From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Association of Smooth Muscle Cell Tissue Factor With Caveolae
By Andre B. Mulder, Jan W. Smit, Victor J.J. Born, Ne1 R. Blom, Marcel H.J. Ruiters, M. Ruud Halie,
and Jan van der Meer
There is still no satisfactory explanation for the lowcatalytic
activity of tissue factor (TF)/factor Vll(a1 complexes towards
coagulation factor X, as found on the apical surface side of
cell layers.It has been hypothesized that TF existsin a latent
form. Layers of cultured human smooth muscle cells, constitutively expressing TF, were immunogold-labeled for TF in
situ and processedfor electron microscopy. We showed
that, besides internalization and accumulation in lysosomallike structures,TF remained associated with noncoated,
flask-shaped microinvaginations of the plasma membrane.
These invaginations were identified as caveolae. In regions
in which intercellular contacts were interrupted, more TFpositive caveolae were observed. Enzymatically detached
smooth muscle cells exhibited a similar enlargement of caveolar structures. Concomitantly, an increase ofcatalytic activity of apically formed TF/Vlla complexes towards factor
X was found on the suspended cells. We speculate that caveolae-associatedTF may function as alatent pool of procoagulant activity, which can rapidly be activated at sites in
which vessel wall integrity is lost.
0 1996 by The American Societyof Hematology.
I
M S , Bagsvaerd, Denmark). Human coagulation factor
X was obtained from Kordia BV (Leiden, The Netherlands). Mouse monoclonal antibodies against humanTF (#4503, #4504, and #4506) were
obtained from American Diagnostica Inc (Greenwich, CT). Nonimmune mouse IgGl was purchased from Becton Dickinson (Mountain
View, CA). Rabbit polyclonal anticaveolin antibody was obtained
from Transduction Laboratories (Lexington, KY). Nonimmune rabbit Igs were from Dakopatts A / S (Glostrup, Denmark). Goat antimouse antibodies coupledto 10-nm gold particles and goat antirabbit
antibodies coupled to S-nm gold particles were obtained from Sanver
TECH (Boechout, Belgium).
Culture of SMCs. SMCs, which had overgrown the endothelial
cultures derived from umbilical cord vessels, were cultured as described
The
SMCs
were
differentiated
from
endothelial
cells by their spindle shape, much larger size, and reactivity
with
an antibody against a-smooth muscle actin, whereas no reactivity
withanantibodyagainstvonWillebrandfactorwasCulture
mediumconsisted of RPM1 1640(GIBCO,Paisley, UK) supplemented with 20% heat-inactivated pooled human serum, 2 mmol/L
L-glutamine, S U/mL heparin, SO pg/mL EC growth factor supplement extracted from bovine hypothalamus;’ 1 0 0 p g h L streptomycin, and 100 IUlmL penicillin.
Ultrastructurul studies. SMCs were cultured in Lab-tek 8 chambedslides (Miles Scientific, Naperville,IL; Mulder et al, manuscript
submitted). Briefly, 30% (wt/vol) gelatin was sterilized
by autoclavation and then chilled to 65°C. Subsequently, small wells of Lab-tek
8 chamber/slideswerecoated
with 20 pL of theviscousgelatin
solution and incubated atroom temperature for 30 minutes, followed
by fixation in O S % glutaraldehydeovernight.Beforeseedingthe
cells,thewellswerewashedthoroughly.
For localizationofTF,
confluent SMC layers were labeled with 50 pg/mL mouse anti-TF
antibodies(#4S03)for 1 hour at 4°C in culturemedium and then
washedandincubatedwithgold(10nm)-labeledgoatantimouse
IgC (20pglmL) in culturemedium for 1hourat
4°C or 37°C.
Other layers were fixed before labeling with antibodies against T F
(a combinationof #4S04 and #4506) at room temperature by immersion in 0.2% glutaraldehyde plus 2% paraformaldehyde in 0.1 mol/
L phosphate buffer at 4°C overnight. To minimize nonspecific binding of the antibodies, the cells were washed in phosphate-buffered
saline (PBS; 0.14 mol/L NaCL, 0.01 moUL phosphate, pH 7.4) with
0.1 S m o l L glycine for 15 minutes and subsequently in a buffer of
0.01 mol/L Tris-HC1, pH 7.4, with2% dry milk solids for 60 minutes
at room temperature. In control samples, nonimmune mouse IgGl
was added instead of anti-TF antibodies.
For quantification ofTF labeling, the percentagesof gold particles
associated with caveolae, with plasmalemma proper, or with coated
pits were determined. For each labeling condition, more than S00
gold particles were scored. Additionally, the percentages of more
than S00 caveolae, which were labeled, were determined.
T IS GENERALLY considered that contact between plasma
factorVII(a)anditsreceptorandcofactor,tissuefactor
(W), is sufficient to initiatecoagulationin v ~ v o . ”TF
~ is a
transmembrane glycoprotein. In the normal state, vascular TF
is located on cells in the adventitia and variably on cells
in
the media.“6 Quiescent endothelium does not show detectable
~ ~ , 2 , 4 , although
.5
induction of endothelial TF has been
it is
Besides regulation
at
the transcriptional
thought that TF activity is modulated by either alterations
in the phospholipid composition of the surface in the proximity of the TF protein’”-” or specific inhibiti~n.’~.”
However, there is still no satisfactory explanation for the
low catalytic activity of TF/factor VII(a) complexes, as
found on the apical surface side of cell layer^.".'^ It has been
hypothesized that TF exists in a latent form.”.”
In this study, we show that TF, constitutively expressed
on layers of cultured human smooth muscle cells (SMCs)?’”
is associated withnoncoated
microinvaginations of the
plasma membrane that are identified as caveolae. Detachment of cells leads to a more open status of these structures,
concomitantly with an increase of TF activity.
MATERIALS AND METHODS
Materials. Ovalbumin(grade V), a-chymotrypsin(frombovine
pancreas, type 11), and gelatin were obtained from Sigma Chemical
CO (St Louis, MO). HEPES was obtained from Biorad Laboratories
(Richmond, CA). Heparin was obtained from Leo Pharmaceutical
Products B.V. (Weesp, The Netherlands). Recombinant human factor VITa was generously providedby Dr T. Jorgensen (Novo-Nordisk
From the Department of Haematology, Division of Haemostasis,
Thrombosis and Rheology, the Central Luboratory of Clinical Haematology, University Hospital, and the Centre for Biomedical Technology, University .f Groningen, The Netherlands.
Submitted October 26, 1995; accepted March 28, 1996.
Addressreprintrequeststo
Andre‘ B. Mulder,MD,Division of
Haemostasis, Thrombosis and Rheology, Department of Haematology, University Hospital Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands.
The publication costs of this article were defrayedin part by page
chargepayment. This article must thereforebeherebymarked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-497l/96/8804-0010$3.00/0
1306
Blood, Vol 88,NO4 (August 15). 1996: pp 1306-1313
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
ASSOCIATION OF SMC-TF WITH CAVEOLAE
1307
For colocalizationof TF with caveolin, unfixed SMC layers were
It was excluded that TF was clustered in and around ca(#4503) at37"C,as
immunogold-labeledwithanti-TFantibodies
veolae only asa result of cross-linking antibodies:' because
described above. The layers were then fixed with 3.5% formaldehydewhen cells were fixed (2% paraformaldehyde and 0.2%gludilutedinPBSOvernightat4"C,followedbypermeabilizationin
taraldehyde) before labeling, gold particles could still be
0.2% Triton X-100 for 10 minutes at room temperature. After washfound associated with caveolae (Fig 2). However, caveolae
ing three times for 5 minutes each withPBS and once with 0.2%
1) and label
were labeled to a much lesser extent (Table
bovine serum albumin in PBS (PBSIBSA) for 10 minutes, the cells
was more often located around instead of physically within
were labeled with 5 pg/mL rabbit antibodies against caveolin for60
caveolae in fixed cells. Therefore, we cannot exclude any
minutes at room temperature in PBSIBSA, washed, and then incueffect of immunolabeling on caveolar localization of TF in
bated with gold (5 nm)-labeled goat antirabbit IgG (20 pg/mL) for
60 minutes in PBS/BSA. In control samples, nonimmune rabbit IgG
unfixed cells, although alternativemechanisms, including
was added instead of the anticaveolin antibody.
lower accessibility of (immunogold-conjugated) antibodies
A modification of a method described for lymphocyte^^^ was used
to caveolar located TF, might have been responsible for the
to examine the cells' ultrastructure. Before embedding, in all cases,
different distribution of gold label in fixed cells.
layers were washed thoroughly and fixed in2% glutaraldehyde. Thin
Further, in line with previously reported data,29 we found
sections werefinallystainedwithuranyl
acetatehethy1 cellulose
in regions of labeled SMC layers in which intercellular conand lead citrate for examination
in a Philips 201 electron microscope
tacts were interrupted more TF-positive caveolae (Fig 3A).
(Philips, Eindhoven, The Netherlands).
Occasionally,
label was present in coated pits or coated vesiTF activity. LayersofSMCsweregrowntoconfluencein24cles at these sites.
well culturedishes(Costar,Cambridge,MA).FactorVIIa(final
Subcellular localization of TFin suspended SMCs. To
concentration, 10 nmol/L) and X (final concentration, 50 nmol/L)
were added in assay buffer (10 mmol/L HEPES, 137 mmol/L NaCI,
further support the detachment-related phenomena, as de4 mmol/L KCI, 1l mmoVL a,D-glucose,5 mg/mL of ovalbumin and
scribed above, we examined cells in suspension. After immu2.5 mmoln CaCI2, pH 7.45). Serial subsamples were subsequently
nogold-labeling of TF at the apical side of SMC layers at
withdrawn from the reaction mixture at 2-minute intervals to mea37"C, cells were washed in isotonic PBS and detached by
surefactor X activation spectroph~tometrically.~'.~~
After 10 mintreatment with 0.05 mg/mL chymotrypsin in PBS for 5 minutes, the cells were washed in assay buffer, detached by brief treatutes at 37°C. Subsequently, the cells wereimmediately fixed
ment with 0.05 mg/mL chymotrypsin for 5 minutes, centrifugated,
and
processed for electron
microscopy (Fig 3B). Accordand resuspended in assay buffer. Subsequently factor X (50 nmol/
ingly, suspended cells exhibited extensive dilated caveolaeL) was added and factor Xa generation was measured, with or withlike structures thatwere preferentially associated with apparout adding additional factor VIIain the assay. Controls consistedof
layers preincubated with inhibitory anti-TF antibodies (50
pg/mL)
ently intracellular located tubular and vesicular structures.
for 30 minutes at 37°C. The viability of the detached cells, tested
Frequently,clusters of caveolaewere seen tosurround a
by lightmicroscopicmeasurementoftrypanblueexclusion,was
central vacuole, a profile characteristic for caveolar enlarge98% L 1%. Cell counts were determined using a Coulter counter.
n~ent.~' These
apparently intracellular located, TF-positive,
Additionally, possible effects of chymotrypsin treatment on the accaveolae-like structures were further characterized by colotivity of TF/factor VIIa complexes were examined. SMC layers were
calization of caveolin3' (Fig 4).
apicallyincubatedwith
factorVIIa,washed,andliftedfromthe
Factor Xa generation by TFJactor Vlla complexes on the
dishes either by chymotrypsin treatment, as described above, or by
surface of layers and suspended SMCs. To determine the
scraping. After centrifugation, cells were lysedby three freezekhaw
functional consequences of cell detachment for TF, layers
cycles (-8OoC/37"C) in assaybufferandsubsequentlytestedfor
factor X activation activity without adding additional factor VIIa in
of SMCs wereincubated with factor VIIa andX and generathe assay.
tion of activated factor X (Xa) was measured (Fig5). Subsequently, these layers were washed and detached, and cells
RESULTS
were resuspended in assay buffer. After the addition offactor
Subcellular localization of TFin layers of SMCs. On
adherent SMCs immunogold-labeled for TF at 4°C and processed insitu for electron microscopy, we found that, besides
Table 1. Quantitative Analysis of SMC Plasmalemma-Associated,
presence on plasmalemma proper and within coated pits,25
Immunogold-LabeledTF Distribution
particles were located within or nearby smaller, noncoated
No. of Gold Particles Bound (Yo)
invaginations of the plasma membrane (Fig 1A). Morphologically, the noncoated invaginations were recognized as caPlasmalemma
Coated
Caveolae
veolae(alsoknownasplasmalemma
vesicles) by their
ProperCaveolae Pits
(96 labeled)
characteristic 50- to 60-nm size and flask shape.26,27When
46.6 4°C
7.2
46.2
57.3
labeling was performed at 37"C, we observed that, besides
37°C
0.4
73.2
26.4
69.2
internalization,gold-labeled TF remained associatedwith
21.3
Fixed
1.3
77.4
19.1
1B).
thenoncoatedplasmamembraneinvaginations(Fig
SMC layers were labeled at 4°C with anti-TF antibodies (#4503),
Even an increase in the relative number of gold particles
washed, and subsequently incubated with gold-labeled antibodies
associatedwithcaveolaecould
be observed(Table
l).
against anti-TF antibodies, either at 4°C or 37°C. Other layers were
Coated pits were consistently devoidof label. Incubation of
fixed overnight with 2% paraformaldehyde and 0.2% glutaraldehyde
layers with nonimmune, mouse IgGinstead of specific antiat 4°C before immunogold labeling of TF (#4504 and #4506) at room
TF antibodies gave no labeling (Fig 1C).
temperature.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
MULDER ET AL
1308
,
-.
-". -. . ..-
-.
".
-
Fig 1. Subcellular localization of TF in SMC layers.
SMC layers were incubated with mouse anti-TF antibodies (#4503) at 4°C. followed by incubation with
gold-labeled goat antimouse antibodies for 1 hour
at 4°C (A) or 37°C (B and C). (A) Afterlabeling at 4°C.
gold particles were associated with coated (arrowhead) as well as with noncoated, flask-shaped invaginations of the apical cell membrane (arrow). (B)
Besides internalization and distributionin
lysosomal-like structures(L) and perinuclear regions (arrowhead), gold-labeled TF remained associated with
the noncoated microinvaginations of the apical
plasma membrane (arrow) after labeling at 37'C. No
labeling of coated pits Icp) was observed.
MC-TF
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
ASSOCIATION OF
WITH CAVEOLAE
1309
Fig 1. (cont'dl. (C) Control layers were incubated
with nonimmune, mouse lgGl instead ofspecific
anti-TF antibodies. Scale bar indicates 100 nm.
X, generation of factor Xa was measured with or without
adding additional factor VIIa in the assay.
Increased activity of apically formed TF/factor VIla complexes could be observed after detachment of the cells, which
could be apically inhibited with anti-TF antibodies. A further
increase of TF activity by adding additional factor VIIa in
the assay of detached cells showed free TF sites that might
have been inaccessible to factor VIIa on the apical side or
located on the abluminal surface side of the cell layers. Mixing experiments, as described by Rao et a13' indicated that
no unbound factor VIIa or antibody was present after washing of the cells (data not shown).
We also excluded the possibility that the increase of activity of the TF/factor VIIa complexes could have resulted from
removal of a cell-associated inhibitor of the TF pathway by
chymotrypsin treatment. Lysate of cells apically incubated
with factor VIIa showed a TF/factor VIIa activity of 3.56 2
0.19 nmolXa/min/lO" cells (means 5 SEM of triplicate
determinations) when cells were liftedfromthe
dish by
scraping versus 3.16 2 0.06 nmolXa/min/lOh cells when
cells were detached by chymotrypsin treatment.
DISCUSSION
Previous studies have shown submaximal catalytic activity of TF/factor VII(a) complexes on the intact apicalsurface
side of cell layers.'".'' Increase of activity can be induced by
various conditions, including exposure of cells to membrane
perturbing agents, such as hydrogen peroxide33or activated
Fig 2. Localization of TF on fixed SMC layers. (A)
SMC layers were fixed in 2% paraformaldehyde plus
0.2% glutaraldehyde before immunogold-labeling
with antibodies
against
TF W4504 plus #4506;
arrows) or (B) with nonimmune mouse IgG1. Scale
bar indicates 100 nm.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1310
MULDER ET AL
J
Fig 3. Localization of TF on partlydetached SMCs
and SMCs in suspension. (A) In regions of SMC layers
in which intercellularcontacts were disrupted, more
TF
(#4503)
-positive caveolae were observed
(arrows). (6) After
immunogold-labeling
of TF
(#4503) at theapical side of SMC layers at 37°C. cells
were washed three times, detached by brief treatment with chymotrypsin, and centrifugated. Subse
quently, the cells were immediately fixed and processed for electron microscopy. Clusters of caveolae
were seen t o surround a central vacuole (asterisks),
a profile characteristic for caveolar
Scale bar indicates l 0 0 nm.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
WITH
ASSOCIATION OF SMC-TF
CAVEOLAE
1311
Fig 4. Colocalization of TF and caveolin. (A) Unfixed SMC layers were immunogold-labeledwith anti-TF antibodies (#4503) at 37°C (arrows).
The layers were then washed
and fixed in 3.5% formaldehyde followedby permeabilization in 0.2% Triton X-100. Subsequently, the cells were
washed and labeled with 5 pg/mL rabbit antibodies against caveolin followed by labeling with gold(5 nml-labeled goat antirabbt IgG (20
pg/mLl larrowheads). (B) There was negligible labeling with 5-nm gold particles when the formaldehyde-fixed cells were incubated with
nonimmune rabbit Igs instead of anticaveolin antibodies (arrowhead). Scale bar indicates 100 nm.
complement,” andby totalcell lyse^.'^'.'"^'^ It is generally
considered that increase of procoagulant activity associated
with cell surfaces can be related to a redistribution of membrane lipids, including availability of anionic phospholipids
in the proximity to TF.“’.I3 Alternatively, it has been shown
that surface-expressed procoagulant TF activity increases
after exposure of cells to proteolytic enzymes and existence
of a cryptic pool of TF has been suggested.lx.’”
In this study, we showed the presence of TF constitutively
expressed on layers of cultured SMCs, within or near noncoated microinvaginations of the plasma membrane, especially in regions in which cell-cell contacts hadbeen disrupted. These microinvaginations were recognized as
~aveolae.’~.”~~’’
Additionally, TF was internalized, probably
through coated pits,” and accumulated in compartments, belonging to theroute of degradation, ie, lysosoma l - l’k
I e Structures.
We further observed in apically labeled and enzymatically
detached SMCs that TF was frequently associated with an
extensive, dilated caveolae-like system. a pattern comparable
to thatseen in layers at sites in whichcell contacts were
disrupted. Most of these caveolae-like structures were apparently located within the suspended cells. However, because
these structures contained caveolin and could be found deep
within SMC layers when TF labeling was performed at 4°C
(data not shown), we think that these structures are most
likely caveolae associated with invaginated plasmalemma
regions.
It canbe hypothesized that loss of all TF, through its
internalization and degradation, might be prevented by excluding TF molecules from coated pits due to association
with caveolar structures. Possibly different transit pathways
of TF could be mediated by cross-linking antibodies, as has
been suggested for GPI-linked proteins,’x or by actions of
other coagulation factors andor their inhibitors. Of special
interest are. besides factor VII(a), factor IX and X, because
different activity profiles have been observed for the latter
two factors..“ Alternatively, although the capability of caveolae to be internalized has been que~tioned.~~.~”
we cannot
exclude (eventual) transfer of caveolar TF to the route of
degradation.3x“’Possible internalization of caveolar-associated TF may have been slowed downor prevented by release
Apical surfow
Total surfaw (-Vlla)
Total s u b
Fig 5. Factor Xa generation by TF/factor Vlla complexes on the
surface of layers and suspended SMCs. Layers of SMCs were incubated with factor Vlla and X and generation of activated factor X
(Xa) was measured (m; apical surface). Subsequently, these layers
were washed, detached, and resuspended in assay buffer. After the
addition offactor X, generation of factor Xa was measured with (total
surface) or without (total surface [-Vllal) adding additional factor
Vlla in theassay. Controls consisted of layers preincubated with inhibitory anti-TF antibodies (#4504; a). Means 2 SEM of triplicate
determinations are shown and are representative of three different
experiments.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1312
MULDER ET AL
of cell tensionin detached cells allowing visualization of
abundant TF-positive caveolar structures (Fig 3).
Finally, we found that catalyticactivity of TF/factor VIIa
complexes, formed on theapical side of SMC layers, increased after bringing cells in suspension, which could be
inhibited by antibodies at the apical side. Obviously, factor
VIIa as well as antibodies against TF are accessible to T F
at the apical surface side, despite the submaximal catalytic
activity of the TF/factor VIIa complexes towards factor X.
These results are consistentwithpriorfindings"
showing
two functionally distinct classesof TF/factor VIIa complexes
on intact cell layers, ie, a small number of complexes exhibiting activity towards factor X and a large number of complexes only showing activity towards factor X after lysis of
the cells. Le et allh also showed
thatinthe
layer setting
both classes exhibited activity towards a peptidyl substrate.
Furthermore, more recent data have also indicated that release of cell tension coincided with increase of T F a ~ t i v i t y . ~ '
It remains to be answered whether caveolar location of
TF has functional consequences. Of special interest in this
context is its possiblecolocalization with othercaveolar
structures, such as a calcium
or tyrosine kinases.s.l~46
On the other hand, specific absence of molecules, such as,
for instance, heparan sulphates4' bearing the inhibitors TF
pathway i n h i b i t ~ r ' ~and
. " antithrombin III,4xmay have implications for TF activity at caveolar sites.
We further speculate that the increase of TF/factor VIIa
catalytic activity towards factorX, after detachmentof cells,
may be related to enlargementof caveolar structures. For
instance, opening of TF-positive caveolar structures may result in an enhanced accessibility of factor X to TFNIIa
complexes. Additionally, the presence of anionicsitesis
important, because factor X needs interaction with anionic
phospholipids forefficient activation by TF/factor VIIa comp l e x e ~ . ~ ~Because
-"
caveolae are devoid of anionic sites,"
it could be possible that flattening of TF-positive caveolae
achieves presence of anionic sites in the proximity of the
TFlfactor VIIa complexes.
In conclusion, we showed that TF, expressed on layers
of cultured human smooth muscle cells, is associated with
caveolae. Furthermore, an enlargement of TF-positive caveolae concomitantly with an increase of activity of apically
formed TF/factor VIIa complexeswas seen after detachment
of the cells.
We speculate thatcaveolae-associated TF mayfunction
as a latent pool of procoagulant activity, which can rapidly
be activated at sites in which vessel wall integrity is lost.
ACKNOWLEDGMENT
We thank Roelie van Wijk for technical assistance and Peter van
de Sijde and Dick Huizinga for preparing the photomicrographs.
REFERENCES
1. Nemerson Y: Tissue factor and hemostasis. Blood 71: 1, 1988
2. Edgington TS, Mackman N, Brand K, Ruf W: The structural
biology of expression and function of tissue factor. Thromb Haemost
66:67, 1991
3. Rapaport SI, Rao LVM: Initiation and regulation of tissue
factor-dependent blood coagulation. Arterioscler Thromb 12: 1 I I I .
I992
4. Drake TA, Morrissey JH, Edgington TS: Selective cellular
expression of tissue factor in human tissues. Implications for disorders of hemostasis and thrombosis. Am J Pathol 134:1087, 1989
S. Wilcox JN, Smith K M , Schwatz SM, Gordon D: Localization
of tissue factor in the normal vessel wall and in the atherosclerotic
plaque. Proc Natl Acad Sci USA 86:2839, 1989
6. Marmur JD, Rossikhina M, Guha A, Fyfe B, Friedrich V,
Mendolowitz M, Nemerson Y , Taubman MB: Tissue factor is rapidly
induced in arterial smooth muscle after balloon injury. J Clin Invest
9 1 :22S3, 1993
7. Faulk WP, Labarrere CA, Carson SD: Tissue factor: Identificationand characterization of cell types in human placentae. Blood
7636, 1990
X. Drake TA, Cheng J, Chang A, Taylor FB Jr: Expression of
tissue factor, thrombomodulin, and E-selectin in baboons with lethal
Escherichia coli sepsis. Am J Pathol 142:1458, 1993
9. Scarpati EM, Sadler J E Regulation of endothelial cell coagulant properties. Modulation of tissue factor, plasminogen activator
inhibitors, and thrombomodulin by phorbol 12-myristate 13-acetate
and tumor necrosis factor. J Biol Chem 264:2070S. 1989
10. Nemerson Y: The phospholipid requirement of tissue factor
i n blood coagulation. J Clin Invest 47:72, 1968
I 1. Bach R, Rifkin DB: Expression of tissue factor procoagulant
activity: Regulation by cytosolic calcium. Proc Natl Acad Sci USA
875995, 1990
12. Drake TA. RufW, Momssey JH, Edgington TS: Functional
tissue factor is entirely cell surface expressed on lipopolysaccharidestimulated human blood monocytes and a constituvely tissue factorproducing neoplastic cell line, J Cell Biol 109:389, 1989
13. Le DT, Rapaport SI, Rao LVM: Studies of the mechanism
for enhanced cell surface factor VIIdtissue factor activation of factor
X on fibroblast monolayers after exposure to N-ethylmaleimide.
Thromb Haemost 72:848, 1994
14. Rao LVM, Rapaport SI: Studies of a mechanism inhibiting
the initiation of the extrinsic pathway of coagulation. Blood 69:645,
I987
1 S. Broze GJ Jr, Warren LA, Novotny WF, Higuchi DA, Girard
JJ, Miletich JP: The lipoprotein-associated coagulation inhibitor that
inhibits the factor VII-tissue factor complex also inhibits factor Xa:
Insight into its possible mechanism of action. Blood 71:33S, 1988
16. LeDT, Rapaport SI, Rao LVM: Relations between factor
VlIa binding and expression of factor VIIdtissue factor catalytic
activity on cell surfaces. J Biol Chem 267:15447,1992
17. Rao LVM, Robinson T, Hoang AD: Factor VIIdtissue factorcatalyzed activation of factors IX and X on a cell surface andin
suspension: A kinetic study. Thromb Haemost 67:654, 1992
I X. Maynard JR, Heckman CA, Pitlick FA, Nemerson Y: Association of tissue factor activity with the surface of cultured cells. J
Clin Invest SS:814, 197.5
19. Maynard JR, Dreyer BE, Stenerman MB, Pitlick FA: Tissuefactor coagulant activity of cultured human endothelial and smooth
muscle cells and fibroblasts. Blood 50387, 1977
20. Mulder AB, Blom NR, Van der Meer J, Smit JW, Halie MR,
Born VJJ: Basal tissue factor expression in endothelial cell cultures
is caused by contaminating smooth muscle cells. Reduction by using
chymotrypsin instead of collagenase. Thromb Res 80:399, 1995
2 I . Mulder AB, Hegge-Paping KSM, Magielse CPE, Blom NR,
Smit JW, Van Der Meer J, Halie MR, Bom VJJ: Tumor necrosis
factor a-induced endothelial tissue factor is located on the cell surface rather than in the subendothelial matrix. Blood 84:1559, 1994
22. Maciag T, Cerundolo .I, Ilsley S, Kelly PR, Forand R: An
endothelial cell growth factor from bovine hypothalamus: Identifica-
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
ASSOCIATION OF SMC-TF WITH CAVEOLAE
tion and partial characterization. Proc Natl Acad Sci USA 765674,
1979
23. Smit JW, Blom NR, Van Luyn MJA, Halie MR: Lymphocytes
with parallel tubular structures: A morphologically distinctive subpopulation. Blut 46:311, 1983
24. Born VJJ, Bertina RM: The contributions of calcium, phospholipids and tissue factor apoprotein to the activation ofhuman
blood coagulation factor X by activated factor VII. Biochem J
265:327, 1990
25. Anderson RGW, Brown MS, Goldstein JL: Role of the coated
endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts. Cell 10:351, 1977
26. Palade GE: Fine structure of blood capillaries. J Appl Physics
24:1424, 1953
27. Yamada E: The fine structure of the gall bladder epithelium
of the mouse. J Biophys Biochem Cytol 1:445, 1955
28. Mayor S, Rothberg KG, Mafield FR: Sequestration of GPIanchored proteins in caveolae triggered by cross-linking. Science
264:1948, 1994
29. Anderson RGW: Caveolae: Where incoming and outcoming
messengers meet. Proc Natl Acad Sci USA 90:10909, 1993
30. Kordylewski L, Goings GE, Page E: Rat atrial myocyte plasmalemmal caveolae in situ. Reversible experimental increases in
caveolar size andin surface density of caveolar necks. Circ Res
73:135, 1993
31. Rothberg KG, Heuser JE, Donzell WC, Ying Y-S, Glenney
JR, Anderson RGW: Caveolin, a protein component of caveolae
membrane coats. Cell 68:673, 1992
32. Rao LVM, Robinson T, Hoang AD: Factor VIIdtissue factorcatalyzed activation of factors IX and X on a cell surface and in
suspension: A kinetic study. Thromb Haemost 67:654, 1992
33. Schorer AE, Rick PD, Swaim WR, Moldow CF: Structural
features of endotoxin required for stimulation of endothelial cell
tissue factor production; exposure of preformed tissue factor after
oxidant-mediated endothelial cell injury. J Lab Clin Med 106:38,
1985
34. Carson SD, Johnson DR: Consecutive enzyme cascades:
Complement activation at the cell surface triggers increased tissue
factor activity. Blood 76:361, 1990
35. Bom VJJ, Van Hinsbergh VWM, Reinalda-Poot HH, Mohanla1 RW, Bertina RM: Extrinsic activation of human coagulation factors IX and X on the endothelial surface. Thromb Haemost 66:283,
1991
36. Anderson RGW: Potocytosis of small molecules and ions by
caveolae. Trends Cell Biol 3:69, 1993
37. Van Deurs B, Holm PK, Sandvig K, Hansen SH: Are caveolae
involved in endocytosis? Trends Cell Biol 3:249, 1993
1313
38. Montesano R, Roth J, Robert A, Orci L: Non-coated membrane invaginations are involved in binding and internalization of
cholera and tetanus toxins. Nature 296:651, 1982
39. Milici A J , Watrous NW, Stukenbrok H, Palade GE: Transcytosis of albumin in capillary ebndothelium. J Cell Biol 105:2603,
1987
40. Tran D, Carpentier J-L, Sawano F, Gorden P, Orci L: Ligands
internalized through coated or noncoated invaginations follow a
common intracellular pathway. Proc Natl Acad Sci USA 84:7957,
1987
41. Parton RG, Joggerst B, Simons K: Regulated internalization
of caveolae. J Cell Biol 127:1199, 1994
42. Carson SD, Perry GA, Pirmccello SJ: Fibroblast tissue factor:
Calcium and ionophore induce shape changes, release of membrane
vesicles, and redistribution of tissue factor antigen in addition to
increased procoagulant activity. Blood 84:526, 1994
43. Fujimoto T: Calcium pump of the plasma membrane is localized in caveolae. J Cell Biol 120:1147, 1993
44. Sargiacomo M, Sudol M, Tang Z, Lisanti M P Signal transducing molecules and glycosyl-phosphatidylinositol-linkedproteins
form a caveolin-rich insuble complex in MDCK cells. J Cell Biol
122:789, 1993
45. Bach R, Konigsberg WH, Nemerson Y: Human tissue factor
contains thioester-linked palmitate and stearate on the cytoplasmic
half-cystine. Biochemistry 27:4227, 1988
46. Zioncheck T F , Roy S, Vehar GA: The cytoplasmic domain
of tissue factor is phosphorylated by a protein kinase C-dependent
mechanism. J Biol Chem 267:3561, 1992
47. Simionescu M, Simionescu N, Silbert JE, Palade GE: Differentiated microdomains on the luminal surface of the capillary endothelium. 11. Partial characterization of their anionic sites. J Cell Biol
90:605, 1981
48. Lawson JH, Butenas S, Ribarik N. Mann KG: Complex-dependent inhibition of factor VIIa by antithrombin 111 and heparin. J
Biol Chem 268:767, 1993
49. Ruf W, Rehemtulla A, Momssey JH, Edgington TS: Phospholipid-independent and -dependent interactions required for tissue
factor receptor and cofactor function. J Biol Chem 266:2158, 1991
50. Krihnaswamy S, Field KA, Edgington TS, Momssey JH,
Mann KG: Role of the membrane surface in the activation of human
coagulation factor X. J Biol Chem 267:26110, 1992
51. Fiore M, Neuenschwander PF, Monissey JH: The biochemical basis for the apparent defect of a soluble mutant tissue factor in
enhancing the proteolytic activities of factor VIIa. J BiolChem
269:143, 1994
52. Simionescu N, Simionescu M, Palade GE: Differentiated microdomains on the luminal surface of the capillary endothelium. I.
Preferential distribution of anionic sites. J Cell Biol 90:614, 1981
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1996 88: 1306-1313
Association of smooth muscle cell tissue factor with caveolae
AB Mulder, JW Smit, VJ Bom, NR Blom, MH Ruiters, MR Halie and J van der Meer
Updated information and services can be found at:
http://www.bloodjournal.org/content/88/4/1306.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.