© 1999 American Heart Association, Inc.
Thrombosis |
Cellular Effects of Heparin on the Production and Release of Tissue Factor Pathway Inhibitor in Human Endothelial Cells in Culture
From the Vascular Biology Laboratory, Weston Centre for Experimental Research, Thrombosis Research Institute, London, United Kingdom.
Correspondence to Dr Cristina Lupu, Thrombosis Research Institute, Emmanuel Kaye Bldg, Manresa Road, Chelsea, London SW3 6 LR, UK. E-mail clupu{at}tri-london.ac.uk
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Abstract—Tissue factor pathway inhibitor (TFPI), the major downregulator of procoagulant activity of the tissue factor–factor VIIa complex (TF · FVIIa), is synthesized and constitutively secreted by endothelial cells (ECs). Here we describe the in vitro effects of heparin on the cellular localization, gene expression, and release of TFPI in human ECs in culture. Both unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH; Fragmin) time-dependently induced a significant enhanced secretion of TFPI, paralleled by a redistribution and increase of TFPI on the cell surface and a decrease of intracellular TFPI. Immunogold electron microscopy showed the presence of clusters of TFPI, both on the plasmalemma proper and within cell-surface opened caveolae/enlarged caveolar profiles. Activation of FX by TF · FVIIa on ECs treated with endotoxin was inhibited by both heparins but to a higher extent by LMWH. Inhibition of protein synthesis by cycloheximide did not reduce the release of TFPI induced by heparin. Long-term incubation (48 hours) resulted in a time-dependent enhanced production of TFPI. After the first 4 to 8 hours, depletion of intracellular TFPI was observed, more significantly with UFH. Northern blot analysis of TFPI mRNA also showed a decrease of the 1.4-kb transcript after 4 hours of incubation with UFH, followed by recovery and an increase over the control level after 24 hours. Incubation of ECs with phorbol ester (PMA) significantly enhanced the secretion of TFPI and increased its activity on the cell surface, probably by preventing invagination of caveolae. Heparin-stimulated release of TFPI decreased significantly in the presence of PMA to a level that was 2.4 times lower than the expected additive value for PMA and UFH separately. Pretreatment of ECs with PMA suppressed a subsequent response to heparin. Altogether, our results suggest that the heparin-induced release of TFPI might involve a more specific mechanism(s) than the previously hypothesized simple displacement of TFPI from the cell surface glycocalyx. We assume that the increased secretion and redistribution of cellular TFPI induced by heparins in ECs in culture can play an important role in the modulation of the anticoagulant properties of the endothelium.
Key Words: tissue factor pathway inhibitor • human endothelial cells • unfractionated heparin • low-molecular-weight heparin • caveolae
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Introduction |
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Endothelial cells (ECs) play a central role in the regulation of hemostasis by ensuring the cellular control of both procoagulant and anticoagulant mechanisms. The anticoagulant and profibrinolytic functions predominate in the quiescent state of the endothelium, thus maintaining blood fluidity (for a review, see References 1 and 21 2 ). Blood coagulation is initiated when factor VII/VIIa (FVII/VIIa) in plasma gains access to tissue factor (TF) at sites of blood vessel injury, and the resulting TF · FVIIa complex activates FX to Xa and FIX to IXa, leading to thrombin generation and the formation of a fibrin clot.3
Although healthy ECs do not express TF constitutively, marked expression of this protein occurs both in vitro after perturbation of ECs with different agonists4 5 6 and in vivo, during sepsis7 or within the tumor vasculature.8 The most physiologically significant inhibitor of the TF · FVIIa complex is the Kunitz-type tissue factor pathway inhibitor (TFPI),9 whose effect becomes manifest with the generation of limited quantities of FXa.10 TFPI uses the tandem Kunitz-type domains in its structure to form a quaternary complex with FXa bound to TF · FVIIa11 and thus prevents further production of FXa and FIXa through the TF-dependent pathway. Recent studies with transgenic mice have shown that TFPI(K1-/-) mice do not survive the neonatal stage, probably owing to unregulated TF · FVIIa hyperactivity, with the consequent consumptive coagulopathy and bleeding, and suggest that human TFPI–deficient embryos may suffer a similar fate.12
The concentration of TFPI in plasma is low (
2 nmol/L). The majority
of the circulating TFPI is associated with lipoproteins13
and represents several carboxy-terminal–truncated forms,
whereas only a minor proportion of TFPI is the full-length, free form
of the molecule.14 15 ECs express and produce TFPI
constitutively and probably contain the major pool of TFPI, residing as
uniform clusters both on the cell surface and within the apical
cytoplasm.16 17 18 19 After acute stimulation with thrombin,
the anticoagulant potency of the ECs toward the TF · FVIIa
complex increases significantly due to TFPI redistribution and enhanced
exposure on the plasma membrane.18 In resting
endothelium, TFPI is exposed on the cell surface via a
glycosylphosphatidylinositol (GPI) link and is targeted by apical
vectorial delivery to restricted plasmalemma microdomains
(caveolae) of particular proteolipidic composition and
function.20 Because the caveolae plasma membrane is devoid
of anionic binding sites,21 particularly heparan sulfate
and/or heparin,22 it is likely that there is at least 1
population of TFPI, the caveolar fraction, which is not weakly bound to
sulfated glycosaminoglycans (GAGs) on the cell
surface glycocalyx after secretion, as has been inferred until now.
Sevinsky et al23 recently described the involvement of
cellular TFPI in the assembly of the TF · FVIIa complex in
caveolae in stimulated ECs; therefore, we can assume that
cell-associated TFPI plays a more important role than the fluid-phase
form of the inhibitor in the maintenance of
hemostatic balance.
Plasma levels of TFPI increase severalfold after in vivo infusion of unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH)24 25 owing to the release of TFPI from readily available endothelial stores.10 Heparin has wide clinical use as an anticoagulant in the initial treatment of acute venous thromboembolism and unstable angina,26 27 its main action being the acceleration of antithrombin-mediated inactivation of thrombin, FXa, and FIXa and the binding of thrombin by heparin cofactor II.28 29
There is compelling experimental evidence showing that TFPI plays an important role in the antithrombotic effect of heparin. The heparin-releasable TFPI is mainly the free, full-length form of the molecule, with higher inhibitory activity toward FXa, enhanced to a greater extent by heparin, than the truncated forms of TFPI that normally circulate in plasma.30 31 Although the heparin-mediated increase of TFPI has been largely documented, most of the results have come from clinical studies and refer mainly to the plasma levels of TFPI. Therefore, the precise mechanisms behind the heparin-induced release of TFPI are still largely unknown. The main purpose of our study was to try to establish the molecular basis of the effect that different heparin preparations may have on the gene expression, synthesis, constitutive secretion, and induced release of TFPI in human ECs in culture.
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Materials
Antibodies and suppliers were as follows. Rabbit anti–recombinant (r) TFPI1–161 IgG was developed in our laboratory, immunoaffinity purified, and tested for specificity (50 µg IgG per mL produced 95% inhibition of TFPI activity in normal human plasma, and the concentration of IgG that still produced 50% inhibition of TFPI activity was
0.6 µg/mL; by
immunofluorescence, the experiments of competition,
eg, staining in the presence of rTFPI, were entirely negative).
Monoclonal anti-caveolin IgG (No. C13620; clone C060 raised against an
11.1-kDa N-terminal fragment of human caveolin) was from
Transduction Laboratories; monoclonal anti-human TF IgG (No.
4509) was from Alpha Labs. Secondary antibodies conjugated to
FITC and Vectashield mounting medium were from Vector
Laboratories Inc. Secondary antibodies conjugated to 5- or 10-nm
colloidal gold were from BioCell Research Labs; protein A coupled to
10-nm gold was from the Department of Cell Biology, University of
Utrecht, The Netherlands; and all other reagents used for
electron microscopy were from TAAB Laboratory Equipment Ltd.
Unfractionated heparin (UFH; Mr 12 kDa,
160 IU/mg) was from Chromogenix AB, and low-molecular-weight heparin
([LMWH], Fragmin; Mr 5.6 kDa, 160
anti-FXa units, 68 anti-thrombin U/mg) was from Pharmacia.
Octyl-ß-D-glucopyranoside was from
Calbiochem-Novabiochem Ltd. Human coagulation FVIIa, FX, and FXa were
purchased from Enzyme Research Labs, Ltd, and chromogenic
substrate S-2337
(N-benzoyl-L-isoleucyl-L-glutamyl-(piperidyl)-glycyl-L-arginine-p-nitroanilide
hydrochloride) was from Quadratech. TRIzol was purchased from Life
Technologies Ltd, Rapid-Hyb hybridization buffer was from Amersham Life
Science Ltd, Gene Screen nucleic acid transfer membrane was from NEN
Research Products, and polyvinylidene difluoride protein
transfer membrane was from Bio-Rad. De–N-sulfated heparin
(deNSH), rabbit brain thromboplastin, cycloheximide (CHX), phorbol
12-mirystate 13-acetate (PMA), as well as secondary antibodies
conjugated with biotin, cell culture media and supplements, endotoxin
(LPS, a lipopolysaccharide extract from Escherichia
coli serotype 0128:B12), PMSF, HEPES, Tris, BSA,
ovalbumin, sodium orthovanadate, and all other reagents were
purchased from Sigma Chemical Co Ltd unless otherwise
stated.
Cell Cultures
We used the immortalized human EC line EA.hy926,32
kindly donated by Dr Cora-Jean S. Edgell (Department of Pathology,
University of North Carolina, Chapel Hill). The cells were grown on
Petri dishes (30-mm diameter), T-75 flasks, or glass coverslips coated
with gelatin, in Dulbecco's modified Eagle's medium containing 4
mmol/L L-glutamine, 15 mmol/L HEPES, 100 U/mL
penicillin, 0.1 mg/mL streptomycin, and 1:10-diluted,
heat-inactivated FBS. Cells were used at 90%
morphological confluence, and the assays were carried out in serum-free
medium supplemented with 1 g/L BSA and 5 mmol/L
CaCl2.
Cell Treatments
ECs were incubated with the heparin preparations described
above, using different concentrations (1 or 10 U/mL for UFH and LMWH or
160 µg/mL for deNSH) and for different periods of time: up to 1 hour
for a short-time effect or up to 48 hours for the long-term effect. In
the experiments designed to study the dependence of the heparin-induced
TFPI release on protein synthesis, ECs were preincubated for 1 hour
with CHX (10 mg/L), followed by either medium only (control cells),
UFH, or LMWH (1 U/mL each) for 30 minutes at 37°C. In other
experiments, ECs were either preincubated with PMA (0.1 µmol/L)
for 1 hour to activate protein kinase C (PKC),33
followed by heparin for 30 minutes, or coincubated with PMA and heparin
for 1 hour at 37°C. When the net increase in TFPI secretion was
calculated, the level of TFPI measured in the control culture medium
was subtracted from that obtained after agonist treatment. The
"expected additive effect" of 2 different agonists was determined
by adding the net amount of TFPI secreted in cultures treated with each
agonist individually. In experiments in which sequential treatments
were done, the effect of the primary agonist on TFPI secretion was
determined using medium alone for the secondary treatment. The residual
response was used as a correction factor when interpreting the effect
of the second agonist during sequential incubations.
For all of the conditions described, supernatants were harvested after incubations and assayed for TFPI antigen and activity, whereas cell monolayers were processed as follows: (1) Cells grown on glass coverslips were rinsed with 0.1 mol/L PBS, pH 7.4, fixed for 1 hour at room temperature with 3% (wt/vol) paraformaldehyde in PBS, and used for immunofluorescence studies. (2) Cells grown in small Petri dishes were used for determination of TFPI antigen or activity on the intact monolayers, as previously described.18 (3) Cells grown in T-75 flasks were used either for extraction of total RNA or for cellular lysate preparation.
Immunofluorescence Studies
We used the indirect immunofluorescence
procedure previously described.18 20 In brief, fixed cells
were incubated with the anti-TFPI IgG for 1 hour at room temperature,
either directly or after permeabilization with 0.2 g/L saponin. All
samples were rinsed, incubated with goat anti-rabbit IgG/FITC, and
mounted with Vectashield on glass slides. Fluorescence
microscopy and digital image collection were performed with a Bio-Rad
MRC 600 confocal laser unit attached to a Nikon Diaphot inverted
microscope (Bio-Rad Microscience Ltd). Samples were analyzed by
optical sectioning through the z axis of the cells, followed
by computer-assisted reconstruction of the images. To allow a
semiquantitative comparison between control and treated cells, the
confocal parameters were kept unchanged during the digital
image collection (neutral density filter No. 2 and diaphragm opening
1/3), images were processed by computer-assisted pseudocolor
banding, and the scale of signal intensities was kept constant
throughout all of the experiments.
Cellular Lysates
After the appropriate incubation, cell monolayers were placed on
ice, rinsed with ice-cold PBS, and scrapped off the flasks in ice-cold
Tris-buffered saline (0.1 mol/L Tris-HCl and 0.15 mol/L NaCl), pH 7.8,
containing 1 mmol/L PMSF, 10 g/L aprotinin, 1 mmol/L sodium
orthovanadate, and 10 mmol/L EDTA. After
centrifugation, cell pellets were resuspended in
ice-cold lysis buffer (Tris-buffered saline containing 10 g/L Triton
X-100, 60 mmol/L octyl-ß-D-glucopyranoside, and the
cocktail of inhibitors), vigorously vortexed, and incubated
for 30 minutes at 37°C with occasional vortexing. After this step,
the insoluble proteins were removed by centrifugation;
the supernatants, representing total cellular lysates, were
collected and frozen.
Western Blotting
Cell supernatants collected after different treatments were
concentrated in Ultrafree-CL filters, and protease
inhibitors were added. From each sample, aliquots
containing equivalent concentrations of total protein (200 ng) were
precipitated with 72% (wt/vol) trichloroacetic acid. Precipitates were
washed with cold acetone, resuspended in Laemmli sample buffer, boiled,
and subjected to nonreducing SDS–polyacrylamide gel
electrophoresis (5% to 15% acrylamide gradient). Proteins
on gels were electrotransferred onto polyvinylidene difluoride
membranes,34 and TFPI was detected with the anti-TFPI IgG,
followed by biotin-conjugated anti-rabbit IgG, streptavidin coupled to
horseradish peroxidase, and peroxidative reaction with
diaminobenzidine.
Immunoelectron Microscopy
We performed both preembedding and postembedding immunogold
localization of TFPI in ECs. For preembedding, after treatment with
heparin or PMA, cell monolayers were fixed for 30 minutes at room
temperature with a mixture of 2% paraformaldehyde and 0.05%
glutaraldehyde in electron microscopy buffer (EMB; 0.1
mol/L sodium phosphate buffer supplemented with 3 mmol/L KCl and
3 mmol/L MgCl2), pH 7.6; rinsed with EMB;
quenched with 0.1 mol/L glycine in EMB; blocked for 30 minutes at
37°C with 10 g/L BSA in EMB; and incubated for 1 hour at 37°C with
anti-TFPI IgG (50 µg/mL) followed by protein A coupled to 10-nm gold
particles (diluted 1:40) in blocking solution. After being washed,
monolayers were fixed for 30 minutes with 2.5%
glutaraldehyde in 0.1 mol/L sodium cacodylate–HCl
buffer, pH 7.4; postfixed for 10 minutes with 1%
OsO4 in the same buffer; scraped off the dishes;
washed; dehydrated as pellets in a graded series of ethanol; and
embedded in epoxy resin.
For postembedding, ECs were fixed for 90 minutes at room temperature with a mixture of 3% paraformaldehyde and 0.05% glutaraldehyde in PBS, dehydrated in an ascending series of ethanol while the temperature was progressively lowered, and embedded in Lowicryl K4M as described.20 Thin sectioning was performed on a Reichert Ultracut microtome (Reichert-Jung Optische Werke), and sections placed on Formvar-coated, 200-mesh nickel grids were immunogold labeled, as described,20 by using a mixture of rabbit anti-TFPI IgG and mouse anti-caveolin IgG, followed by a mixture of secondary antibodies (goat anti-rabbit IgG coupled to 10-nm gold and goat anti-mouse IgG adsorbed to 5-nm gold).
After being counterstained with 1% OsO4, 2.5% uranyl acetate, and lead citrate, the sections were examined with a Philips 201 EM. Controls included omission of the first antibodies or their replacement with nonimmune IgGs.
Northern Blotting
Total RNA was extracted from ECs by an acid-phenol method by
using TRIzol reagent according to the manufacturer's instructions.
Total RNA (30 µg per lane) was size fractionated by gel
electrophoresis in 1.2% agarose–6% formaldehyde gels and transferred
onto a Gene Screen membrane by the capillary blot method.
Prehybridization was performed in Rapid-Hyb buffer for 2 hours at
65°C under agitation. The membrane was then hybridized under the same
conditions in buffer containing 1 to 5 ng/mL
32P-labeled probe. The cDNA probes used were a
601-bp fragment resulting from EcoRI/ClaI
digestion of human full-length TFPI cDNA (a kind gift of Dr G. Broze,
Washington University, St Louis, Mo) and an S26 full-length cDNA as the
housekeeping gene,35 both radiolabeled by random
priming with [
-32P]dCTP. The blots were
washed under increased stringency at 65°C and exposed to x-ray film
at -70°C. Densitometry and comparison between the intensity of the
bands were performed by using the public domain NIH (National
Institutes of Health, Bethesda, Md).
TF-Dependent Activation of FX
For measurement of the proteolytic activity of the TF ·
FVIIa complex toward FX, we used a 2-stage chromogenic
assay.36 ECs were stimulated with endotoxin (10 µg/mL)
in complete medium for 4 hours to express TF, then UFH or LMWH was
added, and incubation was continued for 30 minutes at 37°C. ECs were
rinsed with 10 mmol/L Tris-buffered saline, pH 7.8, containing 1
g/L ovalbumin; incubated for 10 minutes at 37°C with 10
mmol/L EDTA in the same buffer to remove endogenous and
serum-derived coagulation factors; rinsed with buffer A (10 mmol/L
HEPES, 137 mmol/L NaCl, 4 mmol/L KCl, and 11 mmol/L
D-glucose), pH 7.45; and incubated for 30 minutes at 37°C
with 10 nmol/L FVIIa in buffer A containing 10 mmol/L
CaCl2 and 5 g/L ovalbumin. Activation was
initiated by adding 200 nmol/L FX to each dish, and the incubation was
continued for 20 minutes at 37°C with shaking. At defined intervals,
subsamples of 20 µL (duplicates) were withdrawn from the cell
supernatants and transferred into microplate wells containing 20 µL
of 50 mmol/L Tris-buffered saline, pH 8.2, and 10 mmol/L
EDTA. After collection of all samples, chromogenic
substrate S-2337 was added (final concentration 0.3 mmol/L), and
the initial rate of substrate cleavage at 405 nm (mOD U/min) was
measured for 15 minutes at 37°C using a Molecular Devices THERMOmax
microplate reader (Alpha Laboratories Ltd). The amount of FXa generated
was extrapolated from a standard curve prepared with serial dilutions
of human FXa (5 to 50 fmol per well).
In antibody-blocking experiments, cells were preincubated with anti-TFPI IgG (50 µg/mL) or anti-TF IgG (100 µg/mL) for 30 minutes at room temperature before the addition of FVIIa, and the antibodies were also kept in the medium during the incubation with FVIIa. In control experiments, similar incubations of the cells with normal rabbit or mouse IgG fractions did not have any effect on the activation of FX.
Determination of TFPI Antigen and Anticoagulant Activity
TFPI antigen and activity were determined in cell supernatants,
cellular lysates, and on the cell surface, essentially as previously
described18 : the antigen was measured by both direct ELISA
(on cell monolayers) and indirect competitive ELISA (supernatants and
lysates); the activity was determined with a 2-stage
chromogenic assay, based on the ability of TFPI to inhibit
activation of FX by TF · FVIIa in the presence of FXa. Protein
estimation was done using the bicinchoninic acid assay kit (Pierce &
Warriner [UK] Ltd).
Statistical Analysis
All of the experiments described were repeated at least 3 times.
Three or 4 cell-culture dishes were used for each experimental
condition (time point, different concentration of agonists,
simultaneous or consecutive treatments, etc), and the
optical readings made in duplicate or triplicate were averaged
separately for each dish. Results from replicate experiments were
grouped for each experimental condition, and data within groups were
statistically compared between each other by the unpaired t
test and expressed as mean±SD. The mean differences between groups
illustrate comparisons either between different cell treatments at the
same point or at various points within 1 individual treatment and were
considered significant when the P value was 
0.01.
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Effect of Heparin on TFPI After Short-Time Incubation
Increased Release of TFPI
ECs incubated with UFH or LMWH (1 or 10 U/mL) for up to 1 hour released 2 to 3 times more TFPI in the supernatants, measured as either antigen or activity, than did control cells (Figures 1a
and 1b). For each time point,
P was 0.0001 for the mean differences between control and
heparin-treated cells. For UFH, the release of TFPI was both time- and
concentration-dependent (Figure 1a), with P0.0001
for the differences between 2 consecutive time points and
P0.01 for the differences between 1 and 10 U/mL. For LMWH,
the mean differences between time points and different concentrations
were less significant than for UFH.
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The activity of TFPI expressed on the EC surface did not decrease after
incubation with heparin but was enhanced by 20% for UFH and by 25%
for LMWH (P<0.01 for the mean differences; results not
shown). In agreement with this result, ELISA for TFPI on intact EC
monolayers revealed that total TFPI antigen decreased significantly
after heparin incubation, not from the cell surface, but most probably
from intracellular stores (Figure 1c
).
Immunofluorescence Analysis
The quantitative data presented above were matched by the
immunofluorescence results (Figure 2). After 1 hour of incubation with
heparin, the distribution of TFPI in ECs was changed, showing the
"unclustering" of TFPI within patches of strong
fluorescence over the cell surface (magenta, arrows) and
decreased fluorescence for intracellular TFPI (yellow; cf
control cells in row A with LMWH-treated cells in row B).
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Ultrastructural Distribution of TFPI
Resting ECs exhibited small clusters of gold-immunolabeled TFPI
dispersed both on the plasmalemma proper and within or
nearby small, uncoated membrane invaginations, which were positively
identified as caveolae by postembedding double immunolabeling for TFPI
and caveolin (Figure 3a). TFPI was also
found in vesicles/caveolae within the apical cytoplasm (same panel,
arrowheads).
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Preembedding immunostaining for TFPI on UFH- or
LMWH-treated ECs consistently revealed gold labeling on the
cell surface (Figures 3b and 3d), with frequent patches of gold
particles on the plasma membrane (illustrated for LMWH in the inset).
The postembedding double immunolabeling for TFPI and caveolin proved
that TFPI was not displaced from caveolae after treatment with UFH or
LMWH (Figures 3c and 3e). Rows of cell-surface, opened caveolae
or enlarged caveolar profiles underneath the plasma membrane exhibiting
strong immunostaining for TFPI were observed in both
UFH- and LMWH-treated ECs (Figures 3e and 3f).
Activation of FX on EC Monolayers
ECs stimulated with endotoxin for 4 hours promoted activation of
FX by FVIIa in a time-dependent manner (Figure 4). Formation of FXa did not occur on
resting cells, and LPS-treated ECs supported activation of FX as a
consequence of expression of TF, as demonstrated by the lack of FX
activation on cells preincubated with anti-TF IgG (not shown). The rate
of FXa formation decreased after 5 to 10 minutes on control cells but
continued on ECs preincubated with anti-TFPI IgG, thus excluding the
possibility of substrate depletion. For UFH-treated cells, the amount
of FXa detected in the cellular medium after 5 minutes decreased by
30% (P<0.0001) compared with control cells. The rate of
activation was significantly slower and the activation stopped earlier
than on control cells. LMWH produced a similar pattern of FX
activation, although failure to detect the initial increase in FXa
accumulation observed for UFH during the first 5 minutes might suggest
that LMWH is more efficient in reducing FX activation. Accordingly, the
mean differences between UFH and LMWH were significant for each
time-point (P0.0098 for all values). The generation of FXa
proceeded on heparin-treated cells when TFPI was blocked with the
anti-TFPI IgG, which suggests that TFPI directly inhibits the
accumulation of FXa in the cellular medium.
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Effect of Heparin on TFPI After Long-Time Incubation
Secretion of TFPI
Figure 5a illustrates the
time-dependent enhancement of TFPI secretion from ECs during their
incubation with UFH, LMWH, or deNSH for up to 48 hours. The mean
differences between control and heparin-treated ECs were highly
significant (P<0.002), and so were the differences between
2 consecutive time points (P<0.004) for each heparin
preparation tested.
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TFPI in cellular lysates (Figure 5b) decreased after 4 to 8
hours of incubation with UFH (P=0.0075), after which TFPI
gradually increased and exceeded its concentration in control cells
after 48 hours (P=0.015 for the mean difference). The rate
of TFPI secretion was relatively constant for control cells (5 ng
TFPI · mL-1 ·
h-1), whereas for UFH-treated cells, the
production increased 3-fold during the first hour of
incubation, then decreased for the next 8 hours, and increased again at
24 hours (Figure 5c). After 4 hours of incubation with UFH, ECs
failed to respond to a second challenge with UFH (10 U/mL) or calcium
ionophore A23187 (5 µmol/L) to the same extent as did control
cells. As illustrated in Figure 5d, the release of TFPI was
reduced by 56% for UFH and 47% for A23187, which suggests depletion
of intracellular stores rather than desensitization to the homologous
agent.
The Western blot analysis of the TFPI released into the cell
medium showed the presence of a major band (sometimes visible as a
doublet) with an Mr of 40 to 45 kDa (Figure 6a, lane 1). The intensity of TFPI bands
increased time-dependently in samples from heparin-incubated ECs (lanes
2, 3, and 5) compared with the TFPI constitutively secreted by control
ECs (lanes 1 and 4).
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Expression of TFPI Message
As illustrated in Figure 6b, we identified by Northern
blot, for TFPI mRNA in ECs, the 2 transcripts of 4.0 and 1.4 kb
originally described by Girard et al.37
The expression of TFPI was analyzed at different time points
during incubation of ECs with UFH or LMWH for 24 hours. After
densitometric scanning of the gels, the ratio between the band
intensity for each TFPI transcript and S26 was calculated and
represented as a percentage of the appropriate value
obtained for control cells at the same time point. Both preparations of
heparin induced a similar pattern of response (illustrated for UFH in
Figure 6c). The 4.0-kb TFPI transcript remained essentially
unchanged for the period of time tested. In contrast, the intensity of
the 1.4-kb band was reduced after 4 hours (50% for UFH and 30% for
LMWH), after which it showed complete recovery, even increased after 8
hours (90% over control for UFH and 50% for LMWH), and remained at
the same high level after 24 hours.
Cellular Distribution of TFPI
The quantitative data presented above correlate well with
the immunofluorescence studies (Figure 2).
After 8 hours of incubation with UFH or LMWH, the distribution of TFPI
was patchy over the cell surface and concentrated in areas of strong
fluorescence (shown for UFH in Figure 2, row C,
magenta). The intracellular signal appeared faint in most of the cells,
but some cells showed an accumulation of TFPI in
tubulovesicular structures around their nuclei (Figure 2, row C, permeabilized, arrows). After 24 to 48
hours, the distribution of TFPI changed again: the cell surface
immunostaining became stronger as TFPI was concentrated
in large patches of strong fluorescence (shown for UFH in
Figure 2, row D, magenta, arrows), and the staining for
intracellular TFPI was also increased compared with control cells and
accumulated mainly in perinuclear areas (Figure 2, row D,
permeabilized, arrows).
Effect of CHX on TFPI Release
The effect of CHX, as a general protein synthesis
inhibitor, on the secretion of TFPI and its induced release
was studied in experiments in which ECs were preincubated for 1 hour
with medium only (control) or CHX (10 mg/L) and then treated for 30
minutes at 37°C with medium alone, UFH, or LMWH (Figure 7a). As expected, the constitutive
secretion of TFPI decreased by 40% in cells preincubated with CHX
compared with control cells (P<0.0001). In contrast, CHX
pretreatment induced only a minimal and nonsignificant decrease of TFPI
released by ECs during their second incubation with heparin (13%
reduction for UFH, P=0.75, and 20% reduction for LMWH,
P=0.03). The cell surface activity of TFPI was reduced an
equal extent for both control and heparin-incubated cells (35%,
P<0.0001) when ECs were pretreated with CHX (Figure 7b). Moreover, treatment of ECs with heparin after preincubation
with CHX produced the same increase in cell surface TFPI activity as
that observed for control cells.
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Effect of PMA on Heparin-Induced TFPI Release
To gain further insights into the mechanism(s) of TFPI release
induced by heparin, the possible role of PKC was investigated.
Treatment of ECs with PMA for 1 hour causes activation of
PKC33 and inhibits the internalization of caveolae,
leading to their enhanced exposure on the cell surface.38
An electron photomicrograph illustrating the effect of PMA on the
distribution of TFPI in ECs after a 1-hour incubation is
presented in Figure 3g, which shows strong immunogold
staining for TFPI over continuos rows of cell-surface opened
caveolae. Similar to thrombin,18 PMA also enhanced
the release of TFPI in the cell medium (3 times over the
constitutive secretion), decreased the antigen in cellular lysates, and
induced an 40% increase of TFPI activity on the cell surface
(Figures 8b and 8c). Pretreatment of ECs
with PMA inhibited the subsequent heparin-induced release of TFPI, by
60% for UFH and completely for deNSH (Figure 8a).
|
When ECs were coincubated with PMA and heparin for 1 hour, the net
release of TFPI in the PMA-and-heparin–treated sample was 2.4 times
lower than the expected additive value for UFH and PMA tested
individually (Figure 8b). The cell surface activity measured for
TFPI was 2 times lower than the expected additive value for PMA and
heparin and 1.3 times lower for PMA and LMWH (Figure 8c). The
P value was 0.001 for all of the comparisons described in
this paragraph.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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This study analyzed the possible mechanism(s) through which UFH, an LMWH preparation (Fragmin), and deNSH act on the human immortalized EC line EA.hy926 to induce an increased release of TFPI, alteration of the cellular expression and distribution of the inhibitor, and modification of the anticoagulant activity of the cells.
The several-fold increase in plasma TFPI levels after in vivo infusion of heparin has been largely documented,24 25 but the mechanism by which this process occurs has never been deciphered. Based on circumstantial evidence, it was widely accepted that part of the TFPI secreted from ECs became reattached to sulfated proteoglycans in the cell surface glycocalyx through electrostatic bonds between the positively charged C-terminus of TFPI and the negatively charged sulfate groups in GAGs.39 40 Heparin was simply thought to displace TFPI, from its binding sites/GAGs on the EC surface and into the bloodstream, in the form of heparin-TFPI complexes.41 Recently, more pieces of evidence have been gathered to point out a possibly greater significance of the endogenous, cell-associated TFPI than of the circulating form of the inhibitor in maintaining the anticoagulant properties of the endothelium. First, TFPI is located constitutively in ECs within specific glycolipid microdomains/caveolae,20 which represent highly specialized plasmalemma domains performing specific functions: endocytosis and transcytosis,42 43 potocytosis,44 regulation of cell surface–associated proteolysis,23 45 calcium regulation, and signal transduction.44 46 Second, TFPI is exposed on the cell surface through a specific link, the GPI anchor,20 23 and mediates the formation and translocation of TF · FVIIa-FXa complexes in caveolae in activated ECs.23 Third, removal of heparan sulfate from the EC surface with heparitinase or treatment with sodium chlorate to inhibit GAG sulfation does not affect the exposure of TFPI on the cell surface or its anticoagulant activity,20 nor do they prevent the binding of exogenously added rTFPI to the cells.47 Last, in a recent study of immunolocalization of TFPI in ECs, Hansen et al19 detected cellular TFPI on the cell surface and in plasmalemma vesicles, as well as within the endocytic compartment, thus confirming the presence of at least 1 pool of cellular TFPI, probably GPI anchored in the membrane, which can undergo recycling and degradation. On the basis of these considerations and the biochemical and morphological evidence we present herein, we propose that the heparin-induced release of TFPI, at least from ECs in culture, involves more complicated and specific cellular mechanisms than a simple displacement of TFPI allegedly bound to sulfate residues in the cell surface glycocalyx.
As expected, short-time incubation of ECs with heparin resulted in enhanced release of TFPI. The process is both time and concentration dependent and is more significant for UFH than for LMWH, at least at high concentrations (10 U/mL). Inhibition of protein synthesis in ECs does not reduce the effect of heparin, which suggests that the release occurs mostly from preformed TFPI pools, likely to be located intracellularly. Immunofluorescence images also showed that TFPI decreases intracellularly after treatment with heparin and redistributes over the cell surface by unclustering within large patches. We suggest that this patchy redistribution might support the increased TFPI activity and antigen determined by quantitative measurements on intact monolayers. Ultrastructurally, clusters of immunogold-labeled TFPI were frequently found on the plasmalemma surface and in caveolae in ECs treated with heparin, as proved by the postembedding double staining for TFPI and caveolin.
The significance of membrane-bound TFPI is reinforced by results from the experiments in which the TF · FVIIa complex–dependent formation of FXa on LPS-stimulated ECs was studied. The amount of FXa measured in the medium of control cells decreased after 5 to 10 minutes, but the activation reaction continued when ECs were preincubated with anti-TFPI IgG. The ability of heparin-treated ECs to support FX activation is very poor, and after a slight but significant increase of FXa in the medium during the first 5 minutes, the accumulation of FXa stops. It is known that heparin potentiates the inhibition of FXa by TFPI by increasing both the affinity and the rate of reaction, probably through direct acceleration of the interaction between TFPI and FXa, which leads to rapid inhibition of TF · FVIIa by the TFPI-FXa complex.48 For ECs, where free FXa is not efficiently bound by cellular TFPI,23 downregulation of TF · FVIIa activity on the cell surface is dependent on the formation of the quaternary complex with FXa and TFPI and its translocation in caveolae, whose particular composition, notably the lack of anionic binding sites, inefficiently supports the proteolytic function of the TF · FVIIa complex.23 Our data showing that the amount of FXa in the medium of heparin-treated cells decreases below starting point levels, though puzzling at first, can be explained if we consider that the FXa generated initially is used for the formation of the quaternary complex by binding to TFPI in caveolae. From this stage, the complex might undergo endocytosis on the route followed by TFPI,19 20 which would lead to downregulation of the TF · FVIIa activity on the EC surface. Alongside the morphological evidence, results from FX activation experiments reinforce the idea that caveolar TFPI can play an important role in heparin-treated ECs. Accordingly, TFPI in caveolae/enlarged caveolar profiles, which are devoid of anionic sites,21 22 is probably not displaced by heparin, which, on the contrary, seems to induce an enhancement of the number of opened caveolae. We suggest that this can provide ECs with a protected reservoir of potent anticoagulant molecules, stabilized by heparin and probably ready to become functionally active when the ECs are induced to express TF after stimulation.
Long-time incubation of ECs with heparin also induced enhanced secretion of TFPI and a decrease of the level of the inhibitor in cellular lysates. However, the rate of secretion decreased after 4 to 8 hours of incubation with heparin and was enhanced again at 24 hours. The amount of TFPI in cellular lysates also decreased during the first 4 to 8 hours of incubation, suggesting that the intracellular pools of TFPI had become depleted. In addition, ECs treated with heparin for 4 hours failed to respond to a second challenge with heparin or calcium ionophore A23187, with the same release of TFPI as from control cells. In an in vivo study reporting the effect of repeated or continuous intravenous infusion of heparin on the release of TFPI in plasma,49 Hansen et al49 demonstrated depletion of intravascular pools of TFPI, with the major part of the decrease observed after 4 to 8 hours, and suggested that the constitutive synthesis of TFPI was overwhelmed by enhanced secretion of the inhibitor. From our analysis of the expression of TFPI message during the incubation of ECs with UFH or LMWH, we can suggest that the observed depletion might also be due to the decrease of TFPI mRNA, significantly manifested for the 1.4-kb transcript after 4 hours of heparin treatment.
The recovery and increase of intracellular TFPI observed at longer
times might be due to cell-mediated degradation of heparin, although
the enhanced rate of TFPI release may indicate more specific
intracellular effects involving protein synthesis. A study of the time
course of heparin binding to human umbilical vein ECs showed that at
least 3 hours were necessary to reach saturation at 37°C, then part
of the bound heparin underwent endocytosis, and 30% of the
specifically bound heparin remained associated with the cells as an
internalized pool.50 Although the intracellular location
of heparin has not been identified in ECs, some of the internalized
heparin is known to proceed to the nucleus of HeLa cells and
hepatocytes; and additionally, a putative heparin receptor
has been described in ECs.51 Other specific cellular
effects of heparin include the selective inhibition of the
mitogenic stimulation of smooth muscle cells by phorbol
esters and serum, the suggested mechanism being that heparin
selectively represses phorbol ester–inducible activator
protein-1–mediated gene expression by interfering with the binding of
the heterodimeric Fos-Jun/activator protein-1 transcription
factor complex to activator protein-1–like promoter
elements.52 Whether or not heparin can also affect TFPI
expression by an interference with the activator
protein-1–like consensus sequences present in the 5' upstream
region of the TFPI gene53 is a matter of speculation at
this moment and requires further investigation. Comparison between UFH,
LMWH, and deNSH showed that, at least for cultured ECs, there is no
major difference regarding their effect on TFPI. This suggests that the
electric charges and the Mr of heparin are
not major determinants of TFPI release in vitro.
To better understand the mechanism(s) by which heparin induces the
observed modifications of TFPI in ECs, we addressed the question of
whether heparin can interfere with some of the signal transduction
pathways whose machinery is found in caveolae.44 PKC-
and a protein phosphatase are probably the key regulatory enzymes that
control the internalization cycle of caveolae.38 54
PKC- is highly concentrated in caveolae in unstimulated cells and is
constitutively active in this location.55 Treatment of
cells with phorbol esters or agonists that raise the concentration of
diacylglycerol in the cells (thrombin or histamine) further
activates PKC- and inhibits internalization of
caveolae.38 55 In our experience, pretreatment of ECs with
PMA for a short time inhibits subsequent heparin-induced secretion of
TFPI. The coincubation of ECs with PMA and heparin also shows strong
antagonism and suggests that the effects of heparin on
endogenous TFPI in ECs might involve specific cellular
mechanism(s). It is known, for example, that heparin inhibits the
PKC-–dependent pathway of mitogenesis in smooth muscle cells and
fibroblasts, but because it does not directly affect PKC activity or
the phosphorylation step, it is believed that the
heparin block is distal to the activation of PKC.56 On the
other hand, there are other cellular responses elicited by PMA, such as
stimulation of phospholipase D, which can occur independently of
PKC.57 Therefore, further work is needed to elucidate the
nature of the mechanism(s) involved in the release and redistribution
of TFPI in ECs in culture.
The enhanced secretion of TFPI induced by heparin and the modifications of cellular TFPI observed in human ECs in culture, if proven to take place in vivo also, may represent an important mechanism designed to mobilize TFPI from the endothelium to sites of TF exposure with ongoing thrombosis (eg, a damaged vessel wall or TF expressed on monocytes or in atherosclerotic plaque rupture areas)49 or, probably more important, to confer highly regulatory anticoagulant properties to the intact endothelium.
|
Acknowledgments |
|---|
This work was supported by stipends from Pharmacia Upjohn (C.L.), the British Heart Foundation (C.L.), (grant PG/97045), Stanley Thomas Johnson Foundation (F.L.), The Royal Society (C.L., F.L.), and the Thrombosis Research Trust. We thank Dr Cora-Jean S. Edgell (Department of Pathology, University of North Carolina, Chapel Hill) for kindly providing us with the EC line EA.hy926, as well as Dr George Broze, Jr, (Washington University, St Louis, Mo) for the human TFPI cDNA. We highly acknowledge the skilful help of Sally Mill (cell culture).
Received December 1, 1998; accepted January 15, 1999.
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