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Studies on the Acute Release of Tissue-Type Plasminogen Activator From
Human Endothelial Cells In Vitro and in Rats In Vivo:
Evidence for a Dynamic Storage Pool
By Y. van den Eijnden-Schrauwen, T. Kooistra, R.E.M. de Vries, and J.J. Emeis
The process ofacute release of tissue-type plasminogen activator (tPA) is important in locally speeding up fibrinolysis.
Using a sensitive enzyme-linked immunosorbent assay for
tPA, we investigated the acute release of tPA from cultured
human umbilical vein endothelial cells. The addition of
thrombin (0.003 to 3 NIH UlmL) caused the dose-dependent
release of noncomplexed, enzymatically active tPA into the
medium. The amount of tPA released into the medium by
thrombin wassimilar to thedifference in theamounts of tPA
present in extracts from thrombin-treated cells and control
cells. The process of acute release of tPA was complete in 1
minute, whereas the concomitant release of von Willebrand
factor into the medium was
slightly slower (maximum after
3 minutes). By increasing (c.q. decreasing) tPA synthesis, it
was found that the amount of tPA constitutively secreted,
the amount acutely released, and the amount in cell extracts
were increased k.q. decreased) to the same extent. The
same relation was found in vivo. When rats werepretreated
with cholera toxin or retinoic acid to increase tPA synthesis,
plasma levels of tPA were increased, whereas acute release
of tPA, as induced by bradykinin, was increased to thesame
extent. Acutely released tPA and constitutively secreted tPA
were liberated from different pathways in human umbilical
vein endothelial cells; tPA had, relative to the invivo situation, a short residence time in the acutely releasable pathway.
0 1995 by The American Societyof Hematology.
T
between constitutive secretion and regulated secretion of
tPA. Also, we studied the relationship between synthesis and
acute release of tPA.
ISSUE-TYPE PLASMINOGEN activator (tPA) is a key
enzyme in the onset of fibrinolysis. It converts plasminogen to plasmin, which degrades the fibrin component
of a thrombus. tPA incorporated into a clot during coagulation is, in vitro, more efficient in dissolving a clot than tPA
added at a later time.’.*Therefore, the speed and extent of
local delivery of tPA during thrombus formation is important
in speeding up thromboly~is.~
In humans, plasma levels of
tPA increase rapidly after venous occlusion or after the administration of, for example, desmopressin or adrenalin?
There is general agreement that this tPA is derived from the
vascular endothelial cells.5 Secretion of tPA can occur by a
constitutive and a regulated p a t h ~ a y This
. ~ latter pathway,
also called the acute release of tPA, only occurs once receptors on endothelial cells have been triggered.6Until now, the
acute release process has been studied mainlyin animal
models, whereas studies on the acute release of tPA from
human endothelial cells were limited,’,’ presumably because
of the very low tPA concentrations involved. In contrast, the
acute release of von Willebrand factor (vWF), another protein whichis secreted from the endothelium by both a constitutive and a regulated pathway, has been intensively studied
in vitro, probably because of the relatively large amounts that
are secreted in culture.’ However, the recent development of
a sensitive enzyme-linked immunosorbent assay (ELISA)
for tPA antigen has made it possible to study in vitro the
acute release of tPA as well.” The present study was designed to characterize the secretion of tPA from human endothelial cells in vitro, especially with regard to the distinction
From the Division of Vascular and Connective Tissue Research,
Gaubius Laboratory TNO-PG, Leiden, The Netherlands.
Submitted July 28, 1994; accepted January 30, 1995.
Supported by a grant from the Netherlands Heart Foundation.
Address reprint requests to Y. van den Eijnden-Schrauwen, PhD,
Gaubius Laboratory TNO-PG, P.O. Box 430, 2300 A K Leiden, The
Netherlands.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
“advenisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8512-0026$3.00/0
3510
MATERIALS AND METHODS
Materials. Cell culture reagents were from Flow Laboratories
(Irvine, Scotland). Sterile, pyrogen-free, humanserum
albumin
(HSA; 20% wt/vol) was from the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (Amsterdam, The Netherlands). L-methionine was from Life Technologies (Breda, The
Netherlands). 35S-Methionine,labeling grade, was from Amersham
(’s-Hertogenbosch, The Netherlands). Human cy-thrombin, calcium
ionophore-free acid (A23187), sodium butyrate, cholera toxin, bradykinin, and all-trans retinoic acid (RA) were from Sigma (St Louis,
MO). Materials used in the human and rat tPA ELISA and in the
vWF ELISA have been described.’””’ Casein was from Merck
(Darmstadt, Germany), and sodium pentobarbital (Nembutal) from
Sanofi (Paris, France). Mouse monoclonal antibody against fluorescein isothiocyanate (FITC) was kindly provided byDr R. Bos of
our institute.
Dejnitions. Constitutive secretion oftPA (or vWF) is defined
as the amount of tPA antigen (or vWF antigen) secreted during the
30-minute preincubation period in Medium 199 (M199)MSA. The
tPA (vWF) present in the cell extract is defined as the amount of
tPA (vWF) found in control extracts at t = 3 minutes. The thrombininduced acute released tPA (vWF) is defined as the amount of tPA
(vWF) inmediumof thrombin-treated cells minus the amount of
tPA in medium of control cells at t = 3 minutes.
Cell culture. Human umbilical vein endothelial cells (HUVECs)
were isolated using the method of Jaffe et al” and were cultured as
previously describedL4in M199, containing 10% (vol/vol) human
serum (HS), 10% (voUvol)heat-inactivated newborn calf serum, 100
IU/mL penicillin, 100 pg/mL streptomycin, 200 pg/mL endothelial
cell growth factor (ECGF), 2.5 U/mL heparin, and 2 mmoUL Lglutamine (henceforth called total M199 medium) under 5% CO2 at
37°C. Cells were passaged once by trypsin/EDTA treatment at a
split ratio of 1:3 and were cultured to confluencyin 2-cmZ wells
(final density 2 X lo5 cells/well).
Induction of the acute release of tPA in vitro. To exclude my
interference of HS, ECGF, or heparin with the acute release mechanism, HUVECs were cultured in M199 medium, containing 20%
(vol/vol) newborn calf serum, 1 0 0 IU/mL penicillin, 100 pg/mL
streptomycin, and 2 m m o K L-glutamine (“deficient” medium) for
2 hours before an experiment. The 2-hour conditioned media were
collected.” The cells were then washed twice with sterile phosphatebuffered saline (PBS; pH 7.4) and 0.3 mL M199iHSA medium
Blood, Vol 85, No 12 (June 15),
1995:
pp 3510-3517
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351 1
STUDIES ON THE ACUTE RELEASE OF TPA
(M199, containing 0.03% (wt/vol) HSA, 100 IU/mL penicillin, 100
pg/mL streptomycin, and 2 m m o m L-glutamine) was added to the
wells. Thirty minutes later, at t = 0, 15 pL human a-thrombin in
M199iHSA was added to the indicated final concentration (generally
1 NIH U/mL). The same volume of M199iHSA medium was added
to control cells. The media were then collected after 3 minutes,
unless otherwise stated. The cells were washed once with ice-cold
PBS and immediately put on ice. Cell lysates were made by scraping
the cells with a rubber policeman into 0.3 mL PBS containing 0.05%
(vol/vol) Tween 20, 0.01 m o m EDTA, and 0.5% (vol/vol) Triton
X-100. All incubations were performed at 37°C.
Decreasing the constitutive secretion of tPA. To decrease the
constitutive P A secretion, HUVECs were cultured for 0, 2, 4, 8,
and 24 hours in the deficient medium described above.
Increasing the constitutive secretion of IPA. HUVECs were incubated for 24 hours with 0, 0.3, 1, or 3 mmom sodium butyrate"
or with 1 pmoVL RA16 in total M199 medium to increase the constitutive secretion of tPA.
Methionine-labeling of the constitutive and regulated tPA pathways. HUVECs were cultured for 24 hours in total M199 medium,
containing 3 mmoVL sodium butyrate. The cells were then incubated
in total M199 medium that had been made free of methionine by
overnight dialysis against methionine-free M199 and that contained
radiolabeled '%-methionine (20 pCilwel1)and 3 mmoVL sodium
butyrate. A 100-fold excess of unlabeled methionine was added after
0, 1,2, 3, or 4 hours. After a total incubation time of 4 hours, release
was induced; media and cell extracts were prepared, and casein and
EDTA were added as described." The collected media and cell
extracts were incubated overnight at 4°C in microtiter plate wells,
which had been coated with antibodies against tPA as described for
the ELBA procedure." For controls, we used wells that had been
coated with the same concentration of a monoclonal mouse antibody
against FITC. After washing, the bound protein was dissolved with
0.3 moVL NaOH, neutralized with 1.5 m o m HCI, and counted.
Inhibition of the constitutive and regulated tPA pathways byinhibiting protein synthesis. To inhibit protein synthesis, HUVECs were
incubated with 5 pg/mL cycloheximide in total M199 for 0, 0.5, or
1 hour. The acute release was then induced in the presence of 5 pg/
mL cycloheximide (without culturing the cells for 2 hours in deficient medium). In this experiment, 2 pmoVL calcium ionophore
A23187 was used to induce acute release of tPA, because calcium
ionophore A23187 is a receptor-independent stimulant of tPA and
vWF release, which is not dependent on protein synthesis. Also,
A23187 has been shown to induce the acute release of tPA."
Measuring overallprotein synthesis. Overall protein synthesis
was determined by incorporating "S-methionine into endothelial
cell proteins and measuring the amount of 3'S-methioninein the
trichloroacetic acid-precipitable fraction, as described."
Induction of the acute release of tPA in vivo. A control group
of eight male Wistar rats (Iffa-Credo, Someren, the Netherlands),
weighing 200 to 300 g, were fed a standard laboratory diet. To a
second group of eight animals, RA (8 mgkg body weight) was
orally administered daily for 5 days to increase tPA synthesis.16 A
third group was intravenously injected with cholera toxin (500 pg/
kg body weight) 48 hours before an experiment. Rats were anesthetized with Nembutal (60 mgkg intraperitoneally) and were cannulated, and bradykinin (50 pgkg body weight) was injected as a bolus
into the vein of the
penis. Blood was collected through a carotid
artery cannula, and citrated plasma was prepared. Rat tPA antigen
concentrations were determined in citrated plasma by ELISA.I7Animal experiments had been approved by the Animal Experiments
Committee of the Netherlands Organization for Applied Scientific
Research TNO and were in accordance with theguidelines on animal
experimentation presented to the International Committee of Thrombosis and Haemostasis.'*
Assays. Human tPA antigen was measured by ELISA as de-
scribed." Recombinant human one-chain tPA (Activase; Genetech,
San Francisco, CA) wasused for making calibration curves in a
range of 30 to 500 pg/mL. The detection limit in this assay is 10
pg/mL. Both tPA and tPA-PAL1 complexes are detected with equal
efficiency by this ELISA." Rat tPAantigen was measured by ELISA
using rabbit-antirat tPA IgG, as described.17vWF antigen was measured by ELISA, essentially as described," with the following minor
modifications: the rabbit antihuman vWF IgG was diluted 1:2,000,
and 0.1% (wt/vol) of casein was used in all buffers instead of 0.05%
(wt/vol) bovine serum albumin. Human pooled plasma in a range
of 0.078% to 1.25% was used for calibration. Lactate dehydrogenase
activity was determined using a kit, according to the manufacturer's
(Sigma) instructions.
Fibrin zymography." Samples were put immediately in sample
buffer containing 2% sodium dodecyl sulfate (SDS) and were frozen
at -20°C. SDS/9% polyacrylamide slab gels were prepared according to Laemmli.zoGels were then washed, placed on top of a
plasminogen-rich fibrin layer, and incubated for 36 hours at 37°C
as we described in detail elsewhere.''
Datu presentation and units used. The data are presented as
mean 2 SD of three measurements. Human tPA antigen is given as
picograms or nanograms per milliliter, using Activase as standard.
Rat tPA antigen is given as nanograms per milliliter, using rat L,
tPA" as standard. vWF antigen is given as units per milliliter, 100
U being defined as the amount of vWF antigen present in 1 mL of
pooled human plasma.
RESULTS
The regulated (acute) release of tPA in vitro. The constitutive and regulated secretion of tPA was studied in vitro
using first-passage human endothelial cells. In a representative experiment (Fig lA), control cells constitutively secreted 13 pg of tPA per 2 x lo5 cells into the medium over
10 minutes (1.3 pg tPA/min/2 X lo5 cells). This rate of
constitutive tPA secretion resembled the rate of constitutive
tPA secretion during the 30-minute preincubation period (1.6
pg tPA/min/2 X l Os cells) and during the 2 hours preceeding
the experimental period (2.2 pgtPA/min/2 X lo5 cells).
These data suggested that no major changes in constitutive
secretion occurred because of manipulation of the cells during an experiment. On addition of human thrombin (1 NIH
U/mL), the cells very rapidly released tPA into the medium.
The highest increase of tPA occurred during the first minute,
resulting in an additional 25 pg tPA/min/2 X lo5cells in the
medium. The tPA content of the extracts from thrombintreated cells was always lower than the content of the extracts
from control cells (see Fig IA). This difference in tPA content betweenextracts from thrombin-treated cells and control
cells was of the same magnitude as the amount released
into the medium. In five experimental series, the difference
averaged 94% ? 30% (mean ? SD) of the amount of tPA
acutely released by thrombin into the medium at 1 minute.
Because, in vivo, acute release of vWF and tPA are often
observed together, the secretion of v W F was measured in this
experiment as well. vWF was, similar to tPA, constitutively
secreted by control cells, 0.033 U110 min/2 X lo5cells (see
Fig 1B). On addition of thrombin (1 NIH U/mL), vWF was
released slightly slower into the medium than tPA. The increase in vWF was maximal at 3 minutes, at which time an
additional 0.22 U/mL of vWF had been released into the
medium. Figure 1B also shows that the increase of vWF in
the medium resulted in a corresponding decrease of vWF in
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VANDENEIJNDEN-SCHRAUWENETAL
l
250
2
3
4
5
I,
200
150
100
50
0
0
2
4
6
8
10
time (minutes)
2.5
1
I
r
l
.o
l.1
0.0
0
2
4
6
8
10
time (minutes)
Fig 1. Time course of the inductionof the acute release of tPA (A)
and vWF (B) by thrombin. First-passage HUVECs were treated with
1 NIH U/mL of human a-thrombin, or with control M199/HSA medium after a preincubation period on M199/HSA medium in which
48 pg tPA/3O min/2 x l o 5 cells and 143 U vWFl30 min/2 x lo5 cells
were secreted constitutively. Media and cell extracts
of control cells
and thrombin-treatedcells were collected. In (A), the tPA concentration (pg/2 x l o 5cells) in theconditioned media is shown for control
(A)and thrombin-treated cells ( 0 ) .Also shown are tPA concentrations in the corresponding cell extracts of control(A) and thrombinThe difference in tPA concentrations betweencontreated cells (0).
trol and thrombin-treated cell extracts is approximately
equal to the
amount oftPA acutely released. In this experiment, the tPA concentrations in the cell extracts at t = 0 are low as compared with the
other time points. Similarly low values have been observed more
often at random time points
and are not understood. In (B), the vWF
concentration (expressed as U/2 x lo5 cells) is shown for the same
experiment using the same symbols as in (A). All data shown are
mean k SD In = 31.
Fig 2. Zymography of plasminogen activator(PA)-activity in endothelial cells and conditioned medium after thrombin-induced
release
of tPA. The acute release of tPA was induced by 1 NIH U/mL of
human thrombin for 3 minutes. The plasminogen activator activity
in the conditioned media and cell extracts was visualized by fibrin
zymography after SDS/polyacrylamide-gel electrophoresis. Lane 1,
recombinant tPA (Activase); lane 2, conditioned medium of control
cells; lane 3, conditioned medium of thrombin-treated cells; lane 4,
cell extract of control cells (diluted 1:7); and lane 5, cell extract of
thrombin-treated cells (diluted 1:4). The lower bands in lanes 2 t o 5
correspond with the band of free, uncomplexed recombinant tPA
(lane l ) ,which is denoted by the arrow
(+). The upper bands in lanes
2 t o 5 represent tPA complexed t o PAI-1, which is denoted by the
arrowhead (F).AllPA-activitycouldbe
quenched by antibodies
against human tPA (data not shown).
cell extracts. Because the acute release of both tPA andvWF
has reached maximal values at 3 minutes, in subsequent
experiments, the acute release of tPA and vWF was always
determined at 3 minutes. The release of tPA and vWF was
not caused by cell lysis, because the lactate dehydrogenase
content of media was not increased by thrombin and always
remained below 3% of the amount present in the cells.
To study whether free tPA or tPA-plasminogen activator
inhibitor (PAI) complex was acutely released, samples of
media and cellextracts were analyzed by fibrin zymography.
Despite the presence of an approximately fivefold excess of
PAI-I in these media, complex formation isprevented by
a 1: 1 dilution in an SDS-containing buffer of the media,
immediately after a 3-minute stimulation. Figure 2 shows
that only free tPA,but not tPA:inhibitor complex, was
acutely released into the medium on addition of thrombin
(Fig 2, lanes 2 and 3), and that free tPAhad disappeared
from the cell extracts (Fig 2, lanes 4 and 5). Please note that
tPA in cell extracts has the same mobility (ie, the same M,)
as constitutively secreted and acutely released tPA in media
(see Fig 2, lanes 2 through 5 ) .
Dose-dependence of tPA release by thrombin. Figure 3
shows that thrombin increased the amount of tPA (and vWF)
released in a dose-dependent manner. In this experiment, the
constitutive secretion of tPA was 60 pg/2 X I O 5 cells in 30
minutes. In the presence of I NIH U/mL of thrombin, maximal acute release of 40 pg/2 X IOs cells was observed at 3
minutes. From 0.01 NIH U/mL upwards, the acute release
exceeded the constitutive secretion by at least twice the standard deviation. Similar data were obtained for vWF (Fig 3).
To see if cells could release still moretPAon further
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STUDIES ON THE ACUTERELEASE
3513
OF TPA
40
30
Fig 3. Thrombin dose-dependently induces the acute release
of tPA and vWF. Concentrations
of 0 to 3 NIH U/mL of thrombin
were used to induce acute release of tPA and vWF. The
amount of tPA (0; pg/2 x lo5
cells) that was acutely released
intothemediumbythe
indicated concentration of thrombin
and the amount of acutely released vWF (A;
U/2 x I O 5
cells) are shown.
125
-
n
-
20
-
10
-
25
9
0
0
0.1
0
0.01
stimulation after maximal thrombin stimulation, cells treated
with thrombin (1 NIH U/mL for 3 minutes) were subsequently incubated with calcium ionophore A23 187 (1 pmoV
L) for another 7 minutes. Cells treated with thrombin followed by ionophore did not release more tPA than did cells
treated only with thrombin. The doubly stimulated cells released 114% ? 44% (n = 6; n.s.) of tPA, similar to the
amount released by cells treated only with thrombin. Doubly
stimulated cells released in 10 minutes 148% ? 73% (n =
6) of vWF, as compared with cells treated for 10 minutes
with thrombin only.
ConstitutivetPAsecretion and acuterelease of tPA in
various cell isolates. To investigate whether constitutive
tPA secretion (representing tPA synthesis) influenced acute
tPA release in HUVECs, a correlation study was performed
with data from 17 isolates of HUVECs. These 17 isolates
provided naturally occuring variable levels of constitutive
tPA secretion (from 2 to 10 ng/mL/24 h). By linear regression analysis, a correlation between constitutive tPA secretion and acute release of tPA of 0.68 was found (Fig 4). The
percentage of tPA in the cell extract that could be released
averaged 33% with an SD of 17%.
Constitutive tPA secretion and acute release of tPA: Deof tPA. When HUVECs
creasedconstitutivesecretion
were cultured in deficient medium, the levels of the three tPA
parameters constitutive secretion, acute release, and cellular
concentration (expressed as percentages of the level at t =
0) significantly decreased in parallel in time to about 20%
after 24 hours (Fig 5; the absolute values of these parameters
are given in the legend to the figure). These parallel changes
suggest that constitutive secretion, acute release, and cellular
content of tPA are closely linked by some, as yet undetermined, physiological process(es). At 24 hours, the rate of
overall protein synthesis did not differ from that in control
cells (127% +- 18%, n = 6), showing that the viability of
cells cultured in deficient medium had not diminished. Furthermore, vWF release did not differ between cells cultured
in deficient medium and cells cultured in total medium (data
not shown). These data suggest that cells in deficient medium
10
1
thrombin (NIH U/mL)
were as sensitive to thrombin stimulation as were cells cultured in total medium.
Constitutive tPA secretion and acute release of tPA: Increased
constitutive
secretion
of tPA. HUVECs were
treated with 0 to 3 m m o K sodium butyrate or with 1 pmoV
L RA to increase tPA synthesi~.'~.''After 24 hours, acute
release of tPAwas induced. The constitutive secretion of
tPA was enhanced dose-dependently by sodium butyrate,
showing a 20-fold stimulation at 3 mmol/L (Fig 6). These
250
I
T
W
0
0
100
200
300
constitutive tPA secretion
(pg/30 min/2.1 O5 cells)
Fig 4. tPA synthesis and thrombin-induced acute release of tPA
in 17 isolates of HUVECs. In 17 isolates of HUVECs, the acute release
of tPA was induced by 1 NIH U/mL of human a-thrombin, as described in Materials and Methods. The mean constitutive tPA secretion (during 30 minutes) and the mean tPA release 13 minutea after
thrombin-addition) are plotted; the horizontal and vertical bars represent the corresponding SDI In = 3).After linear regression, a correlation of 0.68 was found (n = 17).
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3614
VAN DENEIJNDEN-SCHRAUWENET
l00
80
60
40
20
I
,
,
,
,
,
,
0
4
8
12
16
20
24
0
hours "deficient" medium
Fig 5. The effects of a decreased tPA synthesis on acute release
of tPA. First-passage HUVECs were cultured for 0, 2, 4, 8, and 24
hours indeficientmedium (without ECGF, HS, or heparin).Acute
release was induced with 1 NIH U/mL of human thrombin. The absolute amounts of tPA constitutively secreted, acutely released, and
present in cell
at = Or shown
mean
= 3)' were
47 f 4, 59 k 9, and 240 i: 0 (pg/2 x 10' cells), respectively. The
constitutive tPA secretion (m), the acute release of tPA (AI, and the
amount of tPA in the cell extracts (e) are expressed as percentages
of the concentrations at t = 0 hours.
'
AL
increases in constitutive tPA secretion were parallelled by
increased concentrations of tPA in the cell extracts and by
increased acute release of tPA (Fig 6), again suggesting that
the processes involved are closely linked. RA enhanced the
constitutive secretion twofold, which resulted in a l . 1-fold
increase of tPA in the cell extracts and in a 1.6-fold increase
of acute release of tPA. Increasing the constitutive tPA secretion did notinfluence the timeprofile of acute release,
whereas, inmost experiments using thrombin stimulation
followed by ionophore, all available tPA was released by
thrombin (data not shown). Sodium butyrate and RA did not
change constitutive vWF secretion, the amount of vWF in
cell extracts, or the acute release of vWF.
An acutely releasable tPA pool in HUVECs. The close
correlation between constitutive tPA secretion and acutely
releasable tPA, described above, might suggestacutely
that
releasable tPA is derived from the same pathway as constitutively secreted tPA. If so, the induction of acute release of
tPA should cause a decrease in constitutive secretion over
the subsequent time period. This was studied as follows: at
t = 0, acute release was induced with thrombin; after 3
minutes, hirudin (0.67 pg/mL) was added to the cells to
inactivate thrombin; and the media were collected at 0,3, 10,
60, and 120 minutes after the addition of thrombin. Figure 7
shows that thrombin-treated cells had released 17 pg tPN2
x 105 cells at t = 3 minutes, and that this difference persisted
for the next 2 hours. These data suggested that the releasable
pool was not part Of the constitutive tPA pathway. In a first
attempt to discriminate between tPA following the constitu-
T
180
4.50
120
3.00
60
1S O
i
0
0
0.00
0
1
3
sodium butyrate (mmol/L)
0.3
30
60
90
120
time (minutes)
Fig 7. Thesecretion of tPAover a longerperiod. At t = 0. acute
releasewasinduced
as describedabove,and
the reaction was
stopped at t = 3 minutes with hirudin.The concentration of tPA
Fig 6. Sodium butyrate increasestPAsynthesisandtPArelease.
after 0,3,10,60, and 120 minutes. tPA is continously
First-passage HUVECs were cultured for 24 hours with various con- was maasured
secreted into the medium (A), and, on addkion of thrombin, addicentrations of sodium butyrate. Acute release of tPA was then induced as described in Materlab and Methods. The constitutive tPA
tional tPA is secreted into the medium (e),w
h
c
ih is stillfound in the
Secretion (A),the acute release of tPA (e),and the amount of tPA in
medium after 80 and 120 mlnutes. Data shown are mean f SD (n =
the call extracts (m) are shown as mean f SD (n = 3).
3).
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STUDIES ON THEACUTERELEASE
n
3515
OF TPA
a
0
II
.W
100 II-0
T
0
75
2 25
75
A
Q
) o
Q
-
50
0.0 0.5 1 .O
hours
cycloheximide
25
0
.-E
0
4
2
3
1
hours unlabeled methionine
Fig 8. The residencetime of tPA in the tPA storage pool.HUVECs
that had been cultured for 24 hours in total
M199 medium containing
3 mmol/L sodium butyrate were incubatedfor 4 hoursin totalmethionine-free M199medium containing 20 p C i radiolabeled "S-methionine and 3 mmol/L sodium butyrate. Meanwhile, the cells were
chased for 4,3, 2, 1, or 0 hours with a 100-fold excessof unlabeled
methionine. Release was then induced with 1 NIH UlmL of human
a-thrombin, end the media were collected. After overnight incubation of the media at 4°C in anti-tPA-coated wells of a microtiter
plate, the wells were washed, and the bound protein was dissolved,
neutralized, and counted. The datathree
of independent experiments
are expressed as percentages o f t = 0 (shown as mean t SD), concerning constitutive tPA secretion(A)and acute releaseof tPA (0).In
three independent experiments,the absolute counts found in media
containing constitutively secreted tPA at t = 0 were 143, 297, and
196 disintegrations per minute (dpm)/3O m i d 2 x lo6cells. In three
independent experiments, the absolute counts found in media containing acutely released tPA at t = 0 were 194, 157, and 47 dpm/3
m i d 2 x lo5cells. Aspecificbinding was determinedby adding media
or cell extracts to microtiter plates that were coated with anti-FITC
antibodies. This aspecificbinding was about 6%. as compared with
that for anti-tPA-coated microtiter plates. Theinset shows the con(A)and acute release
of tPA ( 0 )after inhibistitutive secretion of tPA
tion of the protein synthesis with 5 pg/mL cycloheximide in total
M199 for 0, 0.5, or 1 hours. Acute release of tPA was induced by
2 pmol/L calcium ionophore A23187 in the presence of 5 pg/mL
cycloheximide, because calcium ionophore
is a receptor-independent
stimulant that is as effective as thrombin but is notdependent on
protein synthesis. Data shown are mean f SD (n = 3).
tive or regulated pathway, we analyzed the retention time of
tPA. To this end, HUVECs that had been stimulated for 24
hours with butyrate were metabolically labeled with 35Smethionine, chased for 0 to 4 hours with an excess of unlabeled methionine, and then induced to release tPA. Constitutive secretion and acute release of labeled tPA were
determined by overnight binding of radiolabeled material in
the media to anti-tPA-coated microtiter wells. These data
(Fig 8) are expressed as percentages of the amount of radiolabel bound at t = 0 (ie, unchased). Background binding to
anti-FITC coated wells was about 6% for unchased media.
Binding of radiolabeled tPA could not be determined in cell
extracts, because background binding on anti-FITC-coated
plates was too high to permit reliable determination of spe-
cific binding (data not shown). As shown in Fig 8, after
a l-hour chase with unlabeled methionine, the amount of
radioactivity bound from media of unstimulated cells was
less than 50% relative to the amount bound from unchased
medium ofunstimulated cells. In mediafrom thrombin-stimulated cells, the amount of radiolabel additionally released
by thrombin was still 100% relative to the amount additionally released from unchased cells. After 2 hours, both pathways contained about 25% of the amount of label present
in unchased media. Therefore, these data suggest that the
releasable tPA pool is separate from the constitutive secretion pathway. The inset of Fig 8 shows that comparable
data are obtained when protein synthesis is inhibited with
cycloheximide (overall protein synthesis, as measured by
incorporation of radiolabeled methionine, was blocked for
90% after treatment of the cells for 1 hour with cycloheximide). The constitutive secretion of tPA antigen was decreased by 57% after l hour. In contrast, the acutely released
amount of tPA antigen was still 114%, supporting the evidence for a separate intracellular source for tPA. Blocking
of protein synthesis for more than 1 hour severely reduced
the cellular adenosine triphosphate content (data not shown);
thus, experiments were not continued beyond 1 hour.
Acute release of tPA in vivo. To study the relation between constitutive secretion and acute release of tPA in vivo,
rats were pretreated for 2 days with cholera toxin or for 5
days withRA to increase tPA synthesis. After 2 days of
cholera toxin treatment, P A mRNA levels in heart and lung
were significantly increased as were the tPA antigen concentrations, showing increased tPA synthesis in these animals
(data not shown). Treatment of rats with RA also resulted
in increased levels of tPA activity and tPA antigen in several
tissues.'6 Treatment with cholera toxin increased the steadystate plasma levels of tPA 5.4-fold as compared with that
for control animals (ie, t = 0 in Fig 9); treatment with RA
increased tPA plasma levels 1.4-fold (Fig 9). The acute release of tPA was then induced in these animals by bradykinin. As shown in Fig 9, the acute release of tPA had, at 1
minute, increased 1.%fold in RA-treated rats and 3.7-fold in
cholera toxin-treated animals, as compared with thatfor control animals. These data indicate that an increased tPA synthesis gives rise to an increased acute release of tPA in vivo
as well.
DISCUSSION
In this study, we addressed the questions of how acute
release of tPA from human endothelial cells proceeds and
how this release is related to the constitutive secretion of
tPA. Using first-passage HUVECs, we observed, as have
many other^,^ constitutive secretion of tPA into the medium
at a steady rate over many hours. On the addition of thrombin, acute release occurred, as evidenced by the rapid appearance of uncomplexed, enzymatically-active tPA in the medium. The acute release of tPA was very rapid, showing a
maximal increase of tPA within 1 minute. In both these
respects, the acute release reaction in vitro resembled acute
release of tPA as observed in humans in vivo: Release induced by thrombin was dose-dependent. At maximal stimulation by thrombin (2l NIH U/mL), most if not all endothelial tPA was released into the medium, because a second
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
3516
VAN DEN EIJNDEN-SCHRAUWEN ET AL
T
-M
-*-
1
“
*f
l
0
2
4
6
8
10
time (minutes)
Fig 9. Acuterelease of tPA in vivo.Rets were pretreated for 2
days with cholera toxin (m) or daily for 5 days with RA ( 0 )to increase
their tPA synthesis. Acute release of tPA was then induced in pretreated rata and in control rata (AI as described in Materials and
Methods. The concentrationof tPAwas measured in citrated plasma
at 0, 1, 2, 5, 7, and 10 minutes after the injection of bradykinin. The
highest concentration of tPA is found at 1 minute. After 1 minute,
the tPA Concentration decreases because of clearance by the liver.
The vertical bars represent SDs of the mean (n = 8).
stimulation by calcium-ionophore did not result in significantly more tPA release. Concomitantly with tPA, vWF was
also released, as has often, but not always, been observed in
vivo.”,” Compared with tPA, vWF was released slightly
more slowly, showing a maximum at 3 minutes. The time
course of the acute release of tPA and vWF in vitro also
resembled the time course of acute release in the perfused
rat hindleg, which also showed very rapid release of tPA
and a slightly slower release of vWF (as shown by Tranquille
and Erneis’’).
The amount of thrombin-releasable tPA strongly correlated with its rate of constitutive secretion (ie, tPA synthesis)
in HUVECs (Figs 4 through 6 ) . A similar correlation was
found in vivo in rats (Fig 9). Because the constitutive secretion of tPA (tPA synthesis) was coupled, at least quantitatively, so closely with the release of tPA, the question arose
how closely the two pathways are coupled functionally. The
following evidence suggests that both pathways are not identical. Thrombin-induced release of tPA does not diminish
the subsequent constitutive secretion (Fig 7), indicating that
the former is not a precursor of the latter. After metabolic
labeling (Fig 8), released tPA retains label about 1 hour
longer than constitutively secreted tPA. Also, after inhibition
of protein synthesis by cycloheximide, release of tPAis
maintained for at least 60 minutes at control level, whereas
the level of constitutively secreted tPA decreases strongly.
These observations suggest that releasable tPA was delayed
in an additional compartment. This conclusion is supported
by the observation that acutely released P A is not com-
plexed to an inhibitor (Fig 2). Because tPA in the subcellular
matrix and tPA on the plasma membrane are complexed to
PAL 1,23 the tPA released must bederived from an intracellular compartment (see also Fig 2, lane 4),in line with previously published data,” which showed that plasma membrane-bound tPA is not released. Moreover, on subcellular
fra~tionation,’~
the tPA content of an intracellular high-density fraction, free of plasma membrane markers, is diminished after stimulation with thrombin (Schrauwen et al“’ and
Emeis and van den Eijnden-Schrauwen, manuscript in preparation). Therefore, we propose that, after synthesis, tPAis
temporarily stored inan intracellular compartment, presumably consisting of high-density granules. In all these respects,
the pathway followed bytPA destined for acute secretion
resembles that of other secretory proteins,’.25although the
storage pool isless permanent than pools of other stored
proteins.
We observed a close correlation betweenthe rate of synthesis of tPA and the amount of acutely releasable tPA. This
suggests that a fixed percentage of newly synthethized tPA
goes into the tPA storage compartment, regardless of the
rate of synthesis. This has also been described for vWF
tPA that is notacutely released from this pool into the
medium still disappears from the pool, because the time tPA
stays in the pool is only about 1 hour. What happens to this
tPA is not known. We call this tPA that has disappeared
“lost tPA,” because it is not secreted inthe constitutive
pathway (Fig 7) or in the acute release pathway. One possibility is that this lost tPA is deposited in the subcellular
matrix.’* However, we have been unable to detect the required amounts of tPA in the matrices of our cell cultures.
Another possibility is thatthe lost tPAis degraded intracellularly, possibly in a lysosomal compartment, because label
leaves the storage compartment but is not recovered in the
constitutive pathway (Fig 8; compare also Fig 7). In an attempt to find further evidence for this hypothesis, we incubated cells for 4 hours in conditions that diminish lysosomal
enzyme activity (50 pmol/L chloroquine). Under these conditions, the constitutive secretion didnot change, whereas
the amount of tPA in cell extracts did indeed increase. However, this increase could account for only some 10% of the
lost tPA (our unpublished observations).
The storage pool inHUVECs (residence time about 1
hour) was less stable than the tPA storage pool in vivo in
rats, the latter pool being hardly diminished by inhibiting
protein synthesis for 5 hours.” This might explain why endothelial cells in vitro possess a relatively small storage pool
for tPA as compared with that for rats. In cell extracts from
HUVECs about 0.1 ngof tPA/cm* is present, whereas 0.5
to 2 ng of tPA/cm2 has been estimated to be present in vivo
(calculations in Emeis‘).
Acutely released tPA may play animportant role in speeding up thrombolysis. Not only is the tPA thus released itself
enzymatically active, but acute release will also result in a
local tPA concentration much larger than the basal level of
tPAand also thanthat of its inhibitor PAI-1. Thrombin,
which is an effective stimulus for the induction of tPA release (Fig 3), may be the major stimulus for tPA release,
because it will reach high local concentrations near forming
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
STUDIES ON THE ACUTE RELEASE OF TPA
or growing thrombi. Indeed, in vivo in primates, thrombin
formation has been shown to induce acute release of large
amounts of tPA.30In quantitative terms, acute secretion of
50 pg tPA/cmz (as in Fig 6) will result, in a capillary with
a diameter of 6 pm, in a local concentration of 340 ng tPA/
mL. If P A synthesis is increased (Fig 6), up to 900 pg tPA/
cm2 can be released, resulting in a local concentration of 6
pg tPA/mL in that same capillary (compare with Schrauwen
et aI3). This concentration is even higher than the plasma
tPA concentration reached during thrombolytic therapy.
Moreover, tPA present before or during coagulation will be
much more potent in dissolving a thrombus than tPA present
only after thrombus formation has occurred,‘,’ as is the case
during thrombolytic therapy.
The results of this study show that in human endothelial
cells tPA can be acutely released from an intracellular pool,
and that it should be possible to enhance this capacity for
acute tPA release by increasing tPA synthesis. Therefore, in
combination with the possibility” to induce the release of
tPA without inducing the release of vWF, it should be possible to increase local tPA concentrations specifically and to
speed up fibrinolysis/thrombolysis when required.
REFERENCES
1. Brommer EJP: The level of extrinsic plasminogen activator (t-
PA) during clotting as a determinant of the rate of fibrinolysis;
inefficiency of activators added afterwards. Thromb Res 34:109,
I984
2. Fox KAA, Robison AK, Knabb RM, Rosamond TL, Sobel BE,
Bergmann SR: Prevention of coronary thrombosis with subthrombolytic doses of tissue-type plasminogen activator. Circulation
72:1346, 1985
3. Schrauwen Y, de Vries REM, Kooistra T, Emeis JJ: Acute
release of tissue-type plasminogen activator (PA) from the endothelium; regulatory mechanisms and therapeutic target. Fibrinolysis 8:8,
1994 (suppl 2)
4. Chandler WL, LevyWC, Veith RC, Stratton JR: A kinetic
model of the circulatory regulation of tissue plasminogen activator
during exercise, epinephrine infusion, and endurance training. Blood
81:3293, 1993
5. Kooistra T, Schrauwen Y, Arts J, Emeis JJ: Regulation of
endothelial cell t-PA synthesis and release. Int J Hematol 59:233,
1994
6. Emeis JJ: Regulation of the acute release of tissue-type plasminogen activator from the endothelium by coagulation activation
products. Ann NY Acad Sci 667:249, 1992
7. Booth F, Marshall JM, Cederholm-Williams SA: Effect of bradykinin on the exposure of tissue plasminogen activator from human
endothelial cells. Fibrinolysis 2: 107, 1988
8. Booyse F M , Bruce R, Dolenak D, Grover M, Casey LC: Rapid
release and deactivation of plasminogen activators in human endothelial cell cultures inthe presence of thrombin and ionophore
A23187. Semin Thromb Hemost 12:228, 1986
9. Wagner DD: Cell biology of von Willebrand factor. Annu Rev
Cell Biol 6:217, 1990
10. Schrauwen Y , Emeis JJ, Kooistra T: A sensitive ELISA for
human tissue-type plasminogen activator applicable to the study of
acute release from cultured human endothelial cells. Thromb
Haemost 7 1:225, 1994
1 1 . Padr6 T, van den Hoogen CM, Emeis JJ: Distribution of
3517
tissue-type plasminogen activator (antigen and activity) in rat tissues.
Blood Coagul Fibrinolysis l:601, 1990
12. Tranquille N, Emeis JJ: The simultaneous acute release of
tissue-type plasminogen activator and von Willebrand factor in the
perfused rat hindleg region. Thromb Haemost 63:454, 1990
13. Jaffe EA, Nachman RL, Becker CG, Minick CR: Culture of
human endothelial cells derived from umbilical cord veins: Identification by morphological and immunologic criteria. J Clin Invest
52:2745, 1973
14. Van Hinsbergh VWM, Bertina RM, Van Wijngaarden A, Van
Tilburg NH, Emeis JJ, Haverkate F: Activated protein C decreases
plasminogen activator-inhibitor activity in endothelial cell-conditioned medium. Blood 65:444, 1985
15. Kooistra T, Berg J van den, Tons A, Platenburg G, Rijken
DC, Berg E van den: Butyrate stimulates tissue-type plasminogenactivator synthesis in cultured human endothelial cells. Biochem J
247:605, 1987
16. Bennekum A van, Emeis JJ, Kooistra T, Hendriks HFJ: Modulationof tissue-type plasminogen activator by retinoids in rat
plasma and tissues. Am J Physiol 264:R931, 1993
17. Emeis JJ, Hoekzema R, de Vos A F Inhibiting interleukin1 and tumour necrosis factor-a does not reduce the induction of
plasminogen activator inhibitor type- 1 by endotoxin in rats in vivo.
Blood 85:115, 1995
18. Giles AR: Guidelines for the use of animals in biomedical
research. Thromb Haemost 58:1078, 1987
19. Granelli-Piperno A, Reich E: A study of proteases and protease-inhibitor complexes in biological fluid. J Exp Med 148:223, 1978
20. Laemmli UK: Cleavage of stmctural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680, 1970
21. Medh RD. Santell L, Levin EG: Stimulation of tissue plasminogen activator production by retinoic acid: Synergistic effect on
protein kinase C-mediated activation. Blood 80:981, 1992
22. Smalley DM, Fitzgerald JE, O’Rourke J: Adenosine diphosphate stimulates the endothelial release of tissue-type plasminogen
activator but not von Willebrand factor from isolated-perfused rat
hind limbs. Thromb Haemost 70:1043, 1993
23. Kooistra T, Sprengers ED, Van Hinsbergh VWM: Rapid inactivation of the plasminogen-activator inhibitor upon secretion from
cultured human endothelial cells. Biochem J 239:497, 1986
24. Emeis JJ, van den Hoogen CM, Schrauwen Y: An endothelial
storage granule for tissue-type plasminogen activator. Thromb
Haemost 69:609, 1993 (abstr)
25. Kelly FU3: Pathways of protein secretion in eukaryotes. Science 230:25, 1985
26. Reinders JH, Vervoom RC, Verweij CL, van Mourik JA, de
Groot PG: Perturbation of cultured human vascular endothelial cells
by phorbol ester or thrombin alters the cellular von Willebrand factor
distribution. J Cell Physiol 133:79, 1987
27. Mayadas T, Wagner DD, Simpson PJ: von Willebrand factor
biosynthesis and partitioning between constitutive and regulated
pathways of secretion after thrombin stimulation. Blood 73:706,
1989
28. Korner G, Bjornsson TD, Vlodavsky I: Extracellular matrix
produced by cultured corneal and aortic endothelial cells contains
active tissue-type and urokinase-type plasminogen activators. J Cell
Physiol 154456, 1993
29. Tranquille N, Emeis JJ: Protein synthesis inhibition by cycloheximide does not affect the acute release of tissue-type plasminogen
activator. Thromb Haemost 61:442, 1989
30. Giles AR, Nesheim ME, Herring SW, Hoogendoom H, Stump
DC, Heldebrant CM: The fibrinolytic potential of the normal primate
following the generation of thrombin in vivo. Thromb Haemost
63:476, 1990
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1995 85: 3510-3517
Studies on the acute release of tissue-type plasminogen activator
from human endothelial cells in vitro and in rats in vivo: evidence for
a dynamic storage pool
Y van den Eijnden-Schrauwen, T Kooistra, RE de Vries and JJ Emeis
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