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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.
| Abstract |
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Key Words: apolipoprotein B-100 factor III (thromboplastin) factor VII factor X UV spectroscopy
| Introduction |
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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.
| Methods |
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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 FIIIApoB-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).
| Results |
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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.
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Agarose Gel Electrophoresis of the FIIIApoB-100
Complex
The samples containing reconstituted apoB-100, rFIII, and
apoB-100FIII 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 FIIIapoB-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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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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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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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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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.
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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
).
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| Discussion |
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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 |
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| Acknowledgments |
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Received September 11, 1995; accepted February 6, 1996.
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