* Gaubius Laboratory TNO-PG, 2301 CE Leiden, The Netherlands; Electron Microscopy Unit, Clusius Laboratory, University
of Leiden, 2333 AL Leiden, The Netherlands; and § Thrombosis Research Institute, SW3 6LR London, United Kingdom
In previous studies we have shown that, after stimulation by a receptor ligand such as thrombin, tissue-type plasminogen activator (tPA) and von Willebrand factor (vWf) will be acutely released from human umbilical vein endothelial cells (HUVEC). However, the mechanisms involved in the secretion of these two proteins differ in some respects, suggesting that the two proteins may be stored in different secretory granules.
By density gradient centrifugation of rat lung homogenates, a particle was identified that contained nearly all tPA activity and antigen. This particle had an average density of 1.11-1.12 g/ml, both in Nycodenz density gradients and in sucrose density gradients. A similar density distribution of tPA was found for a rat endothelial cell line and for HUVEC. After thrombin stimulation of HUVEC to induce tPA secretion, the amount of tPA present in high-density fractions decreased, concomitant with the release of tPA into the culture medium and a shift in the density distribution of P-selectin.
vWf, known to be stored in Weibel-Palade bodies, showed an identical distribution to tPA in Nycodenz gradients. In contrast, the distribution in sucrose gradients of vWf from both rat and human lung was very different from that of tPA, suggesting that tPA and vWf were not present in the same particle.
Using double-immunofluorescence staining of HUVEC, tPA- and vWf-containing particles showed a different distribution by confocal microscopy. The distribution of tPA also differed from the distribution of tissue factor pathway inhibitor, endothelin-1, and caveolin. By immunoelectronmicroscopy, immunoreactive tPA could be demonstrated in small vesicles morphologically different from the larger Weibel-Palade bodies. It is concluded that tPA in endothelial cells is stored in a not-previously-described, small and dense (d = 1.11- 1.12 g/ml) vesicle, which is different from a Weibel-Palade body.
OF the physiologically occurring plasminogen activators, tissue-type plasminogen activator (tPA)1 is
the most important one in triggering physiological
fibrinolysis and thrombolysis. Transgenic mice in which
the tPA gene has been functionally disrupted (Carmeliet
and Collen, 1996a A similar situation pertains to von Willebrand factor
(vWf), another endothelial cell protein that is synthesized
by, and stored in, the endothelium and is secreted from endothelial cells by both constitutive and regulated secretion
(for review see Reinders et al., 1988 Materials
Nycodenz was obtained from Nycomed AS (Oslo, Norway); sucrose for
density gradients from BDH (Poole, UK). Human Murine monoclonal antibodies against human tPA (clones ESP-4, ESP-5,
ESP-6, PAM-3) and rabbit polyclonal anti-recombinant human tPA IgG
(ADI385R) were purchased from American Diagnostica (Greenwich,
CT). The murine monoclonal anti-tPA antibodies 8C11 and 2B5 were
from Celsus Laboratories (Cincinnati, OH). Rabbit polyclonal anti-
mouse tPA was a gift from Dr P. Carmeliet (Leuven, Belgium). Rabbit
anti-vWf, murine monoclonal anti-vWf IgG, peroxidase-conjugated swine
anti-rabbit Ig, and antibody diluent were from DAKO (Glostrup, Denmark); rabbit anti-caveolin was from Transduction Laboratories (Mamhead Castle, UK). Rabbit anti-human P-selectin (Coughlan et al., 1994 Cells and Tissues
Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase digestion (Jaffe et al., 1973 The rat endothelial cell line RHE, kindly provided by Dr C.A. Diglio
(Wayne State University School of Medicine, Detroit, MI; Diglio et al.,
1988 Rat lung, mouse lung, and mouse diaphragm were obtained from Nembutal-anaesthetized (60 mg/kg i.p.) animals. Human lung was obtained as
anonymous and nontraceable material from lung cancer surgery. Lung
specimens for fractionation were washed free of blood and stored in cold
saline.
Cell and Tissue Homogenization
Lung tissue was finely divided by a McIlwain tissue chopper and suspended in homogenization buffer (5 mM Tris-HCl, 220 mM sucrose, pH,
7.4; 10 ml buffer/gram of tissue). Confluent HUVEC (60 cm2, ~6 × 106
cells) were washed once and scraped into 2 ml homogenization buffer. For
HUVEC homogenates, which contain only low amounts of protein, this
buffer also contained 0.001% Tween-80 to improve tPA recovery (at this
concentration, Tween-80 did not lyse organelles). For the experiments on
thrombin-induced secretion, 300 cm2 HUVEC cultures were used and
scraped into 5 ml buffer. Cells or tissue were homogenized with ten
strokes (1500 rpm) in a glass-teflon Potter-Elvehjem tissue homogenizer,
and the homogenates were then centrifuged at 800 g for 5 min at 4°C. An
aliquot of the supernatant ("total homogenate") was retained to calculate
recoveries, and the remainder was fractionated.
Nycodenz Density Gradient Centrifugation
A Nycodenz solution containing 35% (wt/vol) of Nycodenz was prepared
in 5 mM Tris-HCl buffer (pH 7.4). A Nycodenz stock solution of 30% (wt/
vol) was prepared in homogenization buffer. From this stock solution, solutions of Nycodenz of 27.5 to 5% (in increments of 2.5%) were prepared
in homogenization buffer. A density gradient was made in 14 ml ultracentrifuge tubes, starting with one ml of 35% Nycodenz, and then in eleven
steps of one ml from 30% to 5% Nycodenz, and left to equilibrate overnight at 4°C. Two ml of cell or tissue homogenate was layered on top of
the gradient, and the gradient was centrifuged for 150 min at 40,000 rpm
(202,000 gav) in a Beckman L7-55 ultracentrifuge equipped with a SW-40 rotor, at 4°C. Subsequently, one ml fractions were collected, starting at the
bottom, and stored frozen. The density of the fractions was determined in
a Mettler/Paar DMA 602M density measuring cell, and ranged from 1.03-1.17 g/ml (see Results section). For additional protocol see Wanders et al. (1987) Sucrose Density Gradient Centrifugation
A 46% (wt/vol) stock solution of sucrose (1.375 M) in 5 mM Tris-HCl, pH
7.4, was prepared (for HUVEC, 0.001% Tween 80 was added). From this
stock, sucrose solutions of 43 to 10% (in increments of 3%) were prepared
in buffer. A sucrose density gradient (46-10% sucrose; 12 steps of 1 ml)
was made in 14-ml tubes and left to equilibrate overnight at 4°C. 2 ml of
cell or tissue homogenate was layered on top of the gradient, and the gradient was centrifuged exactly as described above for the Nycodenz gradient. 1-ml fractions were collected, and their densities were measured, as
described above. The density of the sucrose gradient fractions ranged
from 1.03 to 1.16 g/ml.
Assay of Fractions
In the fractions the following parameters were determined, using miniaturized assays in microtiter plates. Where necessary, data were corrected
for interference by the Nycodenz or sucrose content of the fractions. Protein concentration was measured spectrophotometrically by the BCA protein assay procedure as prescribed by the manufacturer (Pierce, Rockford,
IL). tPA activity (Verheijen et al., 1982 Acid phosphatase (substrate: p-nitrophenylphosphate at pH 5.0), alkaline phosphatase (substrate: p-nitrophenylphosphate at pH 10.5), neutral
esterase (substrate: p-nitrophenylacetate at pH 6.6), 5 Western Blotting of P-Selectin
The presence of P-selectin in Nycodenz fractions of HUVEC was determined semi-quantitatively by slot blotting, using a polyclonal rabbit anti-
human P-selectin (Coughlan et al., 1994 Immunocytochemistry
First passage HUVEC were cultured to confluency as described above on
glass coverslips coated with glutaraldehyde-crosslinked gelatin. Once confluent, cells were fixed with 2% freshly depolymerized p-formaldehyde in
PBS containing 5% sucrose (PBS/sucrose) for 1 h at 20°C. Cells were then
washed extensively in PBS/sucrose and briefly permeabilized by treatment with a solution of 0.02% saponin in PBS for 10 min at 18°C. Cells
were then washed in PBS, quenched with 100 mM glycine in PBS, blocked
by treatment with DAKO antibody diluent supplemented with 1% (vol/
vol) normal goat or horse serum for 30 min, and incubated for 1 h at 18°C
with primary antibody diluted in the same blocking solution. As primary antibodies were used, either a cocktail of monoclonal anti-tPA antibodies
(IgG 10 µg/ml) for single labeling or the mixture of these anti-tPA monoclonals with polyclonal rabbit anti-vWf IgG (diluted 1:200) for the double
immunostaining approach. To confirm the labeling pattern, in double labeling experiments a rabbit polyclonal anti-tPA IgG in combination with
monoclonal anti-vWf antibody was used. All specimens were extensively
washed with PBS and incubated for 1 h at 18°C with the secondary antibodies. For single staining we used horse anti-mouse IgG-FITC (diluted 1:100 in PBS) and for double staining a mixture of horse anti-mouse IgG-Texas red and goat anti-rabbit IgG-FITC (both diluted 1:100 in PBS).
Cells were again extensively washed in PBS, briefly rinsed in distilled water, and mounted in Vectashield. The fluorescent specimens were examined with a confocal laser scanning unit (MRC600; Bio Rad, Hemel
Hampstead, UK) attached to a Nikon Diaphot inverted microscope. The
light source was a krypton/argon laser with main lines at 488, 568, and 674 nm. For the visualization of FITC and Texas red staining, K1 and K2 filter
blocks were used. The recorded images were merged to study colocalization. Some samples were analyzed by serial optical sectioning in the Z-axis
of the cells, followed by computer-assisted reconstruction of the images.
Controls to ascertain for the specificity of the binding comprised either
replacement of the first antibodies with normal IgG from the same species or
incubation of the cells with the conjugates only. Staining for double labeling
of tPA and TFPI, endothelin-1, or caveolin, respectively, was performed identically as described for tPA/vWf double labeling, using as antibodies rabbit
anti-TFPI, rabbit anti-endothelin-1, and rabbit anti-caveolin, respectively.
Immunogold Electron Microscopy
For immunogold labeling, HUVEC were grown on Thermanox coverslips,
washed with serum-free medium, and fixed with 3% (wt/vol) p-formaldehyde (freshly prepared) in PBS for 90 min at room temperature. Mouse
lung and diaphragm were fixed by perfusion with 3% p-formaldehyde in
PBS for 10 min, followed by immersion fixation at room temperature for a
further 2 h. Both the cell monolayers and the mouse tissues were dehydrated in an ascending series of ethanol while the temperature is progressively lowered, following the progressive lowering of temperature protocol (for details see Roth, 1989 Density Gradient Centrifugation of Rat Lung on a
Nycodenz Gradient
Rat lung was chosen for the first experiments, since it is, of
all rat tissues, richest in tPA (Padró et al., 1990
Recentrifugation of the two-peak fractions (d = 1.11-
1.12 g/ml, diluted 1:1 with homogenization buffer to reduce density) on an identical Nycodenz gradient resulted
in recovery of tPA and vWf at the density of the original
fractions (Fig. 2 a). To decide whether the distribution of
tPA in the gradient might have been due to binding of particle-free tPA to a particle of d = 1.11-1.12 g/ml, recombinant human tPA (Activase; final concentration 50 ng/ml)
was added to a rat lung homogenate before gradient centrifugation. Subsequently, we separately measured the exogenously added human tPA antigen and the endogenous
rat tPA antigen by species-specific tPA ELISA assays. Rat
tPA was recovered as a single peak at its normal density,
while the human tPA was found distributed diffusely through the gradient (Fig. 2 b). Upon ultracentrifugation
of a lung homogenate in homogenization buffer without
Nycodenz (d = 1.03 g/ml), >90% of tPA and vWf was recovered at the bottom of the tube. After ultracentrifugation in homogenization buffer without Nycodenz but now
containing 1% Triton X-100 to lyse organelles, tPA and
vWf were diffusely distributed through the gradient (not shown). From these data it was concluded that almost all
tPA was present in rat lung homogenates in sedimentable
particles of d = 1.11-1.12 g/ml.
To see whether any of the tPA or vWf could be ascribed
to tPA or vWf in a pathway of ongoing protein synthesis,
rats were pretreated with cycloheximide (2 mg/kg in vivo)
at 3 h before being killed. This procedure inhibits rat protein synthesis fully but does not significantly influence the
size of the storage pool in rat lung (Tranquille and Emeis,
1989 Fractionation of a cell homogenate from a rat heart endothelial cell line (Diglio et al., 1988 Density Gradient Centrifugation of Rat Lung on a
Sucrose Gradient
Using a sucrose gradient of d = 1.03-1.16 g/ml, tPA (both
antigen and activity) was found at the same density (1.11-
1.12 g/ml) as in Nycodenz gradients (Fig. 3). vWf, however, showed a different distribution than in Nycodenz
gradients, as no vWf peak was present in fractions 6 and 7 of sucrose density gradients. This resulted (Fig. 3) in a
clear separation of tPA (peak in fractions 6 and 7) and
vWf (peak in fractions 10 and 11). Marker enzymes for
subcellular fractions, including the mitochondrial marker glutamate dehydrogenase, showed, in sucrose gradients, a
similar distribution as in Nycodenz gradients (not shown).
Density Gradient Centrifugation of Human Lung
Homogenates of human lung were fractionated on a Nycodenz density gradient and on a sucrose density gradient.
The distribution of tPA (antigen and activity) and vWf
was more complex in human lung, and less tPA and vWf
was present in fractions 7 and 8 of the gradients, compared
to rat lung. In sucrose gradients, vWf had practically disappeared from fraction 7 and 8. Of the vWf recovered, 14% ± 3 was found in fractions 7 and 8 in Nycodenz gradients, but
only 2% ± 2 in sucrose gradients (mean ± SD, n = 4; P < 0.01). No such difference between Nycodenz and sucrose gradients was found for the tPA content of fractions 7 and
8 (19% ± 4 in Nycodenz gradients versus 16% ± 4 in sucrose gradients, n = 4, P = 0.33).
Density Gradient Centrifugation of HUVEC
First passage HUVEC (60 cm2/experiment) were fractionated on Nycodenz gradients. In Fig. 4, the averaged distribution of tPA antigen and of vWf is shown for five such
fractionations. Slight differences in density distribution,
especially for tPA, were found between cultures, but the
overall distribution pattern (Fig. 4) resembled that found
in rat lung. For tPA, the major peak was found at d = 1.105 g/ml (fraction 6), with a (variably pronounced) shoulder at d = 1.13 g/ml (fraction 4). A second, smaller
peak was seen at d = 1.06 g/ml (fraction 10). vWf had a
single major peak (d = 1.105-1.115; fractions 5 and 6),
and, in three out of five cultures, a small shoulder at d = 1.06 g/ml (fraction 11). No sucrose density gradient centrifugation was performed on HUVEC.
Thrombin Stimulation of tPA Secretion
For experiments involving thrombin stimulation of regulated secretion (acute release) of tPA, 300-cm2 HUVEC
cultures were used that had been preincubated for 24 h with 1 mM sodiumbutyrate to increase tPA synthesis and
storage (Kooistra et al., 1987
Although the differences in tPA concentration in the
fractions induced by thrombin were relatively small, immunocytochemistry of tPA antigen in these cells showed
that, after 3 min of thrombin stimulation, the granular tPA
staining had disappeared from most cells (Fig. 6), while in
other cells a weak granular staining persisted. This loss of
staining is in agreement with biochemical data, which
showed that, after 1 NIH U/ml thrombin stimulation, no
further acute release of tPA could be obtained by ionomycin (van den Eijnden-Schrauwen et al., 1995
Immunohistochemical Localization of tPA and vWf
in HUVEC
The cellular localization of tPA in HUVEC was visualized
by indirect immunofluorescence labeling and confocal microscopy, as well as at the ultrastructural level by postembedding immunogold labeling and electron microscopy. In
formaldehyde-fixed, saponin-permeabilized HUVEC, tPA
showed, by immunofluorescence, well defined granular staining in the cytoplasm of the cells (Fig. 8 f; compare
with Fig. 6). To enable precise localization of the fluorescence, some specimens were analyzed by serial optical sectioning followed by computer-assisted reconstruction of
the image. Serial optical sectioning along the Z-axis revealed that granular tPA staining was localized in the perinuclear, organelle-rich area as well as in the attenuated areas at the cell edges (Fig. 9).
The granular localization of tPA was confirmed by
postembedding immunogold labeling of ultrathin sections
of HUVEC (Fig. 8, a and b) and of vascular endothelial
cells in mouse lung and diaphragm capillaries (Fig. 8, d
and e). The gold particles delineating tPA were present
throughout the cells, often in clusters over small, fairly
electron-dense vesicles, which in favorable sections could
be seen to be surrounded by a membrane (Fig. 8, b, d, and e). The location of the gold particles suggested that they
were superimposed on the small vesicles that are abundantly present in endothelial cells after routine fixation
and embedding and that are much smaller than Weibel-Palade bodies. The gold particles delineating vWf were detected on much larger, elongated particles, which contained densely packed fibrillar material and had the morphological characteristics compatible with Weibel-Palade
bodies (Fig. 8 c, W-Pb).
No Colocalization of tPA with vWf in HUVEC
To further differentiate between tPA- and vWf-containing
granules in HUVEC, we performed double immunolabeling for tPA and vWf with Texas red- and FITC-labeled
secondary antibodies and simultaneous visualization of
the two proteins by confocal microscopy. To detect
whether the two proteins were colocalized, the two images were merged; any superimposed green and red areas
would then appear in yellow. As illustrated by Fig. 8, f-h
(f, tPA; panel g, vWf), no colocalization of the two antigens was observed (h, in which e and f are superimposed,
so that colocalization of tPA and vWf would show as yellow). Control experiments gave no labeling in all conditions tested (not shown).
No Colocalization of tPA with endothelin-1, caveolin,
or TFPI in HUVEC
Using identical techniques of indirect immunofluorescence and confocal microscopy as described above for
vWf, we looked for colocalization of tPA with three other
endothelial proteins: endothelin-1 and tissue factor pathway inhibitor, which are both secreted by HUVEC, and
caveolin, a component of caveolae (Lisanti et al., 1994).
As shown in Fig. 10, no evidence for colocalization of
these proteins with tPA in HUVEC was obtained. We
show elsewhere (Lupu et al., 1997
Remarkably, the first studies on the subcellular fractionation of plasminogen activators (at that time called "fibrinolysokinase activators," now known to be tPA) from
tissue date 1950 (Tagnon and Palade, 1950 Meanwhile, the Weibel-Palade body has been firmly established to be the storage particle for vWf. This particle,
first described as an endothelial organelle characterized by
specific morphological features (Weibel and Palade, 1964 In clinical and in experimental (in vivo and in vitro)
studies, the regulated secretion (acute release) of tPA and
vWf generally occurs simultaneously (Tranquille and
Emeis, 1989 Our data show that tPA and vWf are not located in a
single storage granule but in granules that can be separated by sucrose density gradient centrifugation and that
can be visualized as separate by light- and electron-microscopical immunocytochemistry. Ultracentrifugation of rat
lung homogenate in buffer showed that tPA (and vWf)
were present in a sedimentable particle, from which the
proteins could be released by Triton treatment. By density gradient ultracentrifugation on a Nycodenz gradient, the
density of the particle was determined to be 1.11-1.12 g/ml.
The distribution of tPA in the Nycodenz gradient was not
due to nonspecific binding of soluble tPA (Fig. 2 b), while
the particle was stable upon recentrifugation (Fig. 2 a). In
previous experiments, we have shown that tissue stores of
tPA, including tPA stores in rat lung, are metabolically
highly stable, hardly showing any decrease 3 h after protein synthesis has been inhibited by cycloheximide (Tranquille and Emeis, 1989 In Nycodenz gradients, the distribution of vWf and tPA
was nearly identical, suggesting that the two proteins could
be present in a single storage granule (Figs. 1, a and b, and
2 a). The equilibrium density of this putative storage granule was, moreover, similar to the equilibrium density of
Weibel-Palade bodies in other types of density gradient,
e.g., Percoll gradients (Reinders et al., 1984 In sucrose gradients, however, the distribution of vWf
differed from that of tPA. tPA showed essentially the
same density distribution as in Nycodenz gradients, but
vWf had disappeared from the d = 1.11-1.12 g/ml fractions (compare Fig. 3 with Figs. 1, a and b, and 2 a). This
observation showed that the two proteins were not stored
in the same granule in rat lung. An explanation for the
density shift of vWf might be destabilization of the Weibel-Palade body by sucrose, leading to collapse, or dehydration by the hypertonic sucrose solutions, leading to a
change in density. The tPA storage particle would then be
immune to these forces. Similar to rat lung, sucrose density centrifugation resulted in human lung in a significant
loss of vWf antigen from the d = 1.11-1.12 g/ml fractions
(from 14% in Nycodenz to 2% in sucrose), without causing a loss of tPA from these fractions (from 19 to 16%).
The distribution of tPA from rat heart endothelial cells
was very similar to that from rat lung. In human lung and
HUVEC (Figs. 4 and 5) the distribution pattern of tPA
and vWf was more complex, although peaks of tPA and
vWf could still clearly be identified at d = 1.11-1.12 g/ml.
Presumably, the presence of other cells (including blood
platelets), more connective tissue in the lung, and more
subendothelial matrix in HUVEC contributed to the more complex patterns observed in homogenates from human
lung and from HUVEC.
Two other small particles have been isolated from endothelial cells. Caveolae (Lisanti et al., 1994; Schnitzer et
al., 1995 Stimulation, by thrombin, of acute secretion of tPA and
vWf from HUVEC (van den Eijnden-Schrauwen et al.,
1995 Thrombin-induced release of tPA was accompanied
(Fig. 7) by a shift to slightly higher densities and an increase in immunoreactivity of P-selectin, a component of
Weibel-Palade bodies (Wagner, 1993 The morphological observations on HUVEC, obtained
using immunocytochemical labeling procedures with confocal microscopy and immunoelectronmicroscopy on thin
sections, showed that tPA was localized throughout the cytoplasm of HUVEC in small vesicular structures, which
differed from Weibel-Palade bodies in size and morphology. Particles staining for vWf were larger and contained a
more electron-dense and fibrillar core. No significant colocalization of tPA- and vWf-containing particles was observed in double immunocytochemical labeling experiments. In summary, the morphological data support and
extend the biochemical data that tPA-containing vesicles
and vWf-containing Weibel-Palade bodies are distinct entities.
Recent observations (van den Eijnden-Schrauwen, 1996,b) and that consequently do not have
tPA circulating in their blood, show a reduced thrombolytic potential and an increased thrombogenic tendency.
Also, clots from which tPA has been removed (functionally by inactivation, or immunologically by immunoabsorbtion) will not lyse (Wun and Capuano, 1985
, 1987
).
Circulating tPA is derived from vascular endothelial cells,
which have been shown to synthesize tPA both in vivo
(Levin and del Zoppo, 1994
; Lupu et al., 1995b
; Padró et
al., 1995
; Schneiderman et al., 1995
) and in vitro (for review see van Hinsbergh et al., 1991
; Emeis et al., 1996
;
Kooistra and Emeis, 1997
). In vivo, tPA can also be demonstrated in vascular endothelial cells by functional and
immunocytochemical techniques, although the precise localization of tPA in endothelial cells is still not settled (for
review see Emeis et al., 1996
). In culture, endothelial cells
constitutively secrete tPA into the medium. Regulated secretion from endothelial cells has been demonstrated as
well, both in vivo (for review see Emeis, 1988
, 1996
; Emeis et al., 1996
) and recently in vitro (van den Eijnden-Schrauwen et al., 1995
, 1997
).
; Wagner, 1990
, 1993
;
Mayadas and Wagner, 1991
; Hop and Pannekoek, 1996
).
In most experimental and clinical situations, tPA and vWf
are secreted simultaneously upon stimulation (Tranquille and Emeis, 1990
; van den Eijnden-Schrauwen et al., 1995
;
for review see Emeis, 1996
). Recent data, however, suggest that the regulated secretion of tPA and vWf are differently regulated, so that the release of the two proteins
can be induced separately (van den Eijnden-Schrauwen et
al., 1997
). For this to be possible, tPA should be stored in a
secretory granule different from the Weibel-Palade body
in which vWf is stored in endothelial cells (for review see Reinders et al., 1988
; Wagner, 1990
, 1993
). No storage
granule for tPA has yet been described, and the possibility
is still open that tPA is also stored in Weibel-Palade bodies. The present study was therefore designed to isolate
and describe the endothelial storage particle for tPA and
to investigate whether this particle was identical to the
Weibel-Palade body, the storage granule for vWf.
MATERIALS AND METHODS
-thrombin, BSA, cycloheximide, N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine and kits
for the determination of lactate dehydrogenase (LDH: No. LD-L 10) and
5
-nucleotidase (No. 253-3) were purchased from Sigma Chemical Co. (St.
Louis, MO). Recombinant human tPA (Activase) was from Genentech
(So. San Francisco, CA), 4-chloro-1-naphtol from Merck (Darmstadt,
Germany), and sodiumpentobarbital (Nembutal) from Sanofi (Paris, France).
)
was kindly donated by Dr M.C. Berndt (Prahran, Australia); rabbit anti-
human TFPI was a gift from Dr C. Lupu (Thrombosis Research Institute,
London, UK); and rabbit anti-human endothelin-1 was a gift from Dr J. Morton (Royal Postgraduate Medical School, London, UK). Texas red-
and FITC-conjugated secondary antibodies, as well as Vectashield fluorescence mounting medium were from Vector Laboratories (Peterborough, UK). Goat anti-mouse and goat anti-rabbit IgG labeled with 5- or
10-nm colloidal gold particles were purchased from Nanoprobes, Inc.
(Stony Brook, NY). Lowicryl K4M embedding medium was from Polysciences (Eppelheim, Germany). All other materials used were of analytical grade.
) and cultured in Medium 199 supplemented with 10% (vol/vol) heat-inactivated newborn calf serum, 10%
(vol/vol) pooled human serum, 100 µg/ml endothelial cell growth factor,
2.5 U/ml heparin, 100 IU/ml penicillin, 100 µg/ml streptomycin and 2 mM
L-glutamine, as described (van Hinsbergh et al., 1985
). Confluent first passage cells were used throughout. Regulated secretion (acute release) of
tPA and vWf from HUVEC was induced in cells that had been preincubated for 30 min in M199 containing 0.3 mg/ml human serum albumin,
L-glutamine, and antibiotics, but no serum. To induce regulated secretion,
15 µl human
-thrombin (final concentration 1 NIH U/ml) was added,
and the medium was collected after 3 min, as described (van den Eijnden-Schrauwen et al., 1995
).
) was cultured in DME containing 10% (vol/vol) fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. For experiments, cells were cultured in 6-well plates and were given fresh medium 24 h before an experiment.
.
) and plasminogen activators inhibitor type-1 (PAI-1) activity (Verheijen et al., 1984
) were determined by
spectrophotometric assay. Human tPA antigen (Schrauwen et al., 1994
),
rat tPA antigen (Emeis et al., 1995
), PAI-1 antigen (Nieuwenhuizen et al.,
1995
), vWf antigen (Tranquille and Emeis, 1990
), and cellular fibronectin
antigen (Friedman et al., 1995
) were measured by ELISA.
-nucleotidase (kit
265-3; Sigma Chemical Co.), glutamate dehydrogenase (using
-ketoglutaric acid and NADH at pH 8.0), LDH (kit LD-L 10; Sigma Chemical
Co.), and angiotensin-converting enzyme (ACE; substrate N-[3-(2-furyl)
acryloyl]-L-phenylalanylglycylglycine by the method of Buttery and Stuart
[1993]) were determined spectrophotometrically, using miniaturized procedures adapted to a microtiterplate reader (Titertek Multiscan MCC/360; Flow
Laboratories, Irvine, Scotland).
). After blotting 25 µl of the fractions on nitrocellulose paper, the paper was blocked with 10 mM Tris-HCl,
pH 7.4, containing 150 mM NaCl, 0.05% Tween 80, and 3% BSA. Subsequently, the paper was incubated with polyclonal rabbit anti-P-selectin
(2.3 µg/ml), followed by incubation with peroxidase-conjugated swine
anti-rabbit Ig, washing, and staining with 2.8 mM 4-chloro-1-naphtol plus
0.01% H2O2 in 20% methanol/80% 200 mM NaCl plus 50 mM Tris-HCl,
pH 7.4. Blots were densitometrically scanned, and the background subtracted. Results will be given as relative OD units per fraction.
). Finally, the samples were embedded
overnight in 100% Lowicryl K4M at
35°C. Polymerization was carried
out by irradiating tissue in gelatin capsules with UV light in a low temperature embedding workstation (MS 5000; Microfield Scientific Ltd., Kingston Bagpuize, UK) for 24 h at
35°C, followed by hardening under UV
light at room temperature for 1 to 2 d. This sectioning was carried out on a
microtome (Ultracut; Reichert-Jung Optische Werke, Wien, Austria), and
thin sections were placed on Formvar-coated 200 mesh nickel grids. For
immunogold labeling we used the postembedding staining protocol previously described (Lupu et al., 1993
). In brief, grids were floated on droplets
of PBS containing 20 mM glycine for 5 min to neutralize any free aldehyde
groups, transferred to PBS containing 1% (wt/vol) gelatine for 10 min to
neutralize "sticky" sites, and washed for 30 min in PBS containing 5%
(vol/vol) nonimmune goat serum (PBS-NGS) to quench nonspecific binding. Subsequently, the sections were incubated for 1 h at room temperature with primary antibodies. For HUVEC, we used a cocktail of murine
anti-human tPA monoclonals or a polyclonal rabbit anti-human recombinant tPA IgG, both diluted 1:50 in PBS-NGS, and monoclonal mouse
anti-human vWf IgG (1:100). For the mouse tissues we have used a polyclonal rabbit anti-mouse tPA diluted 1:50 in PBS containing 1% NGS. After incubation the grids were washed three times in PBS containing 1%
BSA and then incubated for 1 h at room temperature in the same dilution
buffer containing the appropriate gold-labeled secondary antibody.
Thereafter, grids were washed with PBS and distilled water, stained with
uranyl actate and lead citrate, and observed in a transmission electron microscope (EM201; Philips, Eindhoven, The Netherlands).
RESULTS
). In pilot experiments, using a Nydodenz gradient with density
range 1.05-1.27 g/ml, all tPA and vWf was recovered at
densities <1.18 g/ml. In subsequent experiments, therefore, a more shallow gradient (density range 1.03 to 1.17 g/
ml) was employed. In such a gradient (Fig. 1, a-d), tPA
was found as a single symmetrical peak, with a maximum at density 1.11-1.12 g/ml. Both tPA activity and tPA antigen were found at the same position in the gradient (Fig. 1
a). The correlation between tPA antigen and activity in
the 14 fractions was in all four experiments >0.97. In those
two fractions that contained maximal amounts of tPA,
~50% of tPA (Fig. 1 a) and 6-7% of protein (Fig. 1, c and
d) was recovered. vWf antigen also peaked at density 1.11-1.12 g/ml, but in addition showed a second, smaller
peak at density 1.06-1.07 g/ml (Fig. 1 b). Tissue protein,
and most of the marker enzymes measured, showed peak
values in the fractions with densities of 1.03 to 1.08 g/ml
(Fig. 1, c and d). Typically, >70% of the protein was recovered in the these fractions. Maximal alkaline phosphatase and ACE activity (used as plasma membrane marker enzymes) were found around d = 1.05-1.08 g/ml;
acid phosphatase (a marker for lysosomes), neutral esterase, and 5
-nucleotidase (markers for microsomes)
around d = 1.05-1.07 g/ml, and LDH (a marker for the cytosol) ~d = 1.03 g/ml. Only glutamate dehydrogenase activity, a marker enzyme for mitochondria, was recovered
at d = 1.11-1.12 g/ml, the density of maximal tPA and vWf
concentrations. Cellular fibronectin, a marker of connective tissue contamination, was found at density 1.06 g/ml.
Fig. 1.
Gradient centrifugation of rat lung homogenate on a Nycodenz density
gradient. In this and in all
subsequent figures, percentages refer to percentage per
fraction of total activity (or
antigen) recovered in the 14 fractions of a gradient. The
numbers 1-14 of the fractions are displayed on the X
axis. (a) The percentage per
fraction of tPA antigen ()
and the percentage per fraction of tPA activity (
) are
shown as mean ± SD (n = 4). The mean density of the
fractions is also indicated
(
). (b) The percentage per fraction of vWf antigen (
)
is shown as mean ± SD (n = 4). The mean density of the
fractions is given as (
). (c)
The mean (n = 4) percentage
per fraction of cell protein
(
; dashed line), alkaline
phosphatase (
), angiotensin-converting enzyme (
), 5
-nucleotidase (
), and cellular fibronectin (
). (d) The mean (n = 4) percentage per fraction of cell protein (
; dashed line), LDH (
), acid phosphatase (
), neutral esterase (
), and glutamate dehydrogenase (
; dashed line).
[View Larger Versions of these Images (18 + 17 + 17 + 16K GIF file)]
Fig. 2.
(a) A rat lung homogenate was centrifuged on
a Nycodenz density gradient,
and fractions 6 and 7, containing the highest concentrations of tPA and vWf,
were recentrifuged after dilution on an identical Nycodenz gradient. Shown are the
percentage per fraction of
tPA antigen () and vWf antigen (
) in the second density gradient. The fraction
numbers 1-14 are shown on the X axis. The position of the original fractions 6 and 7 of the first gradient is indicated by the line. (b) Human recombinant tPA was added to a rat lung homogenate to a final concentration of 50 ng/ml, followed by centrifugation on a Nycodenz density gradient. Shown on the Y axis are the percentage per fraction of endogenous rat tPA antigen (
) and of exogenously added human tPA antigen (
). The fraction numbers 1-14 are given on the X axis. Rat and human tPA antigen concentrations were determined by species-specific ELISA assays.
[View Larger Versions of these Images (12 + 12K GIF file)]
). Cycloheximide treatment did not significantly influence the distribution of tPA or vWf from rat lung homogenate. Also, the amount of vWf present around d = 1.06 g/ml,
which might have been located in the protein synthesis pathway, was unchanged (data not shown). As discussed
above, no tPA peak is present at this density.
) on a Nycodenz gradient showed that the bulk of tPA (antigen and activity)
was found distributed similarly as in rat lung, though at a
slightly lower density of 1.09-1.10 g/ml (not shown). This
cell line does not synthetize vWf.
Fig. 3.
A rat lung homogenate was centrifuged on a sucrose
density gradient. On the Y axis, the percentage per fraction of
tPA antigen () and of vWf antigen (
) are shown. Note that the
distribution of tPA antigen is practically identical to the distribution on a Nycodenz density gradient (Figs. 1 a and 2). The distribution of vWf on the sucrose gradient is, however, very different,
due to an extensive loss of vWf from the density range 1.10-1.12
g/ml (compare Figs. 1 b and 2). The density of the fractions from
the sucrose gradient is indicated as well (
) and is very similar to
the density profile of Nycodenz gradients.
[View Larger Version of this Image (18K GIF file)]
Fig. 4.
Percentage per fraction of tPA antigen () and of vWf
(
) in Nycodenz gradients after centrifugation of homogenate
prepared from 60 cm2 HUVEC cells. Shown are mean ± SD of
five homogenates from separate cell cultures.
[View Larger Version of this Image (14K GIF file)]
). HUVEC were stimulated
with 1 NIH U/ml of human
-thrombin for 3 min (van den
Eijnden-Schrauwen et al., 1995
), scraped into cold buffer,
and homogenized. Control cultures, not treated with
thrombin, were run in parallel. After thrombin, a loss of
tPA antigen from the high density range of the gradient (fractions 5-8) was observed, compared to the parallel
control cultures. Two typical cultures (out of five) are
shown in Fig. 5, a and b. In the experiment shown in Fig. 5
a, tPA disappeared mainly from density range 1.11-1.12 g/ml
(fractions 6-8), while in the experiment shown in Fig. 5 b,
tPA was reduced in fractions 4-6 (density 1.14 g/ml). The
differences between cultures possibly reflect greater experimental variation in cultured HUVEC than in experiments involving rat lungs (note the small SDs in Fig. 1, a
and b).
Fig. 5.
Distribution of tPA
antigen (pg/ml) from a control HUVEC culture () and
from a culture treated with 1 NIH U/ml of human
-thrombin for 3 min (
). Data from
two separate experiments are
shown (a and b). HUVEC
homogenate was prepared for
each condition from a 300-cm2
HUVEC culture (first passage), pretreated for 24 h
with 1 mM sodiumbutyrate
(see Materials and Methods). Both control and thrombin-treated cells had been obtained from the same primary HUVEC culture. On the X axis the fraction numbers are indicated. The difference in density between corresponding fractions from the thrombin-treated and
control Nycodenz gradient fractionations was always <0.004 g/ml. Note that in Fig. 6 a, tPA is lost mainly from fractions 6-10, while in Fig.
6 b, tPA is lost from fractions 4-8. See text for details.
[View Larger Versions of these Images (14 + 13K GIF file)]
Fig. 6.
Immunocytochemical staining for tPA in first
passage control HUVEC
(left) and in HUVEC after
induction of regulated secretion by 1 NIH U/ml of human -thrombin for 3 min
(right). Cells were fixed and
stained as described in Materials and Methods. The figures show a semiquantitative
measurement of fluorescence
intensity, using pseudocolor
banding. Note that after
thrombin treatment, some
cells have lost all granular
tPA staining, while the other cells show a reduced staining
intensity. Bar, 25 µm.
[View Larger Version of this Image (81K GIF file)]
). By Western
blotting, P-selectin was detected (using the 300-cm2 HUVEC cultures) in the density range 1.10-1.15. After thrombin stimulation, the distribution of P-selectin shifted (one
to two fractions) to higher densities (Fig. 7) and became
more intense.
Fig. 7.
Distribution of P-selectin-reactive material, as determined by Western blotting and densitometric scanning, in Nycodenz gradient centrifugation fractions of homogenates from control () and thrombin-treated (
) HUVEC cultures. Cell culture
and preparatory conditions were as in Fig. 5. Note the increase in
P-selectin immunoreactive material in fractions 2-8 after thrombin treatment.
[View Larger Version of this Image (12K GIF file)]
Fig. 8.
Localization of
tPA and vWf in endothelial
cells: immunogold labeling in
HUVEC of tPA (a and b)
and of vWf (c), and immunogold labeling of tPA in
murine capillary endothelial
cells in lung (d) and diaphragm (e). HUVEC were
fixed in 3% p-formaldehyde
in PBS, and mouse endothelial cells were perfusion
fixed with the same fixative.
The samples were dehydrated at progressively
lower temperature, embedded in Lowicryl K4M resin at
35°C, and immunolabeled on grid with 10-nm colloidal
gold, as described in Materials and Methods. tPA is
found, both in situ and in
vitro, in vesicular structures
with an electron-dense content (a, b, d, and e, arrows). For comparison, a coated
vesicle is indicated on the
surface of a HUVEC (cv).
vWf was detected in structures with the typical characteristics of Weibel-Palade
bodies (c, W-Pb): elongated
structures containing densely
packed fibrilar material. f-h
show double immunofluorescent labeling of HUVEC with
antibodies specific for tPA (f)
and vWf (g). The images collected from both fluorescence channels were superimposed in h. Note that the red
and green granules are clearly different, and that in the superimposed figure of h almost
no yellow (red over green)
grains are observed. Bars:
(a-e) 0.2 µm; (f-h) 25 µm.
[View Larger Versions of these Images (144 + 21K GIF file)]
Fig. 9.
Sequential display of 10 optical sections in the Z-axis
through HUVEC. The cells were double stained for tPA and
vWf, and the two proteins were simultaneously visualized in serial optical sections from the apical (section 1) to the basal (section 10) side of the cells. tPA is detected throughout the cell,
while vWf is distributed mainly in the perinuclear area. Bar, 25 µm.
[View Larger Version of this Image (68K GIF file)]
) that caveolin and TFPI,
but not tPA, colocalize in HUVEC in caveolae.
Fig. 10.
Double staining of first passage HUVEC for tPA (left column, a, d, and g) and either TFPI (b), caveolin (e), or endothelin-1 (h). The panels in the right column represent the superposition of staining for tPA (green) with staining (red) for either TFPI (c), caveolin (f), or endothelin-1 (i). In case of colocalization of two proteins, the resultant superimposed image in the right hand column is yellow.
Note that only in the case of tPA and endothelin-1 (i) in some cells is superimposition observed. For technical details, see Materials and
Methods. Bar, 25 µm.
[View Larger Version of this Image (88K GIF file)]
DISCUSSION
; Lewis and Ferguson, 1950
) and describe that the enzyme activity resides
in the "large granule and microsome" fractions of tissue
homogenates (Lewis and Ferguson, 1950
). Apart from a
few similar studies in the mid-sixties (Lack and Ali, 1964
; Ali and Lack, 1965
; Sugiyama, 1965
; Beard et al., 1968
),
which located tissue PA activity to lysosomes, no major
advances in the localization of tPA in tissues were made.
Recently, it was shown that, in cells transfected with tPA,
tPA is sorted to a granular compartment in the regulated
pathway (Harrison et al., 1996
).
),
was subsequently shown to be the storage organelle for
vWf in endothelial cells (for review see Reinders et al.,
1988
; Wagner 1990
, 1993
; Mayadas and Wagner, 1991
;
Hop and Pannekoek, 1996
).
, Emeis, 1995, van den Eijnden-Schrauwen et
al., 1995
). This might be due to the fact that the cellular
mechanisms involved in tPA and vWf release from endothelial cells are very similar but also due to colocalization
of the proteins in a single particle. The question whether
tPA and vWf are colocalized is of practical importance, because colocalization would make it impossible to induce
selectively the release of either the procoagulant protein
vWf or the anticoagulant protein tPA.
). Cycloheximide treatment did not cause any change in the density distribution of tPA and
vWf either. The large peak of tPA (activity and antigen)
found after density gradient fractionation of lung must
thus be either active tPA stored in a storage pool, or tPA
present in a metabolically inert pool, e.g., extracellular
tPA bound to connective tissue. A contribution from extracellular tPA is, however, most unlikely, because connective tissue components (e.g., cellular fibronectin, see
Fig. 1 d) equilibrate at a much lower density. The tPA
present at d = 1.11-1.12 was, moreover, enzymatically
fully active, while extracellular tPA would have been
present largely as an inactive complex with its inhibitor
PAI-1 (Kooistra et al., 1986
; Fearns et al., 1996
).
; Ewenstein et
al., 1987
).
) are plasmalemmal vesicles characterized by the
presence of, among others, GPI-anchored proteins and caveolin. Recently, a storage particle for endothelin-1 has
been isolated from bovine aortic endothelial cells (Harrison et al., 1995
). It is not likely that tPA storage granules
are identical to caveolae, since the former are much denser
than caveolae and do not contain plasma membrane markers such as alkaline phosphatase (Fig. 1 c), as do caveolae (Lisanti et al., 1994). In addition, by double immunofluorescent staining, tPA- and caveolin-containing structures
were different (Fig. 10). Tissue factor pathay inhibitor
(TFPI) has, in HUVEC, also been localized (Lupu et al.,
1995a
) by immunocytochemistry in a particle, which contains, in addition, caveolin but not tPA (Lupu et al., 1997
),
in agreement with Fig. 10. Together, these data suggest
that the tPA-containing particle does not contain caveolin,
ET-1, and TFPI. We also found no evidence for a colocalization of tPA with ET-1 (Fig. 10 i). The data thus suggest,
though they certainly do not prove, that of the proteins
mentioned above, those four that are secreted from HUVEC (vWf, tPA, ET-1, and TFPI) may be located in different structures, which opens the possibility that endothelial secretory proteins are packaged in separate secretory pathways.
, 1997
) resulted in a loss of tPA from the higher-density fractions (d = 1.10-1.14). This loss coincided with the
appearance of tPA (and vWf) in the culture medium and
thus likely reflects the secretion of tPA (vWf) from its
storage pool. The loss was accompanied by a slight shift to higher densities. Stimulation with 1 NIH U of human
-thrombin for 3 min induces maximal release of tPA
from HUVEC, since neither an increase in thrombin concentration above 1 U/ml nor subsequent stimulation with
the calcium ionophore ionomycin induced any additional tPA secretion (van den Eijnden-Schrauwen et al., 1995
).
Stimulation with 1 U/ml of thrombin was also accompanied by a complete loss of granular tPA staining in some
cells and a severe reduction in tPA staining in the others
(Fig. 6). The quantatively, only moderate disappearance of
tPA from homogenates of thrombin-stimulated cells was
therefore surprising and may reflect the presence, in endothelial cell cultures, of relatively large amounts of tPA outside the secretory pathway.
). Whether the tPA
storage granule also contained P-selectin could not be decided from the data and is still under investigation. The
cell biological substrate of this density shift of P-selectin remains unexplained. It could reflect the fusion of multiple
particles, as described by Richardson et al. (1994)
in rat
endothelial cells after thrombin stimulation in vivo. The
remarkable increase in P-selectin immunoreactivity after
thrombin (Fig. 8) is also not explained but might be due to
an increased accessibility of P-selectin to antibody after
thrombin treatment.
;
van den Eijnden-Schrauwen et al., 1997
) on regulated secretion of tPA and vWf from HUVEC have shown that
several mechanisms are involved in (thrombin-induced)
regulated secretion of tPA and vWf. Regulated secretion
of vWf is mainly dependent upon an increase in intracellular calcium and calcium influx, while regulated secretion of
tPA requires, in addition, the activation of one or more G
proteins (van den Eijnden-Schrauwen et al., 1997
). The
two secretory pathways also show different sensitivities to
actin depolymerization (van den Eijnden-Schrauwen, 1996
).
Such differences in secretory mechanisms presuppose the
existence of two separate storage particles. The present
data provide evidence that two such storage particles indeed exist: the Weibel-Palade body for vWf, and a separate, newly described, particle for tPA.
Received for publication 9 February 1997 and in revised form 11 July 1997.
Address all correspondence to J.J. Emeis, Gaubius Laboratory TNO-PG, P.O. Box 2215, 2301 CE Leiden, The Netherlands. Tel.: (31) 71-518-1451. Fax: (31) 71-518-1904. e-mail: JJ.Emeis{at}pg.tno.nlThis work was supported in part by grants from the Netherlands Heart Foundation (93.126) and the British Heart Foundation (PG/94188).
HUVEC, human umbilical vein endothelial cells; TFPI, tissue factor pathway inhibitor; tPA, tissue-type plasminogen activator; vWf, von Willebrand factor.
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