From the Cardiovascular Biology Research Program,
Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, the § Department of Pathology, and ¶ W. K. Warren
Medical Research Institute, Departments of Medicine and Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma 73104, and the
Departments of
Pediatrics and Cell Biology & Physiology, Washington University School
of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Inflammatory mediators like bacterial
lipopolysaccharide induce monocytes to express tissue factor (TF), the
cell-surface protein that triggers the blood clotting cascade in
hemostasis and thrombotic disease. The physiologic ligand for TF is the
serine protease, factor VIIa (FVIIa), and the resulting bimolecular
enzyme, TF/FVIIa, can be reversibly inhibited by tissue factor pathway inhibitor (TFPI). Culturing monocytic cells in the presence of both
FVIIa and TFPI caused down-regulation of TF expression via reducing its
half-life. To exert this effect, FVIIa had to be competent to bind both
TF and TFPI, and TFPI had to contain the C-terminal domain required for
binding to other cell-surface receptors, including the low density
lipoprotein receptor-related protein (LRP). TF down-regulation by FVIIa
plus TFPI was abrogated by the 39-kDa receptor-associated protein,
which blocks binding of all known ligands to LRP. Furthermore,
treatment with FVIIa plus TFPI caused monocyte TF to colocalize with
Tissue factor (TF)1 is
the integral membrane protein that initiates the blood clotting cascade
in hemostasis and many thrombotic disorders (1). TF allosterically
enhances the enzyme activity of the plasma serine protease, factor VIIa
(FVIIa), and the resulting TF·FVIIa complex activates coagulation
factors IX and X by limited proteolysis. Recent studies have also shown
that TF can act as a signaling receptor upon binding FVIIa (2).
Although TF is normally absent from cells in contact with the plasma,
monocytes can be induced to express cell-surface TF by several
inflammatory mediators, including bacterial lipopolysaccharide (LPS)
(3). Expression of TF on circulating monocytes is thought to drive the
life-threatening coagulopathy observed in sepsis (4, 5) and to cause
thrombosis in other disease states such as unstable angina (6). TF is
also expressed in monocytes/macrophages resident in atherosclerotic
plaques (7, 8), where it can trigger thrombus formation following
plaque fissure or rupture. It is therefore important to understand how
TF expression is controlled in monocytic cells. Several studies have
focused on transcriptional control of TF gene expression (reviewed in
Ref. 9) and mRNA half-life (10). However, much less is known about
post-translational control of TF expression, especially in monocytic cells.
An important physiologic regulator of the enzymatic activity of
TF/FVIIa is the protease inhibitor, tissue factor pathway inhibitor
(TFPI). TFPI circulates in association with lipoproteins and is present
in platelets. In addition, a large pool of TFPI is apparently bound to
the vascular endothelium in vivo, which is releasable by
heparin injection (11). TFPI has three Kunitz-type inhibitor domains,
allowing it to inhibit factor Xa (FXa) and TF/FVIIa simultaneously
(12). Whereas the TFPI·FXa complex is an especially potent inhibitor
of TF/FVIIa, TFPI can also inhibit TF/FVIIa directly. TFPI binds to two
classes of cell-surface proteins, heparan sulfate proteoglycans (HSPGs)
and the low density lipoprotein receptor-related protein (LRP) (13).
LRP is responsible for mediating clathrin-dependent
endocytosis and subsequent degradation of TFPI by hepatoma cells (14).
In addition, TFPI mediates the uptake and degradation of FXa by
fibroblasts and hepatoma cells (15). In cultured vascular endothelial
and smooth muscle cells, TF is localized, via
TFPI-dependent (16) or -independent (17) means, to
caveolae, specialized membrane domains whose phospholipid content
differs significantly from the remainder of the plasma membrane
(18).
In the present study we investigated the role of TFPI in
down-regulation of TF expression in human peripheral blood monocytes and the human monocytic leukemia cell line, Mono Mac 6 (MM6)(19). When
FVIIa and TFPI bound to TF on the surface of monocytic cells, the
half-life of TF was shortened from 3.7 to 1.3 h. Down-regulation of TF expression was dependent upon both FVIIa and TFPI and required that TFPI have an intact C terminus. In contrast to endothelial and
smooth muscle cells, no evidence was obtained in monocytes for
translocation of TF to caveolae or other membrane domains with altered
phospholipid content. However, TF down-regulation by TFPI plus FVIIa in
monocytic cells was dependent upon LRP. We propose that when TF/FVIIa
on the surface of monocytic cells binds TFPI, the resulting
TF·FVIIa·TFPI complex associates with LRP and is translocated to
clathrin-coated pits. TF is thereby internalized and degraded by an
unusual mechanism in which one integral membrane protein (TF) is
bridged, in a ligand-dependent fashion, to a second
integral membrane protein (LRP), the latter promoting
clathrin-mediated internalization.
Reagents and Proteins--
Reagents and supplies were from the
following sources: Histopaque® and protamine sulfate from Sigma;
RPMI-1640, phosphate-buffered saline, and Hank's balanced salt
solution (HBSS) from Mediatech; heparin from Fujisawa Pharmaceutical;
iron-supplemented calf serum from HyClone; AIM-V culture medium from
Life Technologies, Inc.; LPS (Escherichia coli 0111:B4),
bovine serum albumin, FFR-chloromethyl ketone, and
n-octyl-
Recombinant TFPI (20), TFPI13-160 (a C-terminal truncated
form of TFPI (21)), and XK1 (FX/TFPI chimera (22)) were generous gifts
from Dr. George Broze, Jr. (Washington University, St. Louis). The
39-kDa receptor-associated protein (RAP) and rabbit anti-LRP IgG were
prepared as described (23). FX was purified from human plasma and
activated to FXa as described (24, 25). Human plasma FVII was purified
and activated as described (26). FFR-FVIIa was prepared by reaction of
FVIIa with FFR-chloromethyl ketone (1:20 molar ratio) for 30 min at
room temperature in 100 mM NaCl, 50 mM
Tris-HCl, pH 7.5, 0.5% human serum albumin, followed by extensive
dialysis to remove unreacted inhibitor. FFR-FVIIa had less than 1% of
its original amidolytic activity (27). Recombinant E2PD-FVIIa was constructed and purified as described for
E2PD-FX (28) and was a gift from Dr. Pierre Neuenschwander.
TF was purified from human brain by affinity chromatography as
described (29) except that antibody TF9-5B7 was used in place of
TF8-5G9. Recombinant soluble TF was prepared as described (30).
Preparation of polyclonal anti-TF IgG and anti-TF mAb were described
previously (31). The CHO-B cDNA probe was a gift from Dr. R. Walls
of the UCLA School of Medicine (Los Angeles, CA). All proteins except
TFPI, XK1, and RAP were supplemented with 0.5% human serum albumin and dialyzed against sterile phosphate-buffered saline before use in cell
cultures. Stock solutions of TFPI (8 mg/ml) and TFPI13-160 (4.7 mg/ml) were in 2 M urea/phosphate buffered saline and
were diluted directly into culture medium when used. Controls with similarly diluted urea were performed.
Cell Culture--
MM6 cells were grown at 37 °C in 5%
CO2 in RPMI-1640, 10% calf serum supplemented with 50 µg/ml gentamicin. For experiments, cells were rinsed once with
Ca2+,Mg2+-free HBSS and once with AIM-V medium,
and then resuspended in AIM-V, 0.01% calf serum unless otherwise
indicated. MM6 cells were then cultured in multiwell plates. AIM-V has
been used for long term culture of macrophages (32). We found that MM6
cells cultured in AIM-V for 10 days were viable and grew at a rate only slightly lower than cells cultured in RPMI/10% calf serum (data not
shown). Platelet-poor human plasma was prepared by centrifugation of
heparinized blood for 15 min at 300 × g and
recentrifugation of plasma for 15 min at 5,000 × g.
Human peripheral blood mononuclear cells (PBMC) were isolated from
heparinized blood by centrifugation using Histopaque, per the
manufacturer's instructions, then suspended in AIM-V, 0.01% calf
serum, and cultured in multiwell plates or sterile polypropylene culture tubes at 37 °C in 5% CO2. The cells were used
in experiments immediately after isolation.
TF ELISA--
TF was solubilized from cells and culture media by
adding EDTA (7 mM final) and either Triton X-100 (1%
final) or octyl glucoside (60 mM final), after which TF
levels were measured by a sandwich ELISA (30). EDTA was included to
dissociate FVIIa and TFPI from TF, to eliminate competition with
anti-TF antibodies. Control experiments in which FVIIa and TFPI were
added to lysates of TF-expressing monocytes confirmed that these
proteins did not interfere with TF measurement by ELISA in the presence
of EDTA. Statistical significance of differences in ELISA values was
determined using Student's t test.
For measurement of the half-life of TF, cells were treated for 4 h
with LPS and then treated with 10 µg/ml cycloheximide; cultures were
immediately divided; TFPI and FVIIa were added as indicated, and timed
aliquots were removed and processed for ELISA. The effect of
staurosporine on TF levels was investigated by incubating LPS-stimulated cells as above for 3.5 h, adding 2 µM
staurosporine or vehicle (ethanol) and incubating for an additional 30 min. FVIIa and TFPI were then added for 2 h, after which cells
were collected for ELISA.
Western Blot Analysis--
Cells were rinsed with
phosphate-buffered saline, resuspended at 1 × 107
cells/ml in SDS sample buffer with dithiothreitol, and boiled for 5 min. Proteins were resolved on SDS-PAGE and blotted onto polyvinylidene
difluoride membranes, which were then treated with blocking buffer (4%
bovine serum albumin and 1% casein in 50 mM Tris-HCl, pH
7.4, 100 mM NaCl, 0.05% Tween 20). Membranes were subsequently treated with 0.5 µg/ml biotinylated polyclonal anti-TF IgG in blocking buffer for 1 h at 37 °C, rinsed three times
with 50 mM Tris-HCl, pH 7.4, 100 mM NaCl,
0.05% Tween 20, and incubated with NeutrAvidin-horseradish peroxidase
in blocking buffer for 1 h at room temperature. In some
experiments, membranes were co-stained with polyclonal rabbit
anti-caveolin antibody, using horseradish peroxidase-conjugated goat
anti-rabbit as the secondary antibody. Human endothelial cell lysate as
positive control for caveolin staining was provided by the supplier of
the anti-caveolin antibody. Membranes were rinsed as above and treated
with chemiluminescence substrate (3 ml per 54-cm2 membrane)
for 5 min, after which they were exposed to Kodak BIOMAX x-ray film.
RNA Isolation, Blotting, and Hybridization--
RNA was
extracted from 1 × 106 cells using RNAzol according
to the manufacturer's instructions, resolved by electrophoresis on
formaldehyde/agarose gels, and blotted onto polyvinylidene difluoride
membranes. Membranes were hybridized at 65 °C for at least 24 h
with radiolabeled TF or CHO-B cDNA probes and quantified using a
Molecular Dynamics PhosphorImager and ImageQuaNT software, with TF
signals normalized to CHO-B.
Isolation of Triton-insoluble Membrane Fractions--
The
caveolar membrane isolation protocol of Lisanti et al. (33)
was modified as follows: MM6 cells were rinsed with ice-cold phosphate-buffered saline and resuspended in 1% Triton X-100 in MES-buffered saline (25 mM MES, pH 6.5, 150 mM
NaCl) containing 1 mM each NaVO4 and
phenylmethylsulfonyl fluoride. Homogenates were adjusted to 45%
sucrose, overlaid with 4 ml each 35 and 5% sucrose (in MES-buffered
saline), and centrifuged 16-20 h at 39,000 rpm in a Beckman SW41
rotor. Triton-insoluble membranes, including caveolae, float to the
5/35% interface (34). Gradient fractions (1 ml) were assayed for light
scattering at 600 nm, TF content by ELISA, and protein content
(bicinchoninic acid assay). The densest three fractions contained
approximately 500-700 µg of protein, and the lighter fractions
contained Immunofluorescence--
MM6 cells were rinsed with ice-cold HBSS
plus 0.5% bovine serum albumin and incubated with 10 µg/ml
biotinylated goat anti-TF IgG (45 min at 4 °C). After rinsing, cells
were incubated with streptavidin-Oregon Green (45 min at 4 °C),
rinsed again, and fixed in 3.7% paraformaldehyde (10 min at 4 °C).
The fixative was rinsed away, and cells were permeabilized with 0.05%
Triton X-100 in HBSS (10 min at 4 °C). After rinsing, cells were
incubated 45 min on ice with HBSS plus 0.2% bovine serum albumin and
10 µg/ml anti-
The degree of colocalization of TF with Effect of TFPI and FVIIa on TF Expression--
LPS induces TF
expression in cultured peripheral blood monocytes (36) and in monocytic
cell lines such as THP-1 (10) and MM6 (37). In many published studies,
monocytes and monocytic cell lines have been cultured in media
supplemented with serum. In this study, when MM6 cells were cultured in
the serum-free medium AIM-V, we observed that adding very low
concentrations of calf serum (0.01% and below) enhanced TF expression
in response to LPS, whereas higher concentrations of serum (0.05-10%)
reduced TF expression relative to that seen with 0.01% serum (Fig.
1). Low levels of serum may enhance the
responsiveness of MM6 cells to LPS by providing a source of LPS-binding
protein (38), but it was unclear why higher concentrations of serum
caused MM6 cells to express lower levels of TF following LPS
stimulation.
Serum contains FVIIa, the primary ligand for TF, and also TFPI, a
reversible protease inhibitor that binds to the TF·FVIIa complex.
Accordingly, we hypothesized that these molecules may bind to TF on
monocytic cells and promote the down-regulation of TF expression. MM6
cells or PBMC were cultured in AIM-V medium supplemented with 0.01%
calf serum to support optimal TF expression, typically measured 4-6 h
after stimulating the cells with 10 ng/ml LPS. (0.01% serum
contributes negligible levels of FVIIa or TFPI.) Cells incubated with
TFPI after LPS stimulation expressed TF at levels comparable to cells
treated with LPS alone (Fig. 2). Cells treated with FVIIa had very slightly reduced levels of TF. However, MM6
cells and PBMC treated with both TFPI and FVIIa had TF levels that were
typically 50-60% of cells treated with LPS alone (Fig. 2). TFPI can
react both with FXa and with FVIIa bound to TF. In some settings, the
TFPI·FXa complex has a higher affinity for TF/FVIIa than does TFPI
alone (39). However, addition of FXa to TFPI and FVIIa resulted in
levels of TF expression by MM6 cells and PBMC that were similar to
those of cells treated with TFPI and FVIIa without FXa. To simplify
interpretation of results, most of the rest of the experiments in this
study were therefore performed using TFPI and FVIIa without added
FXa.
The time course of TF expression in MM6 cells in response to LPS
stimulation was altered when TFPI and FVIIa were included in the
culture medium. Thus, MM6 cells cultured in the absence of these two
proteins expressed more TF, and did so over a longer period of time,
than did cells treated with a combination of TFPI and FVIIa (Fig.
3). However, Northern blot analysis of
RNA isolated 1 h after LPS stimulation revealed that TFPI and
FVIIa had no effect on TF mRNA levels in MM6 cells (data not
shown), indicating that these ligands affected a later stage of TF
biosynthesis and/or degradation. Accordingly, the effect of TFPI and
FVIIa on the half-life of TF protein in MM6 cells was examined by
inducing TF expression with LPS, after which cycloheximide was added to block further protein synthesis. TF antigen levels were then quantified by ELISA (Fig. 4). Treating cells with
TFPI and FVIIa reduced the half-life of TF protein 3-fold compared with
control cells (mean half-lives of 3.7 h versus 1.3 h; n = 3). In subsequent experiments, the effect of
TFPI and FVIIa on TF expression was evaluated in cells incubated for
6 h, since this time point consistently showed the largest
difference in TF levels with and without treatment.
Effect of TFPI and FVIIa on Detergent Solubility of
TF--
Previous studies showed that TF on the surfaces of endothelial
cells (16) and smooth muscle cells (17) is associated, at least in
part, with caveolae. In endothelial cells, translocation of TF from
Triton-soluble to Triton-insoluble membrane fractions (associated with
caveolae (18)) was induced by the addition of FVIIa and FX and required
endogenous TFPI (16). It is not clear that caveolae per se
exist in monocytes. However, if TFPI and FVIIa can induce the
translocation of TF to Triton-insoluble membrane domains in monocytes
and MM6 cells, it might render this protein undetectable by our ELISA.
We investigated whether the decrease in measured TF levels was due to
insolubility in Triton X-100 in three ways. First, we compared octyl
glucoside versus Triton X-100 lysis of MM6 cells, since
octyl glucoside has been shown to solubilize caveolar proteins (40).
However, TF levels in MM6 cells treated with TFPI and FVIIa and lysed
with octyl glucoside were indistinguishable from cells lysed with
Triton X-100 (data not shown). Second, MM6 cells were treated or not
with TFPI and FVIIa, and cells were lysed by boiling in SDS sample
buffer followed by SDS-PAGE and Western blotting. Consistent with the
ELISA results, TF levels were much lower in lysates from cells treated
with TFPI and FVIIa compared with control cells (data not shown).
Third, association of TF with Triton-insoluble membrane fractions was
examined by lysing MM6 cells with Triton X-100 followed by
fractionation on sucrose density gradients. This method has been used
in other cell types to separate caveolar membranes and their resident
proteins from the remainder of the plasma membrane; the low density,
light scattering fractions contain Triton-insoluble membranes and
caveolin (33). When MM6 lysates were fractionated by density gradient
centrifugation and TF antigen levels were quantified by ELISA or
Western blot, essentially all of the TF was associated with the high
density fractions (fractions 6-9) that contain Triton-solubilized
membrane proteins (Fig. 5). Negligible
amounts of TF antigen (less than 2%) colocalized with the light
scattering, low density fractions under any of the assay conditions. In
these experiments, cells were lysed 15 min after addition of TFPI,
FVIIa, and FXa, since this was sufficient to cause translocation of TF
from high density to low density membrane fractions in endothelial
cells (16). However, MM6 cells lysed 6 h after treatment with TFPI
and FVIIa (with or without FXa) gave equivalent results (data not
shown). Additionally, caveolin could not be detected in MM6 cell
extracts by Western blotting.
Structural Requirements of TFPI and FVIIa for Down-regulation of
TF--
The studies above indicated that TF levels were down-regulated
in monocytes and MM6 cells in response to FVIIa and TFPI, resulting from a decreased half-life of TF protein and not from translocation of
the protein to caveolae or other Triton-insoluble membrane fractions.
We hypothesized that FVIIa and TFPI both bound to TF on the cell
surface and that this binding event altered the half-life of TF.
Alternatively, however, FVIIa and/or TFPI might interact with other
receptors or binding sites on the cell surface which might indirectly
cause the observed reduction in TF half-life.
In order to elucidate the structural requirements for the interaction
between TFPI, FVIIa, and TF responsible for the reduction in TF
half-life, we employed altered forms of FVIIa with modified binding
characteristics. These included active-site blocked FVIIa (FFR-FVIIa),
which binds TF with high affinity but which cannot bind TFPI (41), and
a recombinant, truncated form of FVIIa (E2PD-FVIIa), which
lacks the 4-carboxyglutamate-rich domain and the first EGF-like domain.
E2PD-FVIIa is unable to bind to either the plasma membrane or to TF, as demonstrated by functional assays and dot
blots.2 However, because the
active site of E2PD-FVIIa is intact, it can still bind to,
and be inhibited by, TFPI (data not shown). Unlike cells treated with
TFPI plus FVIIa, cells treated with a combination of TFPI and
FFR-FVIIa, or with TFPI and E2PD-FVIIa, expressed TF at
levels that were indistinguishable from those of control cells (Fig.
6). This indicates that FVIIa must be
competent to bind TFPI and that TFPI·FVIIa complexes must be
competent to bind to TF, in order to cause the observed reduction in TF
half-life.
To examine further the structural requirements for the interaction
between TFPI, FVIIa, and TF responsible for the reduction in TF
half-life, we employed TFPI13-160, a recombinant, truncated form of TFPI lacking both the third Kunitz domain and the
C-terminal region of the protein. TFPI13-160 inhibits the
TF·FVIIa complex as readily as full-length TFPI (42) but is unable to
bind specifically to receptors on hepatoma cells (21). In addition, we
also employed a hybrid protein, XK1, that consists of the light chain
of FXa and the first Kunitz domain of TFPI (22). The first Kunitz
domain of TFPI mediates reversible binding to the TF·FVIIa complex
(43), whereas the light chain of FXa mediates binding to membranes
containing exposed negatively charged phospholipids. Because of these
properties, XK1 is an especially efficient inhibitor of the TF·FVIIa
complex (22). However, like TFPI13-160, XK1 lacks the
C-terminal region of TFPI. Both TFPI13-160 and XK1
inhibited the enzymatic activity of purified TF·FVIIa complexes
in vitro (data not shown).
Unlike cells treated with FVIIa plus TFPI, when LPS-stimulated MM6 or
PBMC were treated with a combination of FVIIa and either XK1 or
TFPI13-160, they expressed TF at levels that were essentially the same as cells treated with LPS alone (Fig.
7). This indicates that inhibition of
TF/FVIIa enzymatic activity by TFPI is not sufficient to down-regulate
TF levels and that the intact C terminus of TFPI is required.
RAP Abrogates TF Down-regulation--
In hepatoma cells, TFPI has
been shown to bind to at least two classes of cell membrane proteins,
HSPGs and LRP (13). In those cells, TFPI binds to LRP with a
Kd of 2.3 nM (14) and to HSPGs with a
Kd of about 30 nM, which outnumber LRP
by about 10-fold (44). It is thought that both receptor systems may
function to clear TFPI from the plasma in vivo (13), but LRP
is required for the cellular uptake and degradation of TFPI by hepatoma
cells in vitro (14). Previous studies have shown that TFPI
must have an intact C-terminal region in order to bind to HSPGs or LRP
(13, 21), matching the requirements found above for mediating TF
down-regulation. We hypothesized, therefore, that the TF·FVIIa·TFPI
complex interacts with HSPGs or LRP via the C-terminal region of TFPI
and that this binding is essential in down-regulating TF expression.
Degradation of TFPI is inhibited by RAP (14), a protein that inhibits
binding and uptake by all known ligands that bind to LRP (45, 46), whereas protamine sulfate inhibits the binding of TFPI to HSPGs (13).
Therefore, in order to determine whether binding of TFPI to HSPGs or
LRP is involved in the down-regulation of TF in monocytic cells, we
conducted experiments in the presence of protamine sulfate or RAP (Fig.
8). Protamine sulfate failed to block the
ability of TFPI plus FVIIa to down-regulate TF expression. In fact, MM6 cells treated with protamine sulfate (with TFPI plus FVIIa) exhibited about 11% lower TF levels relative to cells treated with TFPI and
FVIIa alone (p < 0.005). In hepatoma cells, protamine
sulfate increases by 5-fold the amount of TFPI that can be cross-linked to LRP and enhances LRP-mediated degradation of TFPI (14). On the other
hand, RAP completely abrogated the effect of TFPI plus FVIIa on TF
levels in both MM6 cells and monocytes (Fig. 8). This indicated that
the TF·FVIIa·TFPI complex interacts with LRP in order to effect the
down-regulation of TF levels, possibly via endocytosis with subsequent
degradation of TF.
TFPI and FVIIa Promote the Colocalization of TF with
Since staurosporine can affect multiple cellular processes, we used
dual-label immunofluorescence to examine more directly whether TFPI
plus FVIIa could promote the colocalization of TF with In vitro, endothelial cells and monocytes express TF in
response to a variety of inflammatory mediators, including LPS
(reviewed in Refs. 9 and 51). In vivo, TF expression by
endothelial cells is very rare, even in models of lethal septic shock
and endotoxemia (52-54). On the other hand, TF expression on
circulating monocytes has been well documented in animal models of
bacteremia and LPS treatment and in humans with sepsis (52, 55, 56). In
addition, it is clear that the intravascular coagulation associated with sepsis and endotoxemia is TF-dependent (4, 5). Since these studies strongly argue that induced expression of TF on circulating monocytes drives the coagulopathies observed in sepsis, it
is clearly important to understand how the LPS-induced expression of TF
in monocytes is regulated.
In the present study, we found that expression of TF in monocytes and
MM6 cells in response to LPS was modulated by culturing the cells in
the presence of the plasma proteins, FVIIa and TFPI. Neither FVIIa nor
TFPI by themselves was able to substantially alter the expression of TF
in monocytic cells, but together they caused a 3-fold reduction in TF
half-life. FVIIa is the physiologic ligand for TF, whereas TFPI is a
reversible inhibitor of the enzymatic activity of the TF·FVIIa
complex. Incubating monocytes expressing TF with a combination of FVIIa
and TFPI therefore results in the formation of a trimolecular complex
(TF·FVIIa·TFPI) on the cell surface. The studies presented here
indicate that, in addition to inhibiting the enzymatic activity of the
TF·FVIIa complex, TFPI promotes the permanent removal of cell-surface
TF in monocytic cells via internalization and subsequent degradation.
Previous studies with endothelial and smooth muscle cells have shown
that TF can localize to caveolae and that translocation of TF to
caveolae is promoted by TFPI (16). Localization of TF to caveolae or
similar Triton-insoluble membrane microdomains has not previously been
reported in monocytes. In the present study we were able to demonstrate
the existence of Triton-insoluble membrane fractions in MM6 cells,
although these cells failed to stain with antibodies to caveolin. It is
presently unclear whether or not monocytic cells have caveolae,
although our observations are consistent with previous reports that
peripheral blood monocytes do not express caveolin (57) and that THP-1
cells (a human monocyte-macrophage cell line) do not appear to have
membrane domains with the ultrastructural configuration of caveolae
(58). In any case, we found no evidence for association of TF in MM6
cells with Triton-insoluble microdomains, either in the presence or
absence of FVIIa and TFPI.
TFPI-mediated down-regulation of TF was only observed when cells were
treated with forms of FVIIa that are competent to bind to both TF and
TFPI. TFPI contains multiple Kunitz domains and can inhibit both FVIIa
and FXa simultaneously. Thus, TFPI can form trimolecular complexes with
TF (TF·FVIIa·TFPI) as well as tetramolecular complexes
(TF·FVIIa·TFPI·FXa; see Fig. 10
for a schematic diagram). We found that TFPI-mediated down-regulation of TF was independent of FXa. FX/FXa binds to multiple cell-surface proteins including protease-nexin 1 (59), EPR-1 (60), and Mac-1 (61),
and is mitogenic for some cell types (62). On hepatoma cells and
fibroblasts, FXa also directs the internalization and degradation of
the complex of FXa and TFPI by a mechanism different from that of free
TFPI (15). Since FXa was not essential for observing TF-mediated
down-regulation, and since it has potentially other confounding effects
on cells, we chose to focus most of our studies on the effect of FVIIa
and TFPI on TF expression. However, our studies clearly demonstrate
that TFPI plus FVIIa can mediate TF down-regulation in the presence of
FXa as well.
-adaptin, a component of clathrin-coated pits. Thus, in addition to
reversibly inhibiting TF/FVIIa catalytic activity, TFPI also mediates
the permanent down-regulation of cell-surface TF in monocytic cells via
LRP-dependent internalization and degradation. This
represents an unusual mechanism for receptor internalization, requiring
ligand-dependent bridging of one cell-surface receptor (TF)
to a second cell-surface receptor (LRP), the latter being capable of
clathrin-mediated internalization.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-D-glucopyranoside (octyl glucoside)
from Calbiochem; polyvinylidene difluoride-Plus membranes from Micron Separations; monoclonal anti-
-adaptin and monoclonal and polyclonal anti-caveolin from Transduction Laboratories; Cy-3-conjugated donkey
anti-mouse IgG and donkey serum from Jackson ImmunoResearch; Oregon
Green-conjugated streptavidin from Molecular Probes; horseradish peroxidase-conjugated rabbit anti-goat IgG, horseradish
peroxidase-NeutrAvidin, SuperSignal Ultra (chemiluminescence
substrate), and BCA protein assay kit from Pierce; RNAzol from
Cinna/Biotecx; Random Primed DNA Labeling Kit from Boehringer Mannheim;
and human serum albumin from the American Red Cross (Washington,
D. C.).
100 µg of protein. Proteins were precipitated by 10%
trichloroacetic acid and resuspended in SDS sample buffer. The entire
fraction or 100 µg of protein (whichever was less) was resolved by
SDS-PAGE and Western-blotted as above.
-adaptin mAb, rinsed in HBSS, 0.5% donkey serum,
and then treated with donkey anti-mouse IgG conjugated to Cy-3. After final rinsing, cells were collected onto a slide by cytospin
centrifugation and mounted in 9:1 glycerol:HBSS, pH 8.5. Dual
immunofluorescence detection of TF and
-adaptin was performed with a
Bio-Rad MRC 1024 confocal microscope (Bio-Rad Life Science Group,
Hercules, CA), equipped with a krypton/argon laser. A series of
0.2-µm optical sections in the z axis were analyzed with
Confocal Assistant software written by T. C. Berlje (University of
Minnesota). Images were pseudo-colored and merged using Adobe Photoshop
software (Adobe Systems, Inc., Mountain View, CA). Specificity of
anti-TF staining was demonstrated by adding a large excess (100 µg/ml) of recombinant soluble TF, which reduced the level of staining
to that observed in the absence of primary antibody.
-adaptin was quantified
using an image analysis protocol described elsewhere (35). Briefly, the
original confocal images were converted into binary images with Adobe
Photoshop using a thresholding tool, such that no additional pixels
were introduced into the signal after binarization. For these
experiments, the resolution was approximately 0.2 µm/pixel. The
individual binarized TF images were then multiplied with binarized
-adaptin signals at a 50% scale, so that the TF pixels remained white when they colocalized with
-adaptin pixels, but were gray when
they did not colocalize. Colocalization was calculated as the
percentage of white pixels relative to total pixels (gray plus white).
For these analyses, a total of 6-11 cells and 5609-10,883 pixels were examined.
RESULTS
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Fig. 1.
Effect of serum on TF expression by MM6
cells. MM6 cells at 6 × 105 cells/ml were
cultured in AIM-V medium supplemented with the indicated levels of calf
serum and stimulated for 4 h with 10 ng/ml LPS, after which TF
levels were quantified by ELISA. Data are mean ± S.D. for
triplicate samples from a representative experiment.
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Fig. 2.
TFPI and FVIIa decrease the expression of TF
by MM6 cells and monocytes. MM6 cells at 5.5 × 105 cells/ml (solid bars) or PBMC at 6 × 106 cells/ml (hatched bars) were stimulated with
10 ng/ml LPS and combinations of TFPI (75 nM), FXa (75 nM), and/or FVIIa (50 nM) as indicated. After
6 h, TF levels were quantified by ELISA. Data are mean ± S.E. of at least three experiments, from samples assayed in
quadruplicate. Asterisks indicate values that are
significantly different from control by Student's t test
(** = p < 0.001; * = p < 0.01).
(These experiments also compared the effects of several altered forms
of TFPI and FVIIa. For clarity of presentation and discussion, the
latter results are divided between Figs. 6-8. However, the data in
Figs. 2 and 6-8 came from the same series of experiments, so the
values for cells treated with LPS alone and for cells treated with TFPI
plus FVIIa are shared between these figures.)
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Fig. 3.
Effect of TFPI and FVIIa on the time course
of LPS-induced TF expression by MM6 cells. MM6 cells (5.5 × 105 cells/ml) were stimulated with 10 ng/ml LPS in the
presence ( ) or absence (
) of 75 nM TFPI and 50 nM FVIIa. TF levels were quantified by ELISA. Data are
mean ± S.D. for quadruplicate samples from a representative
experiment.
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Fig. 4.
TFPI and FVIIa shorten the half-life of TF in
MM6 cells. MM6 cells at 5.5 × 105 cells/ml were
stimulated for 4 h with 10 ng/ml LPS, at which time 10 µg/ml
cycloheximide was added to inhibit further TF synthesis (time = 0). The culture was split into two samples, which were either treated
( ) or not (
) with 75 nM TFPI and 50 nM
FVIIa for the indicated times. TF levels quantified by ELISA (mean ± S.D. from samples assayed in quadruplicate) are from a
representative experiment. Mean half-lives calculated from three
separate experiments were 3.7 h for untreated MM6 cells and
1.3 h for cells treated with TFPI and FVIIa.
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Fig. 5.
Lack of association of TF with
Triton-insoluble membrane fractions in MM6 cells. A, TF
content (by ELISA) of sucrose gradient fractions. MM6 cells at 1 × 106 cells/ml were stimulated for 3.5 h with 10 ng/ml LPS and then incubated for 15 min further in the absence
(solid bars) or presence (hatched bars) of 10 nM TFPI, 10 nM FVIIa, and 10 nM FXa
as per Ref. 16. At the end of the incubation, 5 × 107
cells were processed for sucrose gradient centrifugation as described
under "Experimental Procedures," and fractions were collected and
assayed for TF by ELISA. Fraction numbers are indicated on the x
axis, and the approximate positions of 5, 35, and 45% sucrose in
the step gradient are indicated above the graph.
B, light scattering profile of sucrose gradient fractions
from A (in this case, from cells not treated with TFPI,
FVIIa, or FXa). C, TF content (by Western blot) of sucrose
gradient fractions. Sucrose gradient fractions from MM6 cells not
exposed to TFPI, FVIIa, or FXa were processed for SDS-PAGE and Western
blotting as described under "Experimental Procedures" and probed
with anti-TF antibody. The lanes are aligned with the
gradient fractions in A and B. Asterisk indicates
a lane loaded with purified human TF as a positive control. The same
distribution of TF antigen was observed in MM6 cells treated with TFPI,
FVIIa, and FXa (not shown).
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Fig. 6.
FVIIa must be competent to bind to TF and
TFPI in order to down-regulate TF expression in MM6 cells and
monocytes. MM6 at 5.5 × 105 cells/ml
(solid bars) or PBMC at 6 × 106 cells/ml
(hatched bars) were stimulated with 10 ng/ml LPS and the
indicated combinations of TFPI (75 nM) and FVIIa,
FFR-FVIIa, or E2PD-FVIIa (all at 50 nM) for
6 h, after which TF levels were quantified by ELISA. Data are
mean ± S.E. of at least three experiments, from samples assayed
in quadruplicate. Asterisks indicate values that are
significantly different from control by Student's t test
(** = p < 0.001).
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Fig. 7.
The intact C-terminal region of TFPI is
required for down-regulation of TF expression in MM6 cells and
monocytes. MM6 at 5.5 × 105 cells/ml
(solid bars) or PBMC at 6 × 106 cells/ml
(hatched bars) were stimulated with 10 ng/ml LPS and the
indicated combinations of FVIIa (50 nM) and TFPI, XK1, or
TFPI13-160 (all at 75 nM) for 6 h, after
which TF levels were quantified by ELISA. Data are mean ± S.E. of
at least three experiments, from samples assayed in quadruplicate.
Asterisks indicate values that are significantly different
from control by Student's t test (** = p < 0.001).
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Fig. 8.
RAP abrogates the ability of TFPI and FVIIa
to down-regulate TF expression in MM6 cells and monocytes. MM6 at
5.5 × 105 cells/ml (solid bars) or PBMC at
6 × 106 cells/ml (hatched bars) were
stimulated with 10 ng/ml LPS and the indicated combinations of FVIIa
(50 nM) and TFPI (75 nM). Some cultures also
received protamine sulfate (10 µg/ml) or RAP (100 nM)
simultaneously with addition of TFPI and FVIIa. After 6 h, TF
levels were quantified by ELISA. Data are mean ± S.E. of at least
three experiments, from samples assayed in quadruplicate.
Asterisks indicate values that are significantly different
from control by Student's t test (** = p < 0.001).
-Adaptin--
LRP has been localized to clathrin-coated pits in
both F9 (47) and glioblastoma cells (48), so this would be a plausible pathway for internalization and degradation of TF. The protein kinase C
inhibitor, staurosporine, inhibits internalization of various
cell-surface receptors, including LRP (49). When added to cells 30 min
prior to TFPI and FVIIa, 2 µM staurosporine completely abrogated the down-regulation of TF induced by TFPI and FVIIa (data not
shown). This effect of staurosporine suggested that TFPI-mediated loss
of TF might involve internalization, possibly by directing TF to
clathrin-coated pits.
-adaptin, a
component of the adaptor protein-2 complex which associates with
clathrin-coated pits of the plasma membrane (50). MM6 cells were
stimulated with LPS for 3.5 h, after which FVIIa and either TFPI
or TFPI13-160 were added. The cells were incubated for a
further 30 min before being processed for immunofluorescence labeling,
as described under "Experimental Procedures." In control cells (not
treated with FVIIa or TFPI), TF was distributed in a granular pattern
that became more punctate when the cells were treated with a
combination of FVIIa and either TFPI or TFPI13-160 (Fig.
9, top row; green).
When the staining pattern of anti-
-adaptin (Fig. 9, middle
row; red) was merged with the pattern of anti-TF mAb
(green), significant colocalization was observed in cells treated with a combination of FVIIa and TFPI, as evidenced by the
appearance of yellow color (Fig. 9, bottom row).
In contrast, little or no yellow staining was observed in control cells
or in cells treated with a combination of FVIIa and
TFPI13-160 (Fig. 9, bottom row). This was
confirmed by quantifying the degree of colocalization of green and red
pixels in the images as described under "Experimental Procedures."
Control cells had only 3.2 ± 0.3% (mean ± S.E.)
colocalization of green pixels (TF) with red pixels (
-adaptin), and
cells treated with FVIIa and TFPI13-160 had only 9.6 ± 2.0% colocalization. In contrast, cells treated with FVIIa and TFPI
had 26.6 ± 4.4% colocalization of TF and
-adaptin signals.
This indicates that, as with TF down-regulation, the intact C terminus
of TFPI is required to promote colocalization of TF with
-adaptin, a
component of clathrin-coated pits. No fluorescent signal was observed
with monoclonal anti-caveolin staining of these cells (not shown),
consistent with the results above from Western blot analysis of MM6
cell lysates.
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Fig. 9.
TFPI and FVIIa promote colocalization of TF
with -adaptin. MM6 cells were stimulated
with 10 ng/ml LPS for 3.5 h, after which a combination of 50 nM FVIIa and either 75 nM TFPI or 75 nM TFPI13-160 were added, as indicated.
Control cells received neither FVIIa nor TFPI. After 30 min, cells were
collected and incubated with biotinylated polyclonal antibody to TF,
followed by streptavidin-Oregon Green. Cells were then fixed,
permeabilized, and incubated with mAb to
-adaptin, followed by
donkey anti-mouse IgG conjugated to Cy-3. Confocal microscopy,
employing optical sections near the basal portion of MM6 cells,
revealed staining for TF (green, top row) or
-adaptin
(red, middle row). When the images were merged (bottom
row), colocalization of staining for TF and
-adaptin
(yellow) was readily observable in cells treated with TFPI
and FVIIa but not in control cells or cells treated with a combination
of TFPI13-160 and FVIIa. The bar represents 20 µm.
DISCUSSION
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Fig. 10.
Schematic diagram of complexes formed with
LRP. A, the active site of FVIIa (in the TF·FVIIa
complex) can interact with the first Kunitz domain of TFPI
(K1) to form the TF·FVIIa·TFPI complex. This
trimolecular complex then associates with LRP via the C-terminal region
of TFPI (indicated with the letter C). B,
alternatively, TFPI can interact with the active site of FXa via its
second Kunitz domain (K2), and the resulting
TFPI·FXa complex can interact with the TF·FVIIa complex via the
first Kunitz domain of TFPI as in A. The tetramolecular
complex consisting of TF·FVIIa·TFPI·FXa then associates with LRP
via the C-terminal domain of TFPI as in A. We propose that
when either of these receptor-protease-inhibitor complexes binds to
LRP, the entire complex is translocated to clathrin-coated pits for
internalization and degradation of TF. C, multiple copies of
RAP can bind to LRP, blocking binding of all known ligands to this
receptor, including TFPI (reviewed in Ref. 46).
Down-regulation of TF by TFPI required the C-terminal domain of TFPI, since truncated forms of this protein (TFPI13-160 and XK1) were ineffective despite the fact that they are potent inhibitors of TF/FVIIa enzymatic activity. The C-terminal domain of TFPI mediates binding to both HSPGs and LRP, which were therefore candidates for mediating its effect on TF half-life. Protamine sulfate, which competes with TFPI for binding to HSPGs (13), was unable to inhibit the down-regulation of TF by TFPI. Thus, the down-regulation of TF by TFPI does not appear to be dependent upon TFPI interaction with HSPGs.
RAP is a 39-kDa protein that blocks binding of all known ligands, including TFPI, to LRP (45, 46). As LRP mediates the internalization and degradation of TFPI in hepatoma cells (13), we hypothesized that binding of TFPI to the TF·FVIIa complex could result in the loss of TF through LRP-mediated internalization and subsequent degradation. Many cell types express LRP, including peripheral blood monocytes and monocytic cell lines (45, 63, 64). MM6 cells also express LRP, as determined by flow cytometric analysis of cells stained with rabbit anti-LRP IgG (data not shown). Our studies showed that RAP abrogated the effect of TFPI on TF expression levels, consistent with an essential role for LRP in the process of TF down-regulation mediated by TFPI and FVIIa.
Following treatment of cells with a combination of TFPI and FVIIa, TF
colocalized with -adaptin, a component of clathrin-coated pits of
the plasma membrane. We propose that binding of the C-terminal domain
of TFPI to LRP directs the TF·FVIIa·TFPI complex to clathrin-coated pits, where the complex can be internalized (see Fig. 10). This represents an unusual mechanism for receptor internalization and degradation, in which one cell-surface receptor protein (TF) is bridged
via a divalent ligand to a second cell-surface receptor protein (LRP),
the latter being capable of undergoing clathrin-mediated internalization. A situation reminiscent of this exists for the LRP-mediated internalization of the complex of urokinase, plasminogen activator inhibitor type-1, and the urokinase plasminogen activator receptor (uPAR), which also employs a ligand-dependent
bridging mechanism (63). However, whereas the protease·inhibitor
complex bound to uPAR is apparently degraded within lysosomes, the
glycosylphosphatidylinositol-linked receptor (uPAR) is recycled back to
the cell surface within 2 h (65). TF, however, is apparently not
recycled. Although not investigated here, it is likely that FVIIa and
TFPI are also not recycled and that LRP-mediated internalization of TF
results in destruction of the entire receptor-protease-inhibitor
(TF·FVIIa·TFPI) complex by directing it to lysosomes.
The plasma concentration of TFPI is about 2.5 nM, which is
lower than the concentrations employed in this study. However, most of
the TFPI circulating in plasma is truncated at the C terminus, is bound
to lipoproteins, and has very low specific activity (11). In
vivo, a major pool of highly active, full-length TFPI is
apparently bound to the endothelium and is releasable by administration
of heparin (11). Moreover, there is evidence that TF and TFPI may be
expressed simultaneously by LPS-induced or adherent monocytes (54, 66).
Additionally, full-length TFPI is released by platelets upon
stimulation with thrombin (67). Therefore, local concentrations of TFPI
at sites of wounds or infection may be much higher than in plasma.
Therapeutic levels of TFPI shown to be effective in reducing mortality
in animal models of sepsis and other diseases range from 25 to 90 nM (68-70), consistent with the TFPI concentrations employed here. In vivo, TFPI regulates TF/FVIIa function by
inhibiting its enzymatic activity. However, TFPI is a reversible
protease inhibitor, and it is conceivable that TFPI and/or TFPI·FVIIa
complexes may dissociate from cell-surface TF. In this case, the newly
exposed TF molecules would become available to trigger the clotting
cascade again. In the present study, we have described an additional
mechanism for down-regulation of TF function by TFPI. Thus, TFPI (or
the TFPI·FXa complex) binds to the TF·FVIIa complex on cell
surfaces and, via interaction with LRP, causes the entire complex to be translocated to clathrin-coated pits. TF complexes are then
internalized and degraded, resulting in permanent down-regulation of
coagulant activity in monocytes and monocytic cells.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Dr. George Broze, Jr., for gifts of TFPI, TFPI13-160, and XK1 and for valuable advice and suggestions. We also thank Dr. Pierre Neuenschwander for the kind gift of E2PD-FVIIa; Gerald Sedgewick at the Biomedical Image Processing Laboratory, University of Minnesota, for assistance with confocal microscopy; Emma Bianco-Fisher for excellent technical assistance; and Kelsey Kennedy for computer support.
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FOOTNOTES |
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* This work was supported by Thrombosis SCOR Grant P50 HL54502 from the National Institutes of Health (to J. H. M. and R. P. M.), a postdoctoral fellowship from the Oklahoma Affiliate of the American Heart Association (to H. S.), and predoctoral fellowships from the University of Oklahoma M.D./Ph.D. program and the Oklahoma Medical Research Foundation (to A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, 825 NE 13th St., Oklahoma City, OK 73104. Tel.: 405-271-7892; Fax: 405-271-3137; E-mail: morrissey{at}scientist.com.
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ABBREVIATIONS |
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The abbreviations used are:
TF, tissue factor;
TFPI, tissue factor pathway inhibitor;
TFPI13-160, truncated form of TFPI;
FVIIa, factor VIIa;
FFR-FVIIa, active-site
inhibited FVIIa;
FX(a), factor X(a);
HBSS, Hank's balanced salt
solution;
HSPGs, heparan sulfate proteoglycans;
LPS, bacterial
lipopolysaccharide;
LRP, low-density lipoprotein receptor-related
protein;
MM6, Mono Mac 6 cells;
octyl glucoside, n-octyl--D-glucopyranoside;
PBMC, peripheral
blood mononuclear cells;
RAP, receptor-associated protein;
uPAR, urokinase plasminogen activator receptor;
XK1, a chimera consisting of
the first Kunitz domain of TFPI and the light chain of FX;
RPMI, Roswell Park Memorial Institute;
ELISA, enzyme-linked immunosorbent
assay;
PAGE, polyacrylamide gel electrophoresis;
mAb, monoclonal
antibody;
MES, 4-morpholineethanesulfonic acid.
2 M. M. Fiore, P. F. Neuenschwander, and J. H. Morrissey, unpublished observations.
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REFERENCES |
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