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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Tissue factor–mediated endocytosis, recycling, and degradation
of factor VIIa by a clathrin-independent mechanism not requiring
the cytoplasmic domain of tissue factor
Carsten B. Hansen, Charles Pyke, Lars C. Petersen, and L. Vijaya Mohan Rao
Endocytosis and recycling of coagulation factor VIIa (VIIa) bound to tissue
factor (TF) was investigated in baby
hamster kidney (BHK) cells stably transfected with TF or TF derivatives. Cell
surface expression of TF on BHK cells
was required for VIIa internalization and
degradation. Approximately 50% of cell
surface–bound VIIa was internalized in
one hour, and a majority of the internalized VIIa was degraded soon thereafter.
Similar rates of VIIa internalization and
degradation were obtained with BHK
cells transfected with a cytoplasmic domain-deleted TF variant or with a substitution of serine for cysteine at amino
acid residue 245 (C245S). Endocytosis
of VIIa bound to TF was an active process. Acidification of the cytosol, known
to inhibit the internalization via clathrincoated pits, did not affect the internalization of VIIa. Furthermore, receptorassociated protein, known to block
binding of all established ligands to
members of the low-density lipoprotein
receptor family, was without an effect
on the internalization of VIIa. Addition of
tissue factor pathway inhibitor/factor
Xa complex did not affect the internalization rate significantly. A substantial portion (20% to 25%) of internalized VIIa
was recycled back to the cell surface as
an intact and functional protein. Although the recycled VIIa constitutes to
only approximately 10% of available cell
surface TF/VIIa sites, it accounts for
65% of the maximal activation of factor
X by the cell surface TF/VIIa. In summary, the present data provide evidence
that TF-dependent internalization of VIIa
in kidney cells occurs through a clathrinindependent mechanism and does not
require the cytoplasmic domain of
TF. (Blood. 2001;97:1712-1720)
© 2001 by The American Society of Hematology
Introduction
Tissue factor (TF) is the cellular transmembrane receptor for factor
VIIa (VIIa). Fibroblasts, pericytes, smooth muscle cells, and
epithelial cells constitutively express TF, whereas cells in direct
contact with the blood, such as endothelial cells and monocytes,
express TF only when activated by specific pathophysiological
stimuli.1 Injury to the vessel wall or pertubation of endothelium or
monocytes, which could occur in various diseased states, results in
exposure of blood to TF, thereby leading to the formation of
TF/VIIa complexes that trigger the coagulation cascade.2 In
addition to its established role as an initiator of the coagulation
process, TF was recently shown to function as a mediator of
intracellular activities either by interactions of the TF cytoplasmic
domain with the cytoskeleton or by supporting the VIIa-protease–
dependent signaling.3,4 Such activities may be responsible, at least
in part, for the implicated role of TF in tumor development,5-7
metastasis,8-11 and angiogenesis.12-14
Cellular exposure of TF/VIIa activity is advantageous in a crisis
of vascular damage, but it may be fatal when exposure is sustained
as it is in various diseased states. Thus, regulation of TF/VIIa
activity plays a key role in vascular health conditions. Tissue factor
pathway inhibitor (TFPI) is pivotal in regulating TF/VIIa function
by inhibiting its enzymatic activity.15,16 TFPI was also shown to
down-regulate TF/VIIa activity on monocytes17 and fibroblasts18
by a mechanism whereby it induces the internalization and
degradation of the complex upon binding to TF/VIIa. Recent
studies with fibroblasts18 provide evidence for the existence of an
additional, TFPI-independent internalization of TF/VIIa. At present,
it is unknown whether TF cytoplasmic domain is required for
TF-mediated VIIa endocytosis. Although none of the known
sorting sequences for association to clathrin-coated vesicles19 are
present in the cytoplasmic tail of TF, however, it contains a cysteine
(C)245 available for palmitoylation and 3 serine residues susceptible to phosphorylation.1 Both of these posttranslational modifications of TF may result in an altered affinity for membranes and
proteins and thereby affect its subcellular location and the endocytosis pattern.
In the present investigation we examined the role of TF
cytoplasmic domain and C245 in TF-mediated endocytosis of VIIa
by using baby hamster kidney (BHK) cells transfected with wild-type
TF and TF variants. We also evaluated whether the observed VIIa
internalization results from normal membrane turnover or involves an
active internalization process. Our results show that the TF-dependent
internalization of VIIa is an active process that occurs through a
clathrin-independent mechanism and does not require the cytoplasmic
domain of TF. A substantial portion of the internalized VIIa returns to
the cell surface, and this recycled VIIa is functionally fully active.
From the Department of Tissue Factor/Factor VIIa (TF/VIIa) Research, Health
Care Discovery, Novo Nordisk A/S, Maalov, Denmark, and the Department of
Biochemistry, the University of Texas Health Center at Tyler, TX.
Reprints: Lars C. Petersen, Department of TF/ VIIa Research, Novo Nordisk
A/S, Novo Nordisk Park, 2760 Maalov, Denmark; e-mail: [email protected].
Supported in part by grant HL 58869 (L.V.M.R.) from the National Heart, Lung,
and Blood Institute, National Institutes of Health, Bethesda, MD, and a grant
(C.B.H.) from the Danish Academy of Technical Sciences (EF639),
Copenhagen, Denmark.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Submitted May 3, 2000; accepted November 13, 2000.
© 2001 by The American Society of Hematology
1712
BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
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BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
Materials and methods
Reagents
We used the following materials: Dulbecco modified Eagle medium
(DMEM), fetal bovine serum (FBS), trypsin-EDTA (ethylenediamine
tetraacetic acid), and penicillin-streptomycin (all from Gibco-BRL Life
Technologies, Paisley, Scotland); tissue culture flasks and plates (Nunc A/S,
Roskilde, Denmark); other chemicals, reagent grade or better, (Sigma
Chemical, St Louis, MO); monospecific polyclonal sheep antihuman VII
antibodies (Affinity Biologicals, Hamilton, ON, Canada); human factor X
(X) and Xa (Enzyme Research Laboratory, South Bend, IN); and receptorassociated protein (RAP) (gift from Frederik Vilhardt, University of
Copenhagen, Denmark). Recombinant human VIIa,20 VIIa blocked in the
active site with phenylalanyl-phenylalanyl-arginyl chloromethyl ketone
(FFR-VIIa),21 recombinant sTF,22 monospecific polyclonal rabbit antihuman TF immunoglobulin G (IgG),23 and recombinant full-length TFPI24
were prepared as previously described.
Cell culture
The BHK cell line BHK-21 tk-ts13 (CRL 1632; American Type Culture
Collection, Bethesda, MD) was maintained in DMEM (4.5 g/L glucose)
with GlutaMAX 1 supplemented with 10% FCS, 10 IU/mL penicillin, and
100 ␮g/mL streptomycin. Generation of BHK cells that were stably
transfected with full-length TF (BHK(TF)), a cytoplasmic domain–deleted
construct of TF (BHK(TF⌬cyto)), or a substitution of serine for cysteine at
amino acid residue 245 (C245S) construct of TF (BHK(TFC245S)) was
previously described.25
Radiolabeling of proteins
VIIa, FFR-VIIa, and ricin were labeled with iodine-125 (125I) using
IODO-GEN–coated (Pierce, Rockford, IL) tubes and sodium (Na)125I
according to the manufacturer’s technical bulletin and as described
previously.26 Briefly, the labeling reaction was performed in tubes coated
with 10 ␮g IODO-GEN for 4 minutes on ice. The reaction was quenched by
the addition of 1% KI, and free iodine was removed by extensive dialysis
against 10 mM HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic
acid) (pH 7.5) and 150 mM sodium chloride (NaCl). The concentration of
the labeled proteins was determined by measurements of the absorbance at
280 nm. 125I-VIIa retained about 90% to 100% of the functional activity of
unlabeled VIIa.
Internalization of 125I-VIIa
Confluent monolayers of parental BHK cells, BHK cells transfected with
wild-type TF and TF variants, BHK(TF), BHK(TF⌬cyto), and BHK(TFC245S)
were incubated with 10 nM 125I-VIIa for varying times (5 minutes to 4
hours) at 37°C in buffer B, which comprised 10 mM HEPES, 150 mM
NaCl, 4 mM potassium chloride (KCl), and 11 mM glucose (pH 7.5) buffer
containing 5 mM CaCl2 and 1 mg/mL bovine serum albumin. In some
experiments 100 nM RAP, 1 U/mL heparin, or 10 nM of a preformed
TFPI/Xa complex was added prior to the 125I-VIIa addition. At the end of
the incubation period, the supernatant was removed, and the monolayers
were washed 4 times with ice-cold buffer B. The cell surface–associated
125I-VIIa was subsequently eluted after incubation of the cells with a
low-pH (pH 2.5) glycine buffer for 5 minutes at room temperature. The
radioactivity present in the supernatant was considered as VIIa associated
with the cell surface. This assumption has been validated because the
glycine treatment was shown to elute 90% or more of the VIIa associated
with the cells when they were incubated with 125I-VIIa at 4°C for 2 hours.
After removing the glycine eluate, the cells were detached by trypsin/
EDTA solution, and the cell suspension was collected. The radioactivity
present in the cell suspension was measured as internalized VIIa. Identical
results were obtained in some experiments when internalized VIIa was
determined as the amount of radioactivity released to the supernatant after
exposure of the cells (which had been washed with low-pH buffer) to 2 N
INTERNALIZATION AND RECYCLING OF VIIA
1713
NaOH. Nonspecific binding and internalization were determined in parallel
experiments where TF was blocked by preincubating the cells for 1 hour
with 200 ␮g/mL rabbit antihuman TF IgG before adding 125I-VIIa.
TF-specific binding and internalization were obtained by subtracting the
nonspecific binding and internalization from the total binding and internalization. Nonspecific binding and internalization accounted for less than
10% of the total binding and internalization. The rate of internalization was
calculated as d(internalized/surface associated)/dt according to the method
of Wiley and Cunningham.27 The degradation of VIIa was followed by
withdrawal of 20-␮L aliquots from the medium overlying the cells at
selected time intervals and mixing with 200 ␮L 10% vol/vol trichloroacetic
acid (TCA). After centrifugation, degraded VIIa was determined as the
TCA nonprecipitable (TCA-soluble) radioactivity.
Detection of internalized VIIa by immunofluorescence
BHK(TF) cells were cultured in chamber slides (Nunc). Chambers were
incubated with 10 nM VIIa in serum-free medium for 1 hour at 37°C or 4°C.
Control chambers were incubated with cell medium alone. In some
experiments the cells were then exposed to low-pH glycine buffer (pH 2.5)
for 5 minutes to elute surface-associated VIIa. Cells were fixed in 2%
paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes and
then briefly washed in PBS. For immunostaining, slides were preincubated
for 15 minutes in 5% goat serum in tris[hydroxymethyl] aminomethane
(Tris)–buffered saline (TBS) with 0.2% saponin (TBS-S). This and all
subsequent steps were carried out at room temperature.
Primary antibodies (polyclonal rabbit antihuman TF IgG or polyclonal
sheep antihuman VII IgG) were then added in TBS-S at 10 ␮g/mL for 90
minutes, and visualization was performed with either biotinylated swineantirabbit (Dako A/S, Glostrup, Denmark) followed by HRP-streptavidin
and TSA-direct-FITC (trichostatin A–direct–fluorescein isothiocyanate)
(NEN) or with HRP-rabbit-antisheep (Dako) followed by TSA-directFITC. For double-labeling immunolocalization of TF and VIIa, the cells
were first immunostained with anti-TF as described above. The same
samples were then immunostained for VIIa by blocking first for 15 minutes
with TBS-S with 5% rabbit and 5% swine serum followed by incubation in
sheep–anti-VII for 30 minutes, then in biotinylated donkey-antisheep
(Sigma) followed by HRP-streptavidin and TSA-direct-Rhodamine.
Determination of bulk membrane turnover
We added 10 nM 125I-VIIa or 200 ng/mL 125I-ricin to monolayers of
BHK(TF) cells at 37°C. At the end of 5 and 30 minutes of incubation, the
cell surface–associated and internalized ligands were determined. Cell
surface–associated 125I-VIIa was released by incubating the cells with
low-pH glycine buffer for 5 minutes, whereas cell surface–associated
125I-ricin was released by incubating the cells with 0.1 M lactose at 4°C for
1 hour. (Control experiments performed at 4°C showed that these procedures removed cell surface–associated VIIa and ricin with a similar
efficiency, 94.2% and 94.8%, respectively). The data were obtained as the
mean ⫾ SD percent-internalized radioactivity per total cell surface–bound
ligand from 3 independent experiments in triplicate.
Effect of cytosol acidification on VIIa internalization
Confluent monolayers of BHK(TF) cells were pretreated with DMEM (pH
5.5) with and without 10 mM acetic acid for 5 minutes at room temperature.
The cells were then incubated with 10 nM 125I-VIIa for 1 hour, and the cell
surface–associated and internalized VIIa were determined as described
above. Data are calculated as the percentage of internalization in acetic
acid–treated cells relative to nontreated control cells. Internalization of 200
125
ng/mL I-transferrin is shown for comparison. Data are the mean ⫾ SD of
3 independent experiments in triplicate.
Recycling of internalized VIIa and FFR-VIIa
Confluent monolayers of BHK(TF) cells were incubated with 10 nM
for 1 hour at 37°C to allow internalization. The cell surface–
associated radioactivity was then removed by a low-pH glycine wash as
described above. The monolayers were washed 3 times with buffer B to
125I-VIIa
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1714
HANSEN et al
remove residual low-pH glycine, and the cells were subsequently allowed
to stand in buffer B for various times at 37°C for recycling of 125I-VIIa.
Overlying buffer containing the recycled VIIa that was dissociated from the
surface, as intact or degraded, was removed and pooled with radioactive
protein eluted from the cell surface by glycine buffer (pH 2.5). The pooled
samples, comprising the total amount of recycled 125I-VIIa, were then
analyzed for the presence of intact and degraded 125I-VIIa, which were
determined after 10% TCA treatment as the precipitable and nonprecipitable radioactivity, respectively. Following the removal of recycled and
degraded 125I-VIIa, the cells were removed from the dish by trypsin
digestion and counted for radioactivity to determine the remaining internalized 125I-VIIa. A similar procedure was used for recycling experiments with
125I-FFR-VIIa.
Recycled 125I-VIIa in aliquots from the pooled samples was tested for
functional activity after adjusting the pH to 7.4 by adding 100 ␮L pooled
sample to 250 ␮L 50 mM Tris and 150 mM NaCl (pH 8.0). The
Xa-generating activity was measured in a TF/VIIa assay with excess
relipidated TF. Recycled functionally active VIIa was quantified by
comparison, with the activity of a VIIa standard determined under identical
conditions.
Recycling of functionally active VIIa on the cell surface was determined in separate experiments. Following internalization for 1 hour in the
presence of 10 nM VIIa, the cell surface–associated VIIa was removed by
exposure of the cells to a low-pH glycine buffer. Immediately the cells were
washed 3 times with buffer B and then allowed to stand in buffer B. After
varying times, 100 nM factor X (X) was added to the monolayers, and
recycled TF/VIIa complexes were allowed to activate X for 5 minutes. Xa
generation was determined in a chromogenic assay by adding 50 ␮L of the
above mixture to 50 ␮L 50 mM Tris, 150 mM NaCl, 5 mM EDTA, and 1
mg/mL BSA (pH 7.5). The Xa activity was measured in the presence of 1.07
mM Chromozym X. The absorbance at 405 nm was measured continuously
in a microplate reader (Molecular Devices), and initial rates were converted
to Xa concentrations using a Xa standard curve (85 pmol/L to 11 nmol/L).
BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
only a negligible amount of 125I-VIIa was bound and processed by
untransfected BHK cells (Figure 1). Association to the surface of
BHK(TF) cells was half-maximal after approximately 20 minutes
of exposure to 125I-VIIa. Fifty percent of the cell surface–associated
125I-VIIa, which corresponds to approximately 4000 fmol/106 cells,
was internalized within an hour following the treatment. This
corresponds to an internalization constant (k1 ⫽ 0.076 minutes⫺1).
When 10 nM VIIa was offered to the cells, the maximal degradation rate achieved in this TF-dependent VIIa endocytosis was 2650
fmol/106 cells per hour. The amount of VIIa internalized correlated
well with the amount of VIIa offered, up to about 30 nM 125I-VIIa, a
concentration that could saturate TF sites on the cell surface (data
not shown).
Addition of 125I-VIIa in complex with soluble recombinant
TF1-219 to untransfected BHK cells did not change the rate of VIIa
binding, internalization, or degradation (data not shown). These
data document that membrane attachment of TF is needed for the
binding and internalization of VIIa. The kinetics of cell surface
association, internalization, and degradation of FFR-VIIa were
indistinguishable from that of VIIa (data not shown).
Internalization and recycling of cell surface TF
Confluent BHK(TF) cells were incubated with 10 nM VIIa or FFR-VIIa at
37°C for various time intervals (15 minutes to 4 hours) to study the effect of
TF/VIIa internalization on the exposure of the TF receptor on the cell
surface. The cell surface–associated VIIa was removed at the end of the
incubation period by exposure of the BHK(TF) cells to a 5-minute low-pH
glycine buffer wash and 4 subsequent washes with ice-cold buffer B. The
cell surface TF expression at that time was then determined by the 125I-VIIa
binding capacity and the ability to support X activation in the presence of
newly added VIIa. To determine 125I-VIIa binding, the monolayers were
incubated with 125I-VIIa for 90 minutes at 4°C. Then the unbound ligand
was removed, and the cells were washed 4 times with buffer B. The cell
surface–associated 125I-VIIa was obtained after detaching the cells with
trypsin/EDTA and counting the suspension in a gamma counter. To
determine TF levels using X activation assay, 10 nM VIIa and 175 nM X in
buffer B were added to the monolayers. TF functional activity was
measured by the amount of Xa generated at the end of a 15-minute
activation period at 37°C. For this purpose, 25-␮L aliquots were removed
from the well and added to 50 mM Tris, 150 mM NaCl, 5 mM EDTA, and 1
mg/mL BSA for a total of 75 ␮L (pH 7.5). The Xa activity was measured in
the presence of 1.07 mM Chromozym X.
Results
TF-dependent internalization and degradation of VIIa
BHK cells were exposed to 10 nM 125I-VIIa in the presence and
absence of anti-TF IgG, and the amount of TF-specific cell
surface–associated, cell surface–internalized, and cell surface–
degraded VIIa was followed with time. A time-dependent binding
of 125I-VIIa to the cell surface TF followed by internalization and
degradation was observed with BHK(TF) cells. In comparison,
Figure 1. TF-specific binding, internalization, and degradation of VIIa in BHK
cells. 125I-VIIa (10 nM) was added to confluent monolayers of BHK cells (䡺) or BHK
cells transfected with full-length TF (BHK(TF)) (f) that were preincubated for 60
minutes with control buffer or 200 ␮g/mL rabbit antihuman TF IgG at room
temperature. Cell surface association (A), internalization (B), and degradation (C)
were determined at various time points as described in “Materials and methods.”
TF-specific binding, internalization, and degradation were determined by subtracting
the values obtained in the presence of antihuman TF IgG from the values obtained in
its absence. Nonspecific binding of 125I-VIIa to BHK(TF) cells in the presence of
antihuman TF IgG was 10% to 15% of the total binding. A similar amount of 125I-VIIa
was bound to parental untransfected BHK cells in both the absence and presence of
antihuman TF IgG. Data are the mean ⫾ SD of 3 independent experiments in
triplicate.
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BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
INTERNALIZATION AND RECYCLING OF VIIA
1715
Internalization of VIIa demonstrated by
immunofluorescence microscopy
BHK(TF) cells treated with 10 nM VIIa for 1 hour at 37 °C were
analyzed by immunofluorescence microscopy (Figure 2). TF and
VIIa were both immunostained as a diffuse membrane staining and
as bright spots in vesicular perinuclear compartments (Figure
2A,B). Double-labeling experiments revealed that TF and VIIa
were colocalized in most cells (Figure 2C). As expected, cells
incubated with medium alone were negative for VIIa staining
(Figure 3D). Staining was localized primarily to the perinuclear
compartments when the cells were incubated with VIIa at 37°C for
1 hour and subsequently treated with a low-pH glycine wash
(Figure 3B), indicating that cell surface–associated VIIa, but not
internalized VIIa, was removed by this treatment. As shown in
Figure 3C, perinuclear staining, as well as cell surface–associated
staining, was absent when the cells were exposed to VIIa at 4°C to
prevent internalization and subsequently treated with a glycine
Figure 3. Immunofluorescence staining for VIIa or TF. BHK(TF) cells treated with
10 nM VIIa (A-C, G) or buffer (D-F) were immunofluorescence stained with VII
antibodies (A-E) or TF antibodies (F, G). (A) Cells were treated with 10 nM VIIa for 1
hour at 37°C, and the unbound VIIa was removed before fixing the cells. (B) Cells
were treated with 10 nM VIIa for 1 hour at 37°C, the unbound VIIa was removed, and
the cell surface–associated VIIa was eluted with low-pH buffer to illustrate the cell
surface–internalized VIIa. (C) Cells were treated with 10 nM VIIa for 1 hour at 4°C, the
unbound VIIa was removed, and the cell surface–associated VIIa was eluted with
low-pH buffer. (D, E) Cells were treated the same as in panels A and B, respectively,
except that VIIa was not included in the incubation medium. (F, G) Immunostaining of
TF after 1-hour exposure of BHK(TF) cells at 37°C to the control medium and 10 nM
VIIa, respectively.
wash to remove cell surface–associated VIIa. This finding was
consistent with the observation that more than 90% 125I-VIIa
associated to the cell surface of BHK(TF) cells at 4°C could be
displaced by the glycine wash. Immunostaining for TF revealed no
significant difference in staining pattern between cells treated with
buffer (Figure 3F) and those treated with VIIa (Figure 3G). One
should note that very bright staining of intracellular TF masked the
fluorescence of cell surface–stained TF (Figure 3F), thus giving the
false impression that the cells apparently lack TF on their
cell surface.
Figure 2. Double-immunofluorescence staining for TF and VIIa. BHK(TF) cells
were incubated with 10 nM VIIa for 1 hour at 37°C and then stained for VIIa (A) and TF
(B) as described in “Materials and methods.” (C) Pictures of panels A and B have
been merged to highlight areas with colocalization (yellow). Note that most of the cells
show a high degree of colocalization of TF and VIIa. In a subset of these,
colocalization in perinuclear vesicles is quite evident.
Determination of bulk membrane turnover
To determine whether the internalization of VIIa was an active
process or represented a bulk membrane turnover, internalization
of VIIa was compared to the internalization of ricin. Ricin binds to
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1716
BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
HANSEN et al
terminal galactose residues on membrane glycoproteins and glycolipids and is internalized by any structure that pinches off from the
membrane. Thus ricin internalization has been used as a general
marker for bulk membrane turnover.28,29 The initial VIIa internalization rate (32% ⫾ 5% within the first 5 minutes) was approximately
2-fold higher than that of ricin (17% ⫾ 7%). The finding that
endocytosis of VIIa is significantly faster than that of ricin might
indicate that internalization of VIIa is facilitated and does not just
represent bulk membrane turnover.
VIIa is not internalized via LRP in a TFPI-dependent mechanism
Previous studies revealed an endocytosis mechanism in which VIIa
was internalized by LRP in a TFPI-dependent process involving
localization of the TF/TFPI/VIIa complex to clathrin-coated pits.17
To test whether this process was also operative for VIIa endocytosis by BHK(TF) cells, we first studied the effect of cytosolic
acidification. This treatment impedes clathrin-dependent endocytosis, and as expected, pre-exposure of BHK(TF) cells to 10 mM
acetic acid impeded the internalization of transferrin, which is
known to proceed via this pathway. Cytosolic acidification decreased transferrin internalization by 50% in contrast to VIIa
internalization, which was unaffected by this treatment. This
observation suggests that VIIa endocytosis by BHK(TF) cells
proceeds via a clathrin-independent mechanism. In further experiments to rule out the involvement of LRP and TFPI in VIIa
internalization, we studied the effect of RAP and heparin on VIIa
internalization. RAP is known to block the binding of all established ligands to LRP receptors,30 and heparin is expected to
displace TFPI from cell surface heparan sulfate proteoglycans31
and thereby impede TFPI-dependent LRP internalization of the
TF/VIIa complex. Neither RAP nor heparin affected the VIIa
endocytosis (Table 1). In further experiments, TFPI/Xa was
exogenously added to the cells to test the effect of TFPI/Xa on VIIa
cell surface association and internalization. As expected, 10 nM
TFPI/Xa markedly inhibited the cell surface TF/VIIa activity (data
not shown). However, as shown in Table 1, TFPI/Xa had no effect
on VIIa binding and internalization.
TF cytoplasmic domain or its potential acylation at C245 is not
required for TF-dependent endocytosis of VIIa
BHK cell lines were stably transfected with a cytoplasmic-deleted
construct of TF, des(248-263)TF, and with a C245S substitution
construct of TF. The resulting cell lines, BHK(TF⌬cyto) and
BHK(TFC245S), were used to elucidate the role of the cytoplasmic
domain in TF-dependent internalization of VIIa. As previously
described by Sorensen et al,25 the Xa generation activity of the
Table 1. Effect of receptor-associated protein, heparin, and tissue factor
pathway inhibitor/factor Xa on tissue factor–specific binding and
internalization of coagulation factor VIIa
RAP
Cell surface–associated
VIIa, % of control
Internalized
VIIa, % of control
104 ⫾ 5
102 ⫾ 2
Heparin
96 ⫾ 5
106 ⫾ 12
TFPI/Xa
112 ⫾ 8
105 ⫾ 7
Confluent monolayers of BHK(TF) cells were incubated with 10 nM 125I-VIIa for 1
hour in the absence or presence of 100 nM RAP, 1 U/mL heparin, or 10 nM TFPI/Xa.
Complexes of TFPI/Xa were prepared by incubating equimolar amounts of recombinant full-length TFPI and Xa for 30 minutes at 37°C. Data obtained with the cells in the
absence of TFPI/Xa, heparin, or RAP were set to 100%. Data are the mean ⫾ SD of 3
independent experiments in triplicate.
VIIa indicates coagulation factor VIIa; RAP, receptor-associated protein; TFPI/
Xa, tissue factor pathway inhibitor/factor Xa; BHK(TF), baby hamster kidney stably
transfected with full-length tissue factor.
Figure 4. TF-specific binding, internalization, and degradation of VIIa in
BHK(TF), BHK(TF⌬cyto), and BHK(TFC245S) cells. Experiments were performed as
described in Figure 1 with BHK(TF) (■), BHK(TF⌬cyto) (Œ), and BHK(TFC245S) (F). The
data represent the cell surface–associated VIIa (A), internalized VIIa (B), and
degraded VIIa (C). Data are the mean ⫾ SD of 3 independent experiments in
triplicate.
BHK(TF⌬cyto) and BHK(TFC245S) cell lines was comparable to that
of the BHK cell line transfected with full-length TF, BHK(TF),
whereas the VIIa binding capacity of the BHK(TFC245S) cell line
was approximately 50% of that observed with the 2 other TFexpressing cell lines.
Figure 4 shows VIIa cell surface association, internalization,
and degradation observed with BHK(TF), BHK(TF⌬cyto), and
BHK(TFC245S). As expected, less VIIa was bound to the cell surface
of BHK(TFC245S) cells. Still, the rate of the subsequent internalization and degradation reactions observed with the BHK(TF⌬cyto) and
BHK(TFC245S) cell lines was similar to those of BHK(TF) cells.
This suggested that VIIa endocytosis takes place independently of
the cytoplasmic domain and also independently of possible acylation of C245.
In contrast to association and internalization of VIIa, which
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BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
INTERNALIZATION AND RECYCLING OF VIIA
1717
were not affected by addition of TFPI/Xa, TFPI/Xa induced a small
but consistent increase in VIIa-degradation in BHK(TF) cells
(Figure 5). A slightly enhanced VIIa degradation was also evident
with BHK(TF⌬cyto) and BHK(TFC245S) cell lines, indicating that the
TF cytoplasmic domain and its potential modifications played no
role in the TFPI/Xa-dependent enhancement of VIIa degradation.
Recycling of internalized TF/ VIIa
Next we examined whether the internalized VIIa or FFR-VIIa
could be recycled back to the cell surface. Monolayers of BHK(TF)
cells were allowed to internalize 125I-VIIa for 1 hour. The cell
surface–associated radioactivity was completely removed by treating the cells with low-pH glycine, and the cells were allowed to
stand at 37°C for recycling of VIIa. The amount of intracellular
VIIa and the total amount of excreted, intact, or degraded VIIa were
then determined at various time points as described in “Material
and methods.” As shown in Figure 6A, a significant portion (about
20%) of the internalized VIIa pool was excreted as intact (TCAprecipitable) VIIa. Experiments run in parallel suggested that the
recycled TCA-precipitable VIIa was functionally fully active.
Thus, the pooled samples from the overlaying medium and the
low-pH glycine eluate contained an amount of VIIa that, when
quantified by a Xa generation assay with lipidated TF, corre-
Figure 5. Effect of TFPI/Xa on TF-specific VIIa degradation in BHK(TF),
BHK(TF⌬cyto), and BHK(TFC245S) cells. Confluent monolayers of BHK(TF) (A),
BHK(TF⌬cyto) (B), or BHK(TFC245S) (C) cells were incubated with 10 nM 125I-VIIa for 1
hour in the absence (f) or presence (䡺) of 10 nM TFPI/Xa, and the degradation of
VIIa was determined as described in “Materials and methods.” Data are the mean ⫾
SD of 3 independent experiments in triplicate.
Figure 6. Recycling of internalized VIIa. Confluent monolayers of BHK(TF) cells
were allowed to internalize 10 nM 125I-VIIa or 125I-FFR-VIIa for 1 hour. Thereafter, the
cell surface–associated radioactivity was removed by a low-pH glycine wash. After
the glycine wash, the monolayers were washed 3 times with buffer B and then
incubated with buffer B for various times at 37°C for recycling of (A) 125I-VIIa or (B)
125I-FFR-VIIa. Total radioactivity in the internalized pool measured after the first
glycine wash was set to 100%. Subsequent measurement of radioactivity at various
time points of the residual internalized pool (f), recycled intact protein (), and
recycled degraded protein (Œ) was performed as described in “Materials and
methods.” Data are the mean ⫾ SD of 3 independent experiments in triplicate.
sponded well with the amount of TCA-precipitable VIIa (data not
shown). Recycling of TCA-precipitable protein was even more
pronounced with 125I-FFR-VIIa, accounting for 25% to 30% of the
internalized FFR-VIIa (Figure 6B).
Next, we studied the appearance on the cell surface of functionally active VIIa from the internalized pool by measuring the ability
of monolayers to support X activation. In these experiments,
BHK(TF) cells were treated with 10 nM VIIa for 1 hour at 37°C,
cell surface–associated VIIa was removed by glycine wash, and the
appearance of recycled VIIa was monitored directly by the ability
of monolayers to support X activation in the absence of exogenously added VIIa. The Xa-generating activity was compared to
that of monolayers without VIIa preincubation, which exhibited a
negligible activity, and to that of monolayers with 10 nM VIIa
exogenously added, which was set to 100% activity. As shown in
Figure 7, the recycling of VIIa appears to be a rapid process
because functionally active VIIa reappeared on the cell surface as
soon as the cell surface–associated VIIa was removed. It is
interesting to note that the recycled VIIa was capable of generating
as much as 60% to 65% of maximal Xa generation capacity.
Similar experiments were performed with BHK(TF⌬cyto) and
BHK(TFC245S) cell lines, where VIIa recycling was evaluated by
the ability of recycled VIIa on the cell surface to support X
activation. Data from these experiments revealed a recycling
pattern similar to that described in Figure 7 for BHK(TF) cells
(data not shown), suggesting that the cytoplasmic domain of TF
was not obligatory for recycling of VIIa.
The above experiments demonstrated that VIIa was internalized
and recycled to the cell surface. However, the fate of TF in this
process was not evident. Therefore we next examined the amount
of TF exposed to the surface during TF-dependent internalization
of VIIa. Monolayers of BHK(TF) cells were allowed to internalize
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1718
HANSEN et al
Figure 7. Effect of VIIa recycling on X activation. BHK(TF) cells were treated with
10 nM VIIa at 37°C for 1 hour. Cell surface–associated VIIa was then removed by
glycin wash, and the monolayers were washed 3 times with buffer B. Measurement of
subsequent recycling of TF/VIIa activity at different time points was performed by
incubating the cells with 100 nM X for 5 minutes and measuring the amount of Xa
generated (⽧). The activity of cells not preincubated with VIIa and not subjected to
glycine wash (Œ, f) and the activity of cells not preincubated with VIIa but washed
with glycine (‚, 䡺) were measured for comparison at 10 and 240 minutes in the
presence of 100 nM X (Œ, ‚) or in the presence of 100 nM X and 10 nM exogenous
VIIa (f, 䡺).
BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
VIIa internalization and degradation in the kidney cell (or BHK
cell) is strictly dependent upon its binding to cell surface TF.
Cytosolic acidification experiments suggested that VIIa was catabolized by clathrin-independent endocytosis. Further experiments
showed that neither RAP nor heparin affected the TF-mediated
endocytosis of VIIa. This excludes the possible involvement of
LRP and endogenous TFPI in VIIa endocytosis and provides
further support for clathrin-independent endocytosis of VIIa.
Addition of the TFPI/Xa complex resulted in only a slight increase
(approximately 25%) in VIIa degradation.
These data differ from the earlier published data in monocytes17
and fibroblasts,18 which showed a more pronounced effect of TFPI
on TF and VIIa degradation. A possible reason could be that
compared to TF, BHK cells may have only a limited number of
LRP and/or cell surface heparan sulfate proteoglycans which are
needed for internalization of TF/ TFPI/ VIIa/ Xa complexes via
clathrin-dependent mechanism.17,18 In this context it should be
noted that although kidney cells mainly express megalin (another
member of the low-density lipoprotein (LDL) receptor family) and
not LRP, it has been reported that some kidney cells also express
LRP.33 Further, because RAP inhibits the endocytosis mediated by
both LRP and megalin,34 the failure of RAP to inhibit endocytosis
of VIIa in the present study suggests that members of the LDL
receptor family are not involved in TF-mediated endocytosis of
VIIa in kidney cells.
VIIa or FFR-VIIa for varying time intervals from 15 minutes to 4
hour before the cell surface–associated VIIa or FFR-VIIa was
removed by treating the cells with low-pH glycine. Thereafter, the
amount of cell surface–exposed TF was measured either by VIIa
binding or by TF/VIIa functional activity. As shown in Figure 8A,
the total amount of VIIa binding sites on BHK monolayers was
unchanged during internalization of VIIa or FFR-VIIa. In contrast,
however, we observed a transient down-regulation of the amount
of functionally active TF sites during the internalization cycle
(Figure 8B).
Discussion
The binding of VIIa to a number of cells in TF-dependent and
TF-independent reactions has been studied in great detail.1,2 In
comparison, little is known about the fate of cell surface–associated
VIIa and the possible catabolism of cell surface TF/VIIa complexes. Recent studies have described 3 different mechanisms for
VIIa and TF/VIIa internalization. Chang and Kisiel32 described a
TF-independent internalization and degradation of VIIa in human
hepatoma cells. Hamik et al17 found that TF was down-regulated on
monocytes in a reaction involving VIIa, TF, TFPI, and LRP.
Iakhiaev et al18 have provided suggestive evidence that VIIa
complexed with TF on fibroblasts was endocytosed by 2 different
pathways: LRP-independent pathway in the absence of TFPI/Xa
and LRP-dependent pathway in the presence of TFPI/Xa. None of
the above studies addressed the role of cytoplasmic domain of TF
in TF-mediated VIIa endocytosis. In the present study we investigated VIIa-endocytosis in further detail using BHK cells transfected with TF and TF variants.
The data presented in the current study show a significant
internalization and degradation of VIIa in BHK(TF) cells, whereas
there was no detectable internalization and degradation of VIIa
observed in wild-type BHK cells. These data clearly document that
Figure 8. Internalization and recycling of TF. Monolayers of BHK(TF) cells were
allowed to internalize VIIa (f) or FFR-VIIa (Œ) for various times, from 15 minutes to 4
hours. The cell surface–associated VIIa or FFR-VIIa was subsequently removed by
low-pH buffer wash, and the exposure of TF on the cell surface was measured by
either 125I-VIIa binding (A) or the cell surface–associated TF/VIIa functional activity
(B) as described in “Materials and methods.” Data are the mean ⫾ SD of 3
independent experiments in triplicate.
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BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
INTERNALIZATION AND RECYCLING OF VIIA
Although the earlier studies17,18 described above suggest that
TF/VIIa is actively internalized, these studies have not eliminated
the possibility that the observed internalization of TF and VIIa is a
result of and follows normal membrane turnover. The data presented herein provide evidence that TF-mediated endocytosis of
VIIa is an active receptor-mediated process and not the result of a
general turnover of bulk membrane components. The endocytosis
rate of k1 ⫽ 0.076 minutes⫺1, which corresponds to approximately
50% of cell surface–associated VIIa internalized in 20 minutes, is
substantially higher compared to the turnover rate of bulk membrane components, which in general corresponds to 15% to 25%
internalization per hour.35-38 A comparison of internalization of
VIIa and ricin, a marker for membrane turnover,30 provides further
evidence that the rate of VIIa internalization was much higher
compared to the rate of general membrane turnover.
Internalization of receptors for endocytosis via clathrin-coated
pits is coded by specific amino acid sequences in the cytoplasmic
domain.39 Such sequences are not present in the TF cytoplasmic
domain. The sorting sequences for clathrin-independent internalization are not known. Because the cytoplasmic domain of TF
comprises a cysteine and a few serines susceptible to posttranslational modifications, it is possible that these potential modifications
may affect the affinity of transmembrane receptors for membranes,
subcellular location, and interaction with other proteins, which in
turn could affect the internalization and degradation of receptorassociated ligands. However, the data of the present studies show
that the cytoplasmic domain of TF does not play a role in the
TF-dependent endocytosis of VIIa. In this respect, endocytosis of
TF/VIIa complexes is similar to the endocytosis of thrombin/
thrombomodulin (TM) complexes. Internalization of thrombin/TM
complexes was reported to occur predominantly through a clathrinindependent mechanism, and the deletion of the cytoplasmic
domain has no effect on the constitutive internalization of TM as
well as thrombin/TM complexes.40-42
The data on VIIa recycling with BHK(TF) cells (Figure 6)
confirm and extend recent observations in fibroblasts.18 Our data
show that a proportion (approximately 20%) of internalized VIIa
recycles to the cell surface as an intact protein which is fully
capable of activating factor X. It is interesting to note that although
VIIa occupies only about 10% of available TF sites on the cell
surface, recycled VIIa confers the cell with approximately 60% to
70% of its total X-activating capacity (Figure 7). This agrees well
with previous reports showing that the maximal procoagulant
activity of TF-expressing cells is reached at a much lower VIIa
concentration than needed for saturation of an additional pool of
coagulant inactive or “cryptic” TF sites available for binding of
VIIa on the surface.26,43 Although direct evidence for such a
mechanism is missing, these data are consistent with the hypothesis
that VIIa is internalized primarily from the pool of functionally
active TF sites and then recycled back to the same pool representing a preferred residence for VIIa. This model may also explain
1719
other observations, such as a transient decrease in functionally
active TF (Figure 8) and similar internalization rates in
BHK(TFC245S) and BHK(TF⌬cyto) cells (Figures 4 and 5), which
differ substantially in VIIa binding capacity but are equally active
in their capacity to activate X. It is clear, however, that the above
model cannot in itself account for the total endocytotic process of
VIIa including the extent of VIIa internalized and subsequently
degraded. It is possible that uptake and rapid cycling of VIIa are 2
distinct events, where VIIa bound to an active pool of TF follows
rapid recycling of TF without going to a degradation pathway, and
VIIa bound to nonfunctional TF is internalized and directed to a
degradation pathway.
The present data, along with recent reports,17,18 suggest that the
fate of cell surface TF/VIIa complexes might differ with different
cell types. VIIa bound to TF on kidney cells (as suggested by the
present data) is, for example, internalized predominantly through a
clathrin-independent mechanism, and the formation of the inhibitor
complex on the cell surface with TFPI/Xa has no significant effect
on the internalization of VIIa. In contrast, formation of the
inhibited complex (with either TFPI or TFPI/Xa) appears to be a
prerequisite for the internalization of TF in monocytes, and the
internalization and degradation of TF proceeds via a LRP/clathrindependent mechanism.17 Both the above mechanisms are apparently operative in fibroblasts, ie, a substantial amount of VIIa is
internalized in the absence of TFPI/Xa via a LRP/clathrinindependent mechanism, whereas the presence of TFPI/Xa induces
an additional internalization of VIIa by a LRP/clathrin-dependent
pathway.18 Further, the present data obtained with kidney cells
suggest a rapid and preferential recycling of functional TF/VIIa
complexes, whereas no such apparent evidence for preferential
recycling for functional TF/VIIa complexes was found in
fibroblasts.18
In conclusion, our data show that TF-dependent internalization
and degradation of VIIa in kidney cells do not depend on interactions involving the cytoplasmic domain of TF, TFPI/Xa does not
affect the internalization, and the majority of internalized functional
TF/VIIa complexes rapidly recycle back to the cell surface.
Acknowledgments
From Novo Nordisk A/S, Maalov, Denmark, Elke Gottfriedsen and
Berit Lassen, Department of TF/VIIa Research, and Steen Kryger,
Department of Pharmacological Research, provided excellent
technical assistance; Jesper Bøggild Kristensen, Department of
Isotope Chemistry, prepared the iodine-labeled ligands. Prof Bo
van Deurs, Structural Cell Biology Unit, Department of Medical
Anatomy, The Panum Institute, University of Copenhagen, Denmark, and Dr Mirella Ezban, Novo Nordisk, are gratefully acknowledged for their discussion and helpful suggestions.
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2001 97: 1712-1720
doi:10.1182/blood.V97.6.1712
Tissue factor−mediated endocytosis, recycling, and degradation of factor VIIa
by a clathrin-independent mechanism not requiring the cytoplasmic domain of
tissue factor
Carsten B. Hansen, Charles Pyke, Lars C. Petersen and L. Vijaya Mohan Rao
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