Results are
typical of two separate experiments.
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Time-Course Study of the Interaction Between FIII and ApoB-100 by
UV Spectroscopy
By following the absorption of the samples at 270 nm over a period
of time and using a range of concentrations of the reactants, it was
possible to deduce the relative rate of interaction of apoB-100 with
FIII, FVII, and FX. The chosen wavelengths were those that produced the
greatest differences in absorption when examining the spectra at 230 to
400 nm in all three samples tested. The interaction of apoB-100 with
FIII at 270 nm occurred at a faster rate (t
=3.5 minutes) than
that of apoB-100 and FX (t=5 minutes) (Fig 9).
The interaction between apoB-100 and FVII was completed even more
slowly (t=21 minutes). The apparent dissociation constant
(Kd) values for the interaction of apoB-100 with
FIII measured at 270 and 383 nm were 0.03 and 0.171, respectively; with
FX and FVII, these values were 0.08 and 0.97 measured at 270 nm. On the
other hand, the interaction between apoB-100 and FIII, followed by
absorption at 383 nm (corresponding to derivatization of 
-amino
groups), occurred at a slower rate (t=10 minutes) than that
measured at 270 nm (corresponding to aromatic interactions). These
results indicate that the primary interaction between apoB-100 and FIII
occurs more rapidly than the interactions between apoB-100 and FX or
FVII. However, the second interaction between apoB-100 and FIII, which
leads to the inhibition of the latter, occurs at a slower rate than
that between apoB-100 and FX. Therefore, the presence of FX, in
particular, may influence the interaction of apoB-100 and FIII.
Sequence Analysis
The computer-based analysis of the structures of FIII
and the LDL receptor protein (obtained from GenBank) revealed regions
of similar amino acids within the two proteins (Table).
The similarity (77.8%) and identity (44.5%) of the examined peptides
were considerable. Furthermore, both peptide domains were predicted to
have similar secondary structures and to be partially 
-helical
(Fig 10).
|
Discussion
|
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The data presented here indicate that the interaction of
FIII
with apoB-100 occurs in at least two sites. Thus, while the
first
site involves the attainment of a more hydrophobic configuration
or the
formation of hydrophobic interactions
19 20 and occurs
within
20 minutes of incubation, the evidence suggests that the second
interaction
involves negatively charged residues and positive free
amino
groups
20 and occurs over a longer period of time (up
to 60
minutes). This coincides with the progressive inhibition of
FIII
activity by apoB-100, which reached a maximum after 60
minutes. The
incubation of apoB-100 with either FX or FVII also
produced various
modifications to the UV spectra. By monitoring
the interactions between
apoB-100 and FIII, FX, or FVII at various
concentrations of each
coagulation factor, we found that apoB-100
has a higher affinity for
FIII than for the other two clotting
factors. Hence, these data suggest
a model for the formation
of an apoB-100–FIII clotting factor complex
in which, initially,
apoB-100 interacts with FIII. The subsequent
binding of FX and/or
FVII leads to a more rapid induction of a
complex that mediates
the inhibition of FIII activity (Fig 11
).
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Figure 11. Proposed model for the interaction of apoB-100 and
FIII, FX, and FVII. The interaction and the subsequent inhibition of
FIII by apoB-100 from free components (A) follows a certain mechanism
in which FIII is bound and directed to the inhibition site on apoB-100
(B). The subsequent binding of either FX or FVII can accelerate the
inhibition (C). Conversely, the interaction of apoB-100 with either FX
or FVII prior to binding of FIII can obstruct the inhibition of FIII
(D).
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While the sequence of the protein component of FIII (residues 58
through 66) is rich in negative residues, the receptor-binding
domain of apoB-100 is rich in positive and tryptophan
residues.21 22 The peptide region corresponding to the LDL
receptor protein (283 through 291) is predicted to be partly
-helical,23 24 and the domain within FIII (residues
58 through 66) is mainly -helical.25 When the
secondary structures of these two -helical sequences were
reconstructed, it was possible to identify similar motifs within them
(Fig 10
). We propose, therefore, that this domain may be involved in
the interaction and inhibition of FIII by apoB-100. We are currently
investigating the possible function of this domain in the interaction
with apoB-100.
From this study, we conclude that the interaction and inhibition of
FIII by apoB-100 are two separate but interrelated processes.
Furthermore, although the presence of clotting factors X and VII are
not essential for this interaction and, under certain conditions, they
may compete with FIII for the binding sites on apoB-100, they may act
cooperatively in the formation of the inhibited complex under
physiological conditions. The progress of the
inhibition was found to be temperature-dependent, as no inhibition
was observed at 4°C. Also, the interactions that lead to FIII
inhibition seem be to be time dependent. This would explain the
negative results obtained by Aviram and Presser,26 who did
not observe any effect when they assessed FIII activity immediately
after adding LDL to FIII. The blocking of the inhibition that resulted
from preincubation of apoB-100 with either clotting factor X or VII
reinforces the suggestion that FIII and these clotting factors are
bound by apoB-100 in a definite order of priority if inhibition is to
occur. Alternatively, the FIII binding site may be masked by FX or
FVII, thereby preventing inhibition (Fig 11).
The variable effects of the interaction between FIII and apoB-100
that result in either inhibition or enhancement of FIII activity under
different conditions8 9 10 11 15 16 could arise from changes in
the conformation of apoB-100 due to variations in the lipid composition
of the lipoprotein (Reference 2727 and C. Ettelaie et al, unpublished
data, 1995). This indicates a mechanism of interaction that is more
complex than the simple adsorption of the FIII complex to the LDL. We
propose that in vivo the action of FIII is suppressed by its
interaction with the apoB-100 of LDL as well as other FIII
inhibitors, eg, TFPI. This interaction occurs at two sites,
one of which is likely to be within the receptor-binding domain on
apoB-100.
In conclusion, this study indicates that apoB-100 within LDL is
capable of inhibiting the procoagulant activity of FIII via a different
and independent mechanism than that of TFPI and other FIII
inhibitors. In addition, the rate at which the inhibition
by apoB-100 occurs is slower than that by TFPI. Therefore, LDL may play
an important role in the physiological functioning
of FIII and the generation of thrombosis in disease conditions.
|
Selected Abbreviations and Acronyms
|
|---|
| FIII |
= |
factor III (thromboplastin) |
| FVII |
= |
factor VII |
| FX |
= |
factor X |
| rFIII |
= |
recombinant factor III |
| TPFI |
= |
tissue-factor pathway inhibitor |
|
|
Acknowledgments
|
|---|
We would like to acknowledge the support of the Wellcome Trust
and
the British Heart Foundation.
Received September 11, 1995;
accepted February 6, 1996.
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Abstract
Introduction
), the presence of FX and FVII (each 10 U/L) (
), FVII
(10 U/L) only (
), and FX (10 U/L) only (
), and at 4°C
(
) in the absence of FX and FVII.
Samples were removed at intervals over 180 minutes, and residual FIII
activity was measured by means of the one-stage
prothrombin-time assay. The inhibition was calculated as the
percentage of FIII units decreased/original FIII activity. Results
represent SEM of three separate experiments. To avoid
confusion, only one symbol is shown when two or more points coincide.
) (10 U/L) prior to the addition of apoB-100
and monitored for any inhibition over 180 minutes. Results
represent SEM of three separate experiments. To avoid
confusion, only one symbol is shown when two or more points coincide.
, 
, and 
were adjusted according to those used in all the regions predicted to
be 
1,