1. No category

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1992 80: 966-974
Tumor necrosis factor-induced endothelial tissue factor is associated
with subendothelial matrix vesicles but is not expressed on the apical
surface
J Ryan, J Brett, P Tijburg, RR Bach, W Kisiel and D Stern
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Tumor Necrosis Factor-Induced Endothelial Tissue Factor Is Associated With
Subendothelial Matrix Vesicles But Is Not Expressed on the Apical Surface
By Jane Ryan, Jerry Brett, Pim Tijburg, Ronald R. Bach, Walter Kisiel, and David Stern
Cultured endothelial cells can be induced by tumor necrosis
factor/cachectin (TNF) and other cytokines t o synthesize the
procoagulant cofactor tissue factor (TF). Intact monolayers of
TNF-treated endothelial cells showed only minimal TF activity. In contrast, after permeabilization of these monolayers
with detergent (saponin, 0.02%). there was = 10- t o 20-fold
increase in TF-mediated, factor Vila-dependent factor Xa
formation. Extracellular matrix derived from TNF-treated
endothelium, prepared after removing the cells by hypotonic
lysis or ammonium hydroxide (0.1 N), also had similarly
enhanced TF activity. Incubation with a blocking monoclonal
antibody t o TF inhibited the procoagulant activity of both
TNF-stimulated endothelial cells, whether they were intact
or permeabilized, and of their matrices. However, when the
apical cell surface was pretreated with anti-TF antibody,
washed, and then cells were lysed with water or permeabilized with saponin, similar augmentation of TF activity was
still observed, suggesting the presence of a pool of TF t o
which the antibody did not initially gain access. Consistent
with this concept, the presence of TF in the matrix of
TNF-treated endothelial cells was shown by immunoblotting
and morphologic studies; cultured endothelial monolayers
and the native endothelium of aortic segments after exposure t o TNF showed TF in extracellular matrix, associated
with vesicles. In contrast, TF was virtually undetectable on
the apical endothelial surface. Taken together, these findings
suggest that endothelial TF can be present in a cryptic pool
that only gains access t o the blood after alteration in the
integrity of the endothelial monolayer.
0 1992by The American Society of Hematology.
T
interacts. Our results indicate that TNF-induced endothelial TF is not expressed on the apical surface but is in
matrix-associated subendothelial vesicles. These results
suggest an additional level of regulation for endothelial TF
that limits exposure to plasma coagulation components
until noxious stimuli disrupt the integrity of the cell
monolayer.
HE PRINCIPAL ROLE of quiescent endothelium in
regulation of the hemostatic system is to promote
blood fluidity by a combination of mechanisms, including
inhibition of coagulation,' prevention of platelet deposition: removal of any fibrin that might form,3 and maintenance vessel tone! Stimulation of cultured endothelial cells
with cytokines, such as tumor necrosis factor/cachectin
(TNF), leads to a change in their coagulant properties
through changes in cellular anticoagulant and procoagulant
activities/cofactors. An important means through which
TNF brings about these changes, at least in cell culture,
involves induction of the synthesis of the procoagulant
cofactor tissue factor (TF)?-6 Exposure of TF on the
luminal endothelial surface would lead to rapid intravascular coagulation, which is rarely seen in vivo.
In view of the situation in vivo, where TF is localized in
the subendotheIi~m,~-'~
we considered the possibility that
endothelial TF is sequestered from factors VII/VIIa, IX,
and X, the relevant enzyme and substrates with which it
From the Department of Physiology and Cellular Biophysics,
Columbia University, College of Physicians and Surgeons, New York,
NY;the Research Service, VA Medical Center, Minneapolis, MN; and
the Blood Systems Research Foundation Laboratory, Department of
Pathology, University of New Mexico School of Medicine, Albuquerque, NM.
Submitted November 18, 1991; accepted April 22, 1992.
Supported by grants from the Public Health Service (HL42833,
HL42507, HL34625, and HL35246), the Council for Tobacco Research (CTRI 971 and 2101RI), Schultz Foundation, Blood Systems,
Inc, and the New York Heart Association. D.S. completed this work
during the tenure of a Genentech-EIAward from the American Heart
Association.
Address reprint requests to Dr David Stem, Rover Research Laboratory, Department of Physiology and Cellular Biophysics, Columbia
University-College of Physicians and Surgeons, 630 W 168th St, New
York, NY 10032.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with I8 U.S.C. section 1734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-4971/ 92 /8004-0023$3.00/0
966
MATERIALS AND METHODS
Cell culture and aortic segments. Endothelial cells derived from
human umbilical cord veins were prepared by the method of Jaffe"
as modified by Thomton et a1.'* Experiments were performed
within 24 hours of the cells achieving confluence. Cultures were
characterized by indirect immunofluorescence, based on the presence of von Willebrand factor antigen, thrombomodulin activity,
and morphologic criteria.I3 Studies were performed using cultures
(passage 2) that had obtained confluence within 4 to 7 days in 1.75
cm2 wells. To study induction of TF, the growth medium was
aspirated and Medium 199 containing HEPES (10 mmol/L; pH
7.4), penicillin/streptomycin (50 U/mL and 50 pg/mL, respectively), and fetal calf serum (10%; Sterile Systems, Logan, UT) was
added along with indicated concentration of purified recombinant
TNF ( = l o x U/mg; generously provided by Dr P. Lomedico,
Hoffmann-LaRoche, Nutley, NJ). After 6 to 8 hours of incubation
at 37"C, TF activity was assessed as described below. Certain
studies (see Morphologic Studies) also used bovine aortic endothelial cells, grown as previously d e s ~ r i b e d . ' ~ J ~
In studies with calf aortic segments, a 5 to 10 cm portion of the
thoracic aorta was excised within minutes of killing the animal,
placed in Hank's balanced salt solution containing 25 mg/mL
bovine serum albumin, and transported to the laboratory at 21°C.
The adventitia was removed, the segment was placed in a multiwell
template containing growth medium (see above) without heparin
or growth factor, warmed to 37°C and incubated with TNF for 6
hours.16
Coagulationproteins and antibodies. Human factors X and Xa
were purified to homogeneity as described." Monoclonal (IgG1
subclass) and rabbit polyclonal antibody to recombinant human TF
was prepared and characterized as described previously,lXand the
mouse monoclonal or rabbit polyclonal antibodies to bovine TF
were prepared as d e s ~ r i b e d . ' The
~ ~ ~ monospecificity
~
of these
antibodies for tissue factor is presented in each of the references.
Monoclonal antibody to TF was radioiodinated using Enzymobeads (BioRad, Richmond, CA) following the manufacturer's
Blood, Vol80, No 4 (August 15). 1992: pp 966-974
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ENDOTHELIAL TISSUE FACTOR
protocol. The final specific radioactivity was =3 x lo4 cpm/ng.
Rabbit polyclonal anti-TF pathway inhibitor (TFPI) antiserum was
obtained from Dr G. Broze (Washington University, St Louis,
MO). Recombinant human factor VIIa was generously provided by
Dr Ulla Hedner (Novo Industri A/S, Bagsvaerd, Denmark).
TF-mediated activation of factor X was studied after the
incubation period of endothelial monolayers with TNF. Activation
of factor X was examined on intact endothelial monolayers,
monolayers briefly exposed to saponin (0.02% for 10 minutes at
22"C), and extracellular matrix prepared by treatment of the cell
monolayer with either water or ammonium hydroxide (0.1 N), until
cells were no longer visible, followed by extensive washing. In each
case, binding buffer (0.5 mL) containing HEPES (10 mmol/L; pH
7.4), NaCl (137 mmol/L), KCI (4 mmol/L), glucose (11 mmol/L),
CaC12 (2.5 mmol/L), and bovine serum albumin (0.5%) was added
along with factor VIIa (1 nmol/L or the indicated concentration)
and factor X (200 nmol/L). The mixture was incubated at 3 7 T ,
and one aliquot (50 pL) per well of the reaction mixture was
withdrawn at 10, 15, 20, or 30 minutes (unless other times are
indicated), and placed in 50 pL of HEPES (10 mmol/L; pH 7.4),
NaCl (137 mmol/L), KCI (4 mmol/L), and glucose (11 mmol/L)
containing EDTA (10 mmol/L) to stop the reaction. Factor Xa
formation was assessed by monitoring the rate of hydrolysis of the
chromogenic substrate Spectrozyme Xa (0.20 mmol/L; American
Diagnostica, Greenwich, CT,generously provided by Dr Hart) at
22°C using a Vmax reader (Molecular Devices, Menlo Park, CA).
The amount of factor Xa formed was determined from the linear
portion of a standard curve made with known amounts of factor Xa
(0 to 7.5 pmol). Where indicated, cultures were incubated with
anti-TF IgG before the assay for activation of factor X was
performed.
Binding of 1251-anti-TFIgG to endothelial monolayers was
studied using 1.75 cm2wells. After 6 to 8 hours of pretreatment of
endothelial cells with TNF, cultures were washed and incubated
with buffer containing nonimmune mouse IgG (10 pg/mL) and
fetal calf serum (10%) alone or buffer supplemented with a
blocking antihuman recombinant TF monoclonal antibody (6
pg/mL). The latter step, to block TF on the cell surface, was
followed by washing to remove unbound antibody, permeabilization of the monolayer using saponin, or exposure of the matrix by
hypotonic lysis of the cells. Then, endothelial-derived preparations
were incubated with lZI-anti-TF monoclonal (60 ng/mL) alone
(total binding) or in the presence of an 100-fold excess of unlabeled
antibody (nonspecific).
Immunoblotting for tissue factor was performed on endothelial
cell and matrix samples. Cell-lysates were prepared by treatment of
monolayers with ammonium hydroxide. The remaining matrix was
washed with phosphate-buffered saline (PBS), and TF-containing
samples were prepared by scraping with a rubber policeman into
PBS with Triton X-100 (0.1%). Samples were precipitated using
trichloroacetic acid (TCA, 15%), washed twice with ice-cold
acetone, and resolubilized in reduced SDS gel buffer.21 Samples
were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12.5% for lanes 1 to 2 and 7.5% for lanes 3
to 4 in Fig 6), and transferred to nitrocellulose electrophoretically,
using the dry blot system (Polyblot Transfer System; ABN, Emeryville, CA). The nitrocellulose was blocked with PBS-containing
gelatin (3%) at 37°C overnight, and then reacted with rabbit
polyclonal antibody to recombinant human TF (10 pg/mL) in
buffer containing gelatin (1%) overnight at room temperature.**
Bound antibody was detected after incubation with radioiodinated
mouse antirabbit monoclonal antibody (2.5 pg/mL; Sigma).
Morphologic studies. For immunofluorescence, confluent endothelial monolayers were incubated in the presenceiabsence of TNF
(10 nmol/L) for 5 hours at 37"C, and then the monolayers were
967
fixed in 3.5% paraformaldehyde with/without 0.1% NP-40, and TF
was localized using rabbit anti-TF IgG at 37°C for 60 minutes.
Additionally, some monolayers were permeabilized using the same
protocol as for the TF assay. For immunohistochemical localization
of TF at the electron microscopic level, cells grown on Costar
Transwell inserts (Cambridge, MA) were exposed to TNF as
above, fixed in 3.5% paraformaldehyde, washed, incubated with
primary antibody as above, and followed by peroxidase-conjugated
secondary antibody (Sigma) at 37°C for 1 hour. After incubation in
secondary antibody, cells were briefly fixed in 2% glutaraldehyde
and the diaminobenzidine reaction was performed. After development of product, monolayers were postfixed in 2.5% glutaraldehyde, osmicated, and embedded for electron microscopy. Aortic
segments, rapidly procured from animals after killing, were stripped
of adventitia and mounted in a lucite template device allowing
access to the luminal surface, as described previ~usly?~
The
chamber divided the segment into two parts. One part was treated
with TNF in growth medium (as above), and the other was
incubated in normal culture medium alone. At the conclusion of
the experiment, segments were washed in balanced salt solution,
and fixed in 3.5% paraformaldehyde. A biopsy punch was used to
remove tissue samples that were then transferred to fresh fixative
for 18 hours at 4"C, and subsequently dehydrated and embedded in
LR White resin (Polysciences, Warington, PA). Grids were stained
for TF with the rabbit polyclonal antibody, and were shown with
goat-antirabbit IgG conjugated to 12 nm gold particles (Sigma),
and then stained with uranyl acetate and lead citrate for viewing in
the electron microscope."
RESULTS
Quiescent endothelial monolayers had no detectable TF,
based on factor VIIa-dependent factor X activation (Fig 1).
This was true whether cells were tested as intact monolay-
I
Quiescent
TNF-treated
I
Fig 1. Enhanced expression of TF activity on TNF-treated endothelial monolayers after treatment with saponin and ammonium hydroxide. Confluent endothelial cultures were incubated for 6 hours with
TNF (1 nmol/L) or control medium (quiescent), and either assayedfor
TF activity immediately after incubation with TNF (surface), or first
exposed to saponin or ammonium hydroxide (NH,OH), and then
assayed for TF activity. The mean of duplicate determinations is
shown, and is representativeof 10 experiments.
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RYAN ET AL
ers, permeabilized with detergent, or removed by treatment
with ammonium hydroxide. Only minor amounts of TF
activity could be detected on intact cell monolayers 4 to 6
hours after addition of TNF. However, after permeabilization of the cultures by brief exposure to saponin, there was
= 20-fold increase in tissue factor activity similar to increases seen in freeze/thaw preparations of scraped cells
with matrix (data not shown). TF activity of saponin-treated
cells was dependent both on the incubation time and dose
of TNF (Fig 2A and B). In each case, factor Xa formation
was attributable to the presence of TF, as shown by its
inhibition in the presence of anti-TF IgG (data not shown).
To begin to localize TF within the cell monolayer and
underlying matrix, endothelial cells were removed with
ammonium hydroxide to expose the matrix, by methods
hitherto described.z,26 TF activity in matrix preparations
appeared in a time-dependent manner after addition of
TNF. The dependence of matrix-associated TF activity on
TNF concentration roughly paralleled that observed with
saponin-solubilized cells. Hypotonic lysis of TNF-treated
endothelial monolayers, which also exposes the matrix,
resulted in a similar increase in TF activity to that seen with
ammonium hydroxide.
Several possible mechanisms could account for the increase in TF activity observed after ammonium hydroxide/
detergent-treatment of TNF stimulated endothelial cell
cultures, including alterations in the arrangement of phospholipids with exposure of acidic lipids, removal of an
inhibitor, or the presence of a cryptic pool. Recent studies
in fibroblasts have shown that the calcium ionophore
A23187 enhances TF activity.*' Exposure of TNF-treated
endothelial cells to the calcium ionophore A23187 (at 10
and 20 kmol/L) showed only a threefold to fourfold
enhancement of TF activity across a range of factor VIIa
and X concentrations (data not shown). The major inhibitor of the TF pathway, TFPI, is an endothelial product that
could mask TNF-induced TF acti~ity~~-~O;
but preincubation
of endothelial monolayers with neutralizing antibody to
human TFPI showed only minor increases in TF activity
compared with the matrix from the same cells (Fig 3).
When TNF-treated endothelial cultures were exposed simultaneously to both the anti-TFPI antibody and to the calcium
ionophore, the combined effect of both agents was no more
Fig 3. The effect of anti-TFPI antibody on TF activity of intact,
TNF-stimulated monolayers. Confluent monolayers were incubated
with TNF (1 nmol/L) for 6 hours, washed in incubation buffer, and
assayed for TF activity after one of the following treatments: (A)
dissolution of the cell monolayer by exposure to ammonium hydroxide followed by washing three times in incubation buffer; preincubationoftheintactmonolayerfor 1hourwith(B)l/lW.(C)1/500,or(D)
1/1000dilution of the antiserum followed by washing; (E)incubation
of the intact monolayer with nonimmune rabbit serum followed by
washing; and (F) no further manipulation of the monolayers except
washing. Means f SEM of triplicate determinations are shown, and
the experiment was repeated twice.
than a threefold to fourfold increase above the baseline
observed in nonpermeabilized TNF-treated cultures.
To test whether enhanced TF activity after disruption of
the TNF-treated endothelial monolayer was caused by TF
apoprotein initially exposed on the cell surface, whose
activity was modulated under our experimental conditions
(such as detergent solubilization and/or hypotonic lysis
resulting in phospholipid rearrangement), or whether additional, initially cryptic, TF was exposed by these treatments,
intact, TNF-treated endothelial monolayers were preincu-
Dose Response
Time Course
Fig 2.
(A) Time course and (8) dose-dependence
of TNF-induced endothelial cell TF activity: compari-
0
2
4
6
Time, hr
8
son of intact cells, saponin-permeabilized cultures,
and ammonium hydroxide-prepared matrix. Confluent endothelial monolayers were incubated with
TNF, and TF activity was assessed by studying factor
Vlla-dependent factor Xa formation, as described in
the text. TF activity was determined on intact endothelial cell monolayers (A),monolayers exposed to
saponin (O), and monolayers treated with ammonium hydroxide).( after the indicated time of exposure to TNF (1 nmol/L, A) or after 6 hours of incubation with the indicated concentration of TNF (6).The
results are representative of three experiments.
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ENDOTHELIAL TISSUE FACTOR
969
bated with anti-TF antibody to block cell surface antigen.
Then, TF activity, as well as exposure of new TF antigen,
was assessed after permeabilization or exposure of the
matrix. Control experiments established that each of these
treatments did not disrupt interaction of the antibody with
TF. After blocking of TF on the surface of TNF-treated
endothelial cells with anti-TF antibody, permeabilization
with saponin resulted in enhanced levels of factor Xa
formation, compared with controls not exposed to TNF
(Fig 4A, I to IV). Consistent with the view that the anti-TF
IgG was not able to gain access to endothelial TF unless the
cells had been permeabilized, addition of this antibody
after saponin treatment blocked TF activity (Fig 4A, IV).
Similar results were observed in TNF-stimulated cultures
exposed to hypotonic lysis. Hypotonic lysis enhanced TF
activity even when TF on the apical surface of the cell had
been previously blocked by addition of anti-TF IgG (Fig 4B,
VI to X). When anti-TF antibody was incubated with cell
preparations after lysis under hypotonic conditions, TF
activity was blocked (Fig 4B, IX). These data, suggesting
that permeabilization of TNF-treated endothelial cells, as
well as exposure of their matrix, provides access to the
induced TF, were supported by the results of radioligand
binding studies with anti-TF antibody. Specific binding of
1251-anti-TF IgG was enhanced on permeabilized, TNFtreated endothelial cultures, and on their matrices, compared with intact cells (data not shown).
The results of these coagulation physiology and antibody
binding experiments led us to localize TF in the endothelial
cell and underlying matrix after exposure of the cultures to
TNF (Fig 5). By immunofluorescence, there was little
evidence of TF antigen on the apical surface of untreated
controls (Fig 5A) or nonpermeabilized TNF-treated monolayers (Fig 5B). In contrast, positive staining was evident
when TNF-treated endothelial monolayers were first permeabilized; staining of vesicular structures was evident, as well
as some diffuse staining, possibly representing intracellular
TF and/or antigen associated with the basal surface of the
cell (Fig 5C and D). Extracellular localization of TF was
confirmed by electron microscopy of nonpermeabilized
cells (Fig 5E), which showed TF antigen especially in the
subendothelium associated with vesicle-like structures. Negligible amounts of T F were observed on the cell surface of
TNF-treated endothelial cultures using the same ultrastructural techniques.
Our morphologic and coagulation physiology studies
suggested that TF was present in functional form in the
extracellular matrix. As expected, immunoblots from endothelial cell lysates and matrix-derived from control cultures
showed no bands with anti-TF antibody. In contrast, with
lysates or matrix preparations derived from TNF-treated
endothelial cultures, a major immunoreactive band was
present corresponding to = 43 Kd (Fig 6).
Enzymology studies were performed to determine parameters of factor VIIa-mediated activation of factor X by
cultures permeabilized with saponin and matrix-preparations derived from TNF-treated endothelial cells (Fig 7).
Although it was difficult to accurately assess parameters of
factor Xa formation on intact monolayer preparations,
using the permeabilized cells and matrix from TNF-treated
cultures, the half-maximal rate of factor Xa formation
occurred at factor X and VIIa concentrations of 50 nmol/L
and 0.05 nmol/L, respectively. Vmax was greatest on the
saponin permeabilized cells, presumably because of the
A
I
II
111
IV
VI
VI1
Vlll
IX
x
Fig 4. Enhanced expression of TF activity on endothelialcultures treated with saponin or subjected to hypotoniclysis: effect of antibody to TF.
(A) Confluentendothelialmonolayerswere first exposed to TNF (1 nmol/L) for 8 hours, washed in binding buffer, and then subjected to one of the
following treatments: I. no treatment; II, culture preincubated with anti-TF monoclonal antibody; 111, culture treated with saponin; IV, culture
treated with saponin. washed, incubated with anti-TF monoclonal antibody, and then assayed for TF; and V, culture preincubatedwith anti-TF
monoclonal antibody, washed, treated with saponin, and assayedfor TF. After these treatments, TFactivitywas assessedas described in the text.
(B) The study was performed using the same general protocol as above. except that saponin treatment was replaced by hypotonic lysis in
experiments Vlll through X. In each case, the concentration of anti-TF monoclonal antibody 6 pg/mL, and the incubationperiod with the antibody
was 30 minutes at room temperature. Results are the means of duplicate determinations,and are representativeof three experiments.
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RYAN ET AL
970
Fig 5. lmmunolocalization of
TF in cultured endothelial cells
exposed t o TNF. (A) Endothelial
cells incubated in medium alone
(no TNF) were fixed and permeabilized in 3.5% paraformaldehyde containing 0.1% NP-40
stained for TF by immunofluorescence as described in the text.
(6) Monolayers of endothelium
treated with TNF (10 nmol/L for
6 hours) were fixed and stained
for TF without permeabilization
or (C) with permeabilization. ( 0 )
TNF-treated monolayers were
treated according t o the identical protocol with 0.02% saponin
as used in the coagulation physiology studies, and stained for TF.
(E) Cultured endothelial monolayers grown on membranes were
treated with TNF, and TF was
visualized with peroxidase in the
electron microscope. The arrowheads depict sites of reaction
product deposition. Original
magnification Athrough D, ~ 6 5 0 ;
E, bar = 500 nm.
contribution of both intracellular and matrix-associated TF.
Matrix preparations from hypotonically lysed cells showed
a lower Vmax, probably resulting from the removal of some
intracellular TF.
Although T F expression by endothelial cells in culture
has been shown with endotoxin and several inflammatory
cytokines, including TNF and interleukin-l,S.6*-'lit has bcen
more difficult to show endothelial T F in vivo.R To begin to
bridge the gap between studies in cell culture and the in
vivo setting, experiments were performed with the native
endothelium of bovine aortic vessel segments. After incubation with TNF, immunoperoxidase studies found TF antigen in a pattcrn rcscmbling that observed in cultured
endothelial cells (Fig 8A and B), ie, the antigen was mostly
localizcd to the endothelial cell layer and subendothelium.
In the electron microscope, using gold conjugated anti-TF
antibodies, T F was shown to be present predominately in
subendothelial vesicle-like structures (Fig 8D). This local-
ization of TF was not seen in untreated control endothelial
cultures (Fig 8C),which also produced some matricial
vesicles, but were devoid of demonstrable TF.
DISCUSSION
Previous studies have shown that exposure of cultured
endothelial cells to the cytokine TNF leads to the synthesis
and expression of TF, a cofactor considered to be the major
initiator of coagulation in vivo.32 Exposure of T F on the
vessel wall in vivo would be expected to correlate closely
with activation of coagulation and fibrin formation on the
vessel surface. However, fibrin formation localized to the
luminal surface of an intact vessel is not a common
occurrence, even in inflammatory lesions in which cytokines
are present.-'-'In this context, after the intravenous infusion
of large amounts of interleukin-1 into rabbits, very little TF
activity was detectable on the surface of aortic segments,
and only occasional fibrin in close association to the vessel
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ENDOTHELIAL TISSUE FACTOR
97 1
- 200
i
69-
46-
-92
1
.
- 69
-46
30-
I
2
3
4
Fig 6. Characterization of TNF-induced, endothelial cell-derived TF
associated with the matrix by immunoblotting. Confluent endothelial
cultures were incubated with TNF (1 nmollL) or buffer alone for 6
hours, and the cell monolayer was removed by exposure t o ammonium hydroxide. Matrix was then solubilized in SDS-containing'Jample buffer, and immunoblotting was performed iollowed reduced
SDS-PAGE and transfer of material on the gel t o nitrocellulose
membranes: lane 1, SDS cell extract from unstimulated cultures; lane
2, solubilized matrix from control cultures; lanes 3 and 4 represent the
same samples, respectively, derived from TNF-treated endothelial
cultures. The bars indicate the migration of standard proteins run
simultaneously in an adjacent lane corresponding t o molecular weights
of 92 Kd (phosphorylase 6). 69 Kd (bovine serum albumin), 46 Kd
(ovalbumin), 30 Kd (carbonic anhydrase), 21 Kd (soybean trypsin
inhibitor), and 14 Kd (lysozyme).
surface was found.34It has been suggested that T F production by endothelium in vivo does not occur, or if it does, its
expression is at levels difficult to detect.H.3sS36However,
evidence showing the presence of T F associated with the
endothelium in inflamed placental vessels"' and in certain
vascular beds after the infusion of endotoxids.36 led us to
consider the alternative hypothesis that the activity of
endothelial TF is tightly controlled.
In this report, wc show that, in response to TNF, T F
produced by cultured and by native endothelium in situ is
not expressed to a significant extent on the luminal cell
surface, based on morphologic and functional studies.
Although our findings might at first appear to contradict
previous reports, including our own, concerning the expression of TF by stimulated endothelial ~ e l l s , in~ the
' ~ ~current
~
experiments we have taken special precautions to insure
the integrity of the monolayer before determining the TF.
Cultures were not scraped or subjected to any treatment
that might alter viability or detach the cell monolayer from
the growth surface, such as EDTA-containing buffer or
local anesthetics, before the T F determination. Furthermore, formation of thrombin and fibrin, both of which have
been shown to alter integrity of the endothelial monodid not occur to an appreciable extent, because all
studies were performed in thc presence of only purified
factors VIIa and X. Under these conditions, TF antigen and
activity was barely detectable on the surface of TNFstimulated cells.
Initially we considered it possible that an inhibitor such
as TFP12X-3i1
might be inhibiting T F that was present on the
cell surface, or that TF might not be optimally active
because of its phospholipid milieu. However, when TNFtreated endothelial cultures were exposed to either neutralizing anti-TFPI antibody and/or the calcium ionophore
Dependence on Factor Vlla
Dependence on Factor X
0
.k
4
matrix
I
. .
0.1 0.2
0.4
0.6
0.8
[Factor Vlla], nM
1.0
40
80
120
160
200
240
280
[Factor XI, nM
Fig 7. Parameters of TF-factor Vllbmediated activation of factor X on TNF-treated intact endothelial cells (cells), after permeabiliration w k h
saponin (saponin), or after preparation of the matrix (matrix). Endothelial cells were exposed t o TNF (1 nmol/L) for 6 hours, and then factor Xa
formation in the presence of either factor Vlla (1 nmol/L) and the indicated concentration of (A) factor X, or (B) in the presence of the indicated
concentration of factor Vlla and factor X (200 nmol/L) was studied. Results are shown on intact endothelial cell monolayers (A),monolayers
exposed t o saponin (O),and monolayers treated with ammonium hydroxide (m). Factor Xa formation, the mean of duplicate determinations, was
determined as described in the text, and is representative of three experiments.
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
RYAN ET AL
972
--
E,.
J
.
''
-
A"
A23187,27a maximum increase of only threefold to fourfold
in TF activity was observed. Rather, the potential procoagulant activity of TF was only realized after permeabilization
of the cells with a low concentration of saponin, or removal
of the cell monolayer, even when cell surface TF activity
had been previously blocked by anti-TF antibody (the latter
observation indicates that TF was not likely to have been on
the cell surface at the start of the experiment). Immunolocalization studies showed TF antigen in cultured endothelial cells and aortic segments was associated with vesiclelike structures in the subendothelial matrix, but not on the
apical cell surface to an appreciable extent. Furthermore,
matrix-associated TF appeared to be intact, by Western
blotting and in functional studies showing factor VIIamediated activation of factor X. Taken together, these data
suggest that TF produced by endothelium in response to
TNF is not expressed at a site that facilitates optimal
interaction with its substrates and enzyme, but rather is
sequestered in the matrix, within the cell and possibly on
the basal surface. However, after disruption of the endothelium and/or exposure of the subendothelium, TF activity is
expressed. These observations are in accordance with
recent findings of Carson et a14' that activation of the
complement cascade on the surface of fibroblasts, in which
Fig 8. TNF-mediated induction of TF in the native endothelium of bovine aortic segments.
Aortic segments were incubated
in (A and C) medium alone or (E
and D) medium containing TNF
(10 nmol/L for 5 hours), and then
TF antigen was localized in the
light microscope using immunoDeroxidase (sections were counterstained with hematoxylin) (A
and B), or in the electron microscope using secondary antibodies coupled to colloidal gold (C
and D). The basal aspect of the
endothelial cells are along the
upper margins (*). Arrow heads
depict sites of localization of gold
particles. Inset represents higher
resolution of the area depicted
by arrowheads to the right. Original magnification, x325 (A and
B); bar = 500 nm (C and D).
TF antigen is constituatively produced, leads to enhanced
TF activity. In this context, earlier studies of Schorer et a14?
showed that exposure of endotoxin-treated endothelial
monolayers to hydrogen peroxide resulted in a large increase in TF activity in parallel with destruction of the
monolayer.
The presence of TF in subendothelial matrix, and release
of vesicles containing TF activity from cellular surfaces has
been previously reported. Weiss et a17observed TF activity
on inverted vessel strips denuded of endothelium. A specialized situation is the observation of Almus et a143that TF is
present in Wharton's jelly of the umbilical cord, and can
penetrate fenestrations of the muscularis media of the
umbilical vein. Shedding of TF-rich vesicles has been
observed from the surface of tumor cells, fibroblasts, and
monocytes."-47 Our results on TNF-treated endothelial
cells link these previous observations in the context of
vessel wall biology by indicating that TF-containingvesicles
can be released into the subendothelium. These TFcontaining vesicles are released only or chiefly at the basal
cell surface and, in accordance with the previous report by
Brox et a!$* we recovered only minimal TF activity from
conditioned media, even after concentration by trapping on
filters. This may represent an example of unidirectional
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
ENDOTHELIALTISSUE FACTOR
973
export of endothelial cell proteins, such as has been
observed with release of urokinase-typeplasminogen activator.@
The results of our study indicate that after the synthesis
of TF by either TNF-treated native endothelium in situ or
cultured endothelial cells, additional mechanisms control
the expression of its full functional activity. Extrapolation of
these results to the in vivo setting would suggest that
damage by agents that bring about permeabilization of the
endothelium and/or loss of endothelial monolayer integrity, would be important for actual expression of TF activity
synthesized by endothelium in response to inflammatory
cytokines.
ACKNOWLEDGMENT
We thank S. Rover for his generous contribution. Dr Gabriel
Godman provided invaluable suggestions throughout the course of
this work.
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