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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
In vitro and in vivo effects of tPA and PAI-1 on blood vessel tone
Taher Nassar, Sa’ed Akkawi, Ahuva Shina, Abdullah Haj-Yehia, Khalil Bdeir, Mark Tarshis, Samuel N. Heyman, and Abd Al-Roof Higazi
Tissue type plasminogen activator (tPA)
is a key enzyme in the fibrinolytic cascade. In this paper we report that tPA
contains 2 independent epitopes that exert opposite effects on blood vessel tone.
Low concentrations of tPA (1 nM) inhibit
the phenylephrine (PE)–induced contraction of isolated aorta rings. In contrast,
higher concentrations (20 nM) stimulate
the contractile effect of PE. The 2 putative
vasoactive epitopes of tPA are regulated
by the plasminogen activator inhibitor-1
(PAI-1) and by a PAI-1–derived hexapeptide that binds tPA. TNK-tPA, a tPA variant
in which the PAI-1 docking site has been
mutated, stimulates PE-induced vasoconstriction at all concentrations used. The
stimulatory, but not the inhibitory, effect
of tPA on the contraction of isolated aorta
rings was abolished by anti–low-density
lipoprotein receptor–related protein/␣2macroglobulin receptor (LRP) antibodies.
Administering tPA or TNK-tPA to rats regulates blood pressure and cerebral vascu-
lar resistance in a dose-dependent mode.
In other in vivo experiments we found
that the vasopressor effect of PE is more
pronounced in tPA knockout than in wildtype mice. Our findings draw attention to
a novel role of tPA and PAI-1 in the
regulation of blood vessel tone that may
affect the course of ischemic diseases.
(Blood. 2004;103:897-902)
© 2004 by The American Society of Hematology
Introduction
Tissue type plasminogen activator (tPA) is one of the 2 principal
plasminogen activators in mammals. The activation of plasminogen by tPA or urokinase plasminogen activator (uPA) leads to the
generation of the potent proteolytic enzyme plasmin, which cleaves
blood clots; tPA⫺/⫺ mice accumulate fibrin in several organs,1 and
their capacity to cleave exogenous clots,1 including pulmonary
microemboli, is decreased.2
Several groups have previously shown that in addition to its
capacity to bind fibrin,3 tPA binds to cells within the blood vessel
wall.4 According to these studies, tPA binds to at least 2 receptors
on the cell surface, one of which was identified as plasminogen
activator inhibitor-1 (PAI-1).4,5
Another receptor in the vasculature that plays an important role
in the metabolism of tPA is the low-density lipoprotein receptor–
related protein/␣2-macroglobulin receptor (LRP), which mediates
the internalization and degradation of tPA.5-7 The binding of tPA to
LRP is enhanced by PAI-1, which, in addition to neutralizing the
catalytic activity of tPA, increases its internalization and degradation by cells.8
In an experimental stroke model, the infarcted area in tPA⫺/⫺
mice was smaller than in wild-type (WT) animals,9 indicating that
endogenous tPA plays a deleterious role in the development of the
damage induced by stroke. In contrast to the decreased sizes of
ischemic cerebral infarcts seen in tPA⫺/⫺ mice, the sizes of infarcts
in plasminogen activator inhibitor type-1 knockout mice (PAI1⫺/⫺) were increased.10 However, in urokinase plasminogen activator knockout mice (uPA⫺/⫺) no change in infarct size was found10;
therefore, the combined data obtained from uPA⫺/⫺ and PAI⫺/⫺
mice suggest that the negative role of tPA in stroke development
may not be related to the activation of plasminogen. Additional
results also show that exogenous tPA increases the size of the
infarcted area caused by stroke.9,10 More recent studies have shown
that tPA induces vasodilatation of isolated cerebral blood vessels,
an effect that was considered responsible for the intracranial
bleeding that can follow thrombolytic treatment with tPA.11
In spite of the widespread use of tPA as a thrombolytic agent in
acute myocardial infarction (MI) and its growing use in acute
ischemic stroke, little is known about its ability to regulate
vasoactivity. An effect on vasoactivity may play a critical role in
the deleterious effect seen during the development of stroke or in
the bleeding that may appear after the administration of tPA to
patients with acute MI or stroke. Therefore, our aim was to study
the effect of tPA on blood vessel contraction and dilatation and to
attempt to elucidate the molecular basis of this effect. Our findings
show that tPA has 2 independent epitopes with opposite effects on
blood vessel tone; the activity of each epitope is abolished by
PAI-1. We also report that phenylephrine causes a greater increase
in blood pressure in tPA⫺/⫺ mice than in WT animals and that
administering tPA to rats regulates blood pressure and cerebral
vascular resistance.
From the Departments of Clinical Biochemistry and Medicine, Hadassah
Hospital, Mt Scopus, Israel; Interdepartmental Unit and School of Pharmacy,
Hadassah University Hospital and Hebrew University-Hadassah Medical
School, Jerusalem, Israel; and the Departments of Pathology and Laboratory
Medicine, University of Pennsylvania, Philadelphia.
Reprints: Abd Al-Roof Higazi, Department of Pathology and Laboratory
Medicine, University of Pennsylvania, 513A Stellar-Chance, 422 Curie Blvd,
Philadelphia, PA 19104; e-mail:[email protected].
Materials and methods
Materials
Recombinant tPA (rtPA) and tPA variant TNK-tPA were obtained from
Genentech (South San Francisco, CA). In addition, rtPA was obtained from
Submitted May 29, 2003; accepted August 21, 2003. Prepublished online as Blood
First Edition Paper, September 25, 2003; DOI 10.1182/blood-2003-05-1685.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by National Institutes of Health grants HL60169, HL66442, and
HL67381.
© 2004 by The American Society of Hematology
BLOOD, 1 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 3
897
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898
BLOOD, 1 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 3
NASSAR et al
American Diagnostica (Greenwich, CT). Phenylephrine (PE) and L-nitroarg-methyl ester (L-NAME) were purchased from Sigma (St Louis, MO).
tPA⫺/⫺, uPA⫺/⫺, and uPAR⫺/⫺ mice on a C57/black background and
wild-type C57/black mice were purchased from Jackson Laboratories (Bar
Harbor, ME). Four-month-old male mice were used in all studies. Recombinant 39-kDa receptor-associated protein (rRAP) was generously provided
by Dr D. Strickland (American Red Cross, Rockville, MD). Anti-LRP antibodies
were also provided by D. Strickland and by American Diagnostica. The peptides
(EEIIMD and REIIMD) were synthesized as previously described.12 Human
umbilical vein vascular smooth muscle cells (SMCs) were the kind gift of Dr D.
Cines (Department of Pathology and Laboratory Medicine, University of
Pennsylvania) and were prepared as previously described.6
Methods
Contractile response of isolated aortic rings. Experiments were performed as previously described.13-15 Mice were killed by exsanguination.
Thoracic aortas were removed with care to avoid damage to the endothelia,
dissected free of fat and connective tissue, and cut into transverse rings 5
mm in length.16 To study the role of endothelial cells in the performed
experiments, the aorta rings were gently rotated on a stainless steel rod to
remove the endothelium (denuded aorta). To record isometric tension, the
rings were mounted in a 10-mL bath containing oxygenated (95% O2; 5%
CO2) solution of Krebs-Henseleit (KH) buffer (144 mM NaCl, 5.9 mM
KCl, 1.6 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3,
and 11.1 mM D-glucose). The rings were equilibrated for 1.5 hours at 37°C
and maintained under a resting tension of 2 g throughout the experiment.
Each aortic ring was then contracted by adding PE or endothelin in stepwise
increments (from 10⫺10 M to 10⫺5 M). In other experiments, tPA or
TNK-tPA was added at the indicated concentrations (see figure legends) 15
minutes before the addition of PE. To exclude any effect of the vehicle of
tPA or TNK-tPA, control experiments were performed using dialyzed tPA
and TNK-tPA; no difference between the dialyzed and nondialyzed tPA or
TNK-tPA was found. When indicated, the nitric oxide (NO) synthase
inhibitor L-NAME (1 ␮M final concentration) was added 10 minutes before
tPA or TNK-tPA. In each experiment, rings exposed to KH buffer alone
were analyzed in parallel. Isometric tension was measured with a force
displacement transducer and was recorded online using a computerized
system (ExperimentiaÆ, Budapest, Hungary).
The half-maximal effective concentration (EC50) was calculated by
measuring the response of aorta rings (y-axis) to increasing concentrations
of PE (x-axis). The lines that intersect with the y- and x-axes were drawn to
determine the concentration of PE that induces 50% of its maximal effect.
Each EC50 was calculated as the mean ⫾ SD of 3 separate experiments.
Blood pressure recording in mice. Mean arterial blood pressure
(MABP) was measured as previously described.13-15 Mice were anesthetized by intraperitoneal administration of 10 mg/kg⫺1 ketamine and 50
mg/kg xylazine. One cannula was placed in the left carotid artery to record
the mean arterial blood pressure, and a second, through which PE (0.3 ␮g
per animal) or saline was administered, was placed in the right jugular vein.
Blood pressure was monitored continuously through a transducer placed in
the left carotid cannula using the CARDIOSYS computerized system
(ExperimentiaÆ).
Hemodynamic studies in rats
Sprague-Dawley rats (353 ⫾ 4 g) were used for all experiments and were
fed on regular chow with free access to drinking water. The study was
conducted in accord with the National Institutes of Health Guide for the
Care and Use of Laboratory Animals. The rats were anesthetized with
thiobutibarbital (Inactin) (100 mg/kg intraperitoneally), and catheters were
placed in the trachea (PE-250), external jugular vein, femoral artery, and
vein (PE-50). Each animal was kept in a prone position, and normal saline
was infused throughout the experiment at a rate of 5 mL/h. Mean arterial
blood pressure was continuously monitored by a pressure transducer system
connected to the arterial line. The skin above the Bregma suture was
removed, and the periosteum and the external plate of the calvarium were
slightly eroded. A laser Doppler flow probe (Perimed, Stockholm, Sweden)
was placed perpendicularly to the bone 2 mm lateral and posterior to the
Bregma, on the right side, for the determination of cortical cerebral blood
flow. Online recordings of blood pressure and cerebral blood flow were
stored, displayed, and analyzed with the use of a computerized system
(MacLab/8; Analog Digital Instruments, Castle Hill, NSW, Australia).
Mean values for each determinant were analyzed over 1-minute periods.
Changes in regional cerebral vascular resistance were extrapolated from the
relative changes in blood pressure divided by the relative changes in blood
flow as described in Heyman et al.17 All values are expressed as the
percentage of baseline measurements.
Experimental design
Following a baseline 30-minute period with stabilized hemodynamics, rats
(n ⫽ 7-10 per group) were injected with tPA (2-10 mg/kg) alone or combined
with the PAI-1–derived hexapeptide EEIIMD (0.5 mg). A third group was
injected with TNK-tPA (2-10 mg/kg) in 0.5 mL, infused over 1 minute.
In a complementary second set of experiments, we studied the effect of
tPA on carotid blood flow. Anesthetized rats were prepared as detailed
above, but the measurement of cerebral cortical blood flow was substituted
with the direct determination of blood flow at the common carotid artery
using a perivascular transonic ultrasonic volume flow sensor (T-106;
Transonic Systems, Ithaca, NY).
Binding, internalization, and degradation of tPA
Sodium iodide I 125 tPA or TNK-tPA was radiolabeled and characterized as
described previously.18 SMC cells were grown to confluence in 48-well
Falcon multiwell tissue culture plates (Becton Dickinson, Lincoln Park, NJ)
to a final density of approximately 5 ⫻ 104 cells/well in Dulbecco modified
Eagle medium (DMEM) media (Bet Haemek Israel), supplemented with
10% fetal calf serum (FCS). Cells were prechilled to 4°C for 30 minutes and
washed twice with KH buffer. They were then incubated with KH buffer
containing 20 nM 125I-tPA for 2 hours at 4°C. Washing the cells 4 times with
binding buffer to remove unbound ligand terminated the incubation.
Radiolabeled ligand bound to the cell surface was released by adding 50
␮M glycine-HCl, pH 2.8 as described previously.12,18 Nonspecific binding
was determined by measuring cell-associated radioactivity in the presence
of 500 nM unlabeled tPA. Specific binding was defined as the difference
between total and nonspecific binding.
To measure internalization, after the cells were incubated with 125I-tPA
for 2 hours at 4°C and washed 4 times with binding buffer to remove
unbound ligand, the cells were warmed to 37°C for another hour, and
radiolabeled ligand bound to the cell surface was released with glycineHCl, pH 2.8. After releasing cell surface–bound ligand, internalized tPA
was determined by solubilizing the cells with 0.1 N NaOH and measuring
the cell-associated radioactivity. To measure degradation, the media were
removed after 18-hour incubation with radiolabeled ligands at 37°C,
trichloroacetic acid was added to a final concentration of 10%, the
precipitated protein was separated by centrifugation, and the radioactivity
in the supernatant was measured as previously described.12,18
Statistical analysis
In vivo data are presented as mean ⫾ SEM. One-way analysis of variance
(ANOVA) with post-hoc Newman-Keuls analysis and paired t test were
performed for the evaluation of repeated within-group hemodynamic
measurements. Statistical significance was set at P less than .05.
Ex vivo experiments were performed in triplicate and were repeated a
minimum of 3 times on different tissues. All data are presented as
mean ⫾ SD of the 3 experiments.
Results
tPA stimulates or inhibits contraction of aorta rings in a
dose-dependent manner
To study the involvement of tPA in the regulation of blood vessel
tone under physiologic or therapeutic conditions, we examined its
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BLOOD, 1 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 3
effect on the contraction of isolated rat aorta rings. As shown in
Figure 1, 1 nM tPA inhibited PE-induced vasoconstriction, with
EC50 increasing from 34 to 351 nM PE. The inhibitory effect of tPA
on PE-induced vasoconstriction is in line with the data published
by Cipolla et al.11 When endothelin was used instead of PE, tPA had
a similar effect (not shown).
Because 1 nM tPA is within the physiologic range and is far below
therapeutic concentrations, we examined the effect of a higher dose of
tPA on PE-induced vasoconstriction. As shown in Figure 1, 20 nM tPA
produced an effect opposite that seen at 1 nM; 20 nM tPA enhanced the
vasoconstriction induced by PE, decreasing the EC50 of PE from 34 to
1.6 nM. At higher concentrations (50 nM) tPA induced a similar
provasoconstrictive effect (data not shown).
tPA exerts its effects by acting on vascular endothelial cells
Because the endothelial cell layer plays a critical role in the
regulation of blood vessel vasoactivity, we examined the effect of
tPA on denuded aorta rings. We found no effect of tPA on the
contraction of denuded aorta rings or on aorta rings pretreated with
the NO synthase inhibitor L-NAME (data not shown).
TNK-tPA, a variant with stimulatory but not inhibitory effect on
blood vessel contraction
TNK-tPA is a bioengineered variant of tPA that has a longer
half-life than tPA,19 an 80-fold greater resistance to PAI-1 than tPA,
and a 14-fold greater relative fibrin specificity.19,20 In vitro,
TNK-tPA is 8- and 13-fold more potent than tPA toward whole
blood and platelet-enriched clots, respectively.19 In spite of these
great advantages of TNK-tPA over tPA in vitro, TNK-tPA had no
significant advantage over rtPA in clinical studies.21-23
To elucidate the basis for the discrepancy between TNK-tPA
activity in vitro and in patients, we studied its vasoactivity. Figure 2
shows that low concentrations of TNK-tPA had an opposing effect
to that of tPA on the contraction of aorta rings. The vasoconstriction
induced by PE was stimulated to a similar extent by 1 nM TNK-tPA
and by 20 nM tPA (compare with Figure 1). Figure 2 shows also
that the stimulatory effect of TNK-tPA on PE-induced vasoconstriction was greater when its concentration was increased from 1 to 20
nM; 20 nM TNK-tPA decreased the EC50 from 34 to 0.63 nM. As in
the case of tPA, TNK-tPA failed to induce any effect in the
vasoconstriction of denuded aorta rings or aorta rings pretreated
with L-NAME (not shown).
Figure 1. Effect of tPA on PE-induced contraction of isolated mouse aorta rings.
Contraction of isolated aorta rings was induced by increasing concentrations of PE in
the absence of tPA (Œ) or in the presence of 1 nM (f) or 20 nM tPA (䡺). Values are
presented as mean ⫾ SD.
tPA REGULATES SMOOTH MUSCLE CELL CONTRACTION
899
Figure 2. Effect of TNK- tPA on PE-induced contraction of isolated mouse aorta
rings. Contraction of isolated aorta rings was induced by increasing concentrations
of PE in the absence of TNK- tPA (Œ) or in the presence of 1 nM (f) or 20 nM TNK-tPA
(䡺). TNK-tPA at both concentrations enhanced PE-induced vasoconstriction.
PAI-1 inhibits tPA-mediated vasoactivity
To understand the molecular basis for the differences between the
vasoactivity of TNK-tPA and tPA, we asked whether the PAI-1
docking site, corresponding to amino acids 296 to 299,24,25 that had
a tetra-alanine substitution (K296A/H297A/R298A/R299A) in
TNK-tPA,19,26 was involved in tPA vasoactivity. To answer this
question, we examined the effect of PAI-1 (the natural ligand of the
docking site) on tPA vasoactivity. Figure 3A shows that both the
tPA provasodilatation and provasoconstrictive effects are inhibited
by equimolar concentrations of PAI-1.
Amino acid residues 350 to 355 in PAI-1 regulate tPA
vasoactivity
In addition to the docking site, PAI-1 interacts with the catalytic
site of tPA. To determine in a more specific way the role of the
PAI-1 docking site in the vasoactivity of tPA, we examined the
effect of the PAI-1–derived hexapeptide EEIIMD, which corresponds to the amino acid residues 350 to 355 of PAI-1 (the epitope
in PAI-1 that interacts with the tPA docking site24,27). Figure 3A
shows that 2 ␮M PAI-1–derived peptide, like full-length PAI-1,
prevented the provasodilatory and the provasoconstrictive effects
of rtPA. The control peptide REIIMD, which has almost no effect
on PAI-1 binding to uPA or on uPA vasoactivity,12,25 did not affect
tPA-mediated vasoactivity (not shown). The PAI-1–derived peptide
EEIIMD had no effect on the catalytic activity of tPA (not shown),
but it inhibited the binding of PAI-1 to tPA (Figure 3B).
PAI-1 is known to increase the LRP-mediated internalization
and degradation of tPA; in view of the ability of the PAI-1–derived
peptide to mimic the PAI-1 effect on tPA vasoactivity, we asked
whether it also affects tPA metabolism. Figure 3C shows that the
degradation of radiolabeled tPA is increased by PAI-1 and by
PAI-1–derived peptide, although the effect of EEIIMD was markedly lower than that of PAI-1. As shown in Figure 3C, the
degradation of tPA in the presence or absence of PAI-1 or EEIIMD
was inhibited by anti-LRP antibodies but not by irrelevant IgG (not
shown). PAI-1 and the PAI-1–derived peptide EEIIMD affected the
internalization of radiolabeled tPA by cells in a manner comparable
to that of tPA degradation (not shown). These results are in line
with our previous report on the effect of the PAI-1–derived peptide
on uPA internalization and degradation.12 The data presented in
Figure 3C also show that TNK-tPA degradation is LRP-mediated,
but at a much slower rate than WT tPA; furthermore, EEIIMD, like
irrelevant IgG (not shown), had no effect on TNK-tPA degradation.
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900
NASSAR et al
BLOOD, 1 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 3
antibodies had no effect on the provasodilatory effect obtained
at low concentration of tPA (1 nM). In contrast, anti-LRP
antibodies abolished the provasoconstrictive effect obtained at
20 nM tPA. Furthermore, in the presence of anti-LRP antibodies,
20 nM tPA induced vasodilatation comparably to that induced
by 1 nM tPA (Figure 4), supporting the existence of 2
independent tPA receptors and the conclusion that the vasodilatation is LRP-independent. Figure 4 also shows that anti-LRP
antibodies have no effect on the contraction of aorta rings in the
absence of tPA. rRAP induced an effect comparable to that of
anti-LRP antibodies (data not shown).
Exogenous tPA affects systemic blood pressure and cerebral
vascular resistance in rats
Figure 3. Effect of PAI-1 and PAI-1–derived peptide on the vasoactivity of tPA.
(A) EC50 of PE on the contraction of isolated aorta rings was determined in the
absence (Control) or presence of tPA 1 nM (tPA 1 nM) or 20 nM (tPA 20 nM), tPA with
an equimolar concentration of PAI-1 (tPA 1 nM ⫹ PAI-1), (tPA 20 nM ⫹ PAI-1), tPA
with 2 ␮M EEIIMD (tPA 1 nM ⫹ EEIIMD), (tPA 20 nM ⫹ EEIIMD), 20 nM PAI-1 (PAI-1),
or 2 ␮M EEIIMD (EEIIMD) alone. (B) Inhibition of the binding of PAI-1 to tPA by the
PAI-1–derived peptide. The binding of PAI-1 (1 nM) to immobilized tPA was
determined in the absence (cont.) or presence of 2 ␮M EEIIMD (EEIIMD). Equimolar
concentration of tPA (tPA) served as a positive control. (C) PAI-1–derived peptide
enhances LRP-mediated degradation of tPA. The degradation of WT tPA (tPA) or
TNK-tPA (TNK-tPA) by SMCs was determined in the absence or presence of PAI-1
(PAI-1) or PAI-1–derived peptide (EE) and of anti-LRP antibodies (anti-LRP) (as
specified in the labels).
After showing the effect of tPA and its inhibitor (PAI-1) on
the contraction of isolated aorta rings, the next step was to
examine the vasoactivity of the plasminogen activator and PAI-1
in-vivo. Figure 5 shows that the intravenous administration of 2
mg/kg tPA to anesthetized rats induced a rapid and partially
reversible decline in blood pressure that was most pronounced
by 3 minutes. When the animals were given the same amount
of tPA together with 0.1 mg of the PAI-1–derived peptide
EEIIMD, no significant hemodynamic change was detected.
Injection of saline alone induced no change in rat blood pressure
(not shown).
Taking into consideration the potential deleterious role of tPA in
an experimental stroke model in which the infarcted area in tPA⫺/⫺
mice was smaller than in WT animals,9 we asked whether tPA
regulates cerebral circulation. Figure 5 shows that despite the
decrease in blood pressure, cerebral blood flow was not significantly affected by tPA. Figure 5 also shows that tPA decreased
cerebral vascular resistance significantly; this finding could explain
the maintenance of cerebral blood flow in the presence of a
decrease in blood pressure.
Using the alternative method in which carotid blood flow was
monitored, tPA induced a modest, although consistent, vasoactive
response; the calculated vascular resistance declined by
3.5% ⫾ 0.3% below baseline, within 2 to 3 minutes of tPA
administration (n ⫽ 19; P ⬍ .0002). When tPA was administered
together with 0.1 mg EEIIMD, no significant change in vascular
resistance was detected, as in the results shown in Figure 5.
The data presented in Figure 5 show that under the described
experimental conditions, in vivo the vasodilatory effect of tPA is
dominant between the 2 opposite effects seen ex vivo. It was,
therefore, of interest to examine in vivo the vasoactivity of
TNK-tPA, the tPA variant that ex vivo had only a vasoconstrictive
effect (Figure 2). Figure 5 shows that TNK-tPA (2 mg/kg) had no
vasodilatory effect in vivo and that its administration even induced
LRP mediates the provasoconstrictive effect of tPA
The data presented in Figure 3 indicate that PAI-1 inhibits the
vasoactivity of tPA and increases its degradation. LRP is one of
the major receptors of tPA in the liver and vasculature and
mediates a significant part of tPA clearance from the circulation.28 PAI-1 is known to modify the interaction of tPA with
LRP.7,8 LRP is also involved in signal transduction, including
Ca⫹⫹ mobilization,29 and it mediates uPA and defensin14,15
vasoactivity. We, therefore, examined the involvement of LRP
in tPA-induced vasoactivity. Figure 4 shows that anti-LRP
Figure 4. Inhibition of tPA-induced vasoconstriction, but not vasodilatation, by
anti-LRP antibodies. EC50 was calculated from the response of mouse aortic rings
to increasing concentrations of PE and was determined in the absence or presence of
1 or 20 nM tPA and in the absence (u) or presence of anti-LRP antibodies (䡺).
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BLOOD, 1 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 3
tPA REGULATES SMOOTH MUSCLE CELL CONTRACTION
901
Figure 7. Effect of endogenous tPA on the contraction of isolated aorta rings.
Contraction of aortic rings from WT or tPA⫺/⫺ mice was determined in the presence of
increasing concentrations of PE, and the EC50 of PE was calculated. (Mean ⫾ SD,
*P ⬍ .0001 vs tPA⫺/⫺.)
Endogenous tPA participates in the regulation
of blood pressure
Figure 5. Effect of tPA on rat hemodynamics. Blood pressure and cortical cerebral
blood flow were monitored in anesthetized rats given intravenous tPA alone (tPA) or in
combination with the PAI-1–derived peptide EEIIMD (tPA ⫹ EEII) or TNK-tPA
(TNK-tPA). Cerebral vascular resistance was extrapolated from the corresponding
changes in blood pressure and regional flow. Values are presented as mean ⫾ SEM.
In tPA-treated rats but not in the other 2 groups, blood pressure and cerebral vascular
resistance significantly declined. (*P ⬍ .05 vs baseline, ANOVA).
a slight and statistically insignificant increase in blood pressure
during the first 5 minutes. Moreover, cerebral vascular resistance
did not decline significantly (Figure 5).
The inability of each subtype of tPA to induce an increase in
blood pressure prompted us to ask whether the circulating concentrations were in the effective range. To answer this question, we
determined the concentrations of radiolabeled tPA in the circulation, as previously described.2 We found that 3 minutes (when the
maximal effect of tPA was achieved) after the injection of 2 mg/kg
WT tPA, the concentration in the blood reached 166 nM. Taking
into consideration the possibility that an endogenous ligand(s) may
compete for the receptor mediating the vasoconstrictive effect of
tPA, we increased the dose of tPA to 10 mg/kg. Figure 6 shows that
at this concentration, WT tPA lost its vasodilatory effect and instead
caused a statistically insignificant increase in blood pressure,
whereas TNK-tPA induced a significant increase in the MABP of
approximately 14% (n ⫽ 10; P ⬍ .0002).
Figure 6. Effect of tPA on blood pressure of rats. Blood pressure was determined
in anesthetized rats 3 minutes after intravenous injection of saline (columns 1 and 4),
2 or 10 mg/kg tPA (columns 2 and 3, respectively), or 2 or 10 mg/kg TNK-tPA
(columns 5 and 6, respectively). (Mean ⫾ SD, *P ⬍ .0002 vs baseline.)
Having shown the effect of exogenous tPA on blood vessel tone in vitro
and in vivo, we next asked whether endogenous tPA plays any role in the
regulation of blood vessel vasoactivity. Figure 7 shows that aorta rings
from tPA⫺/⫺ mice had a greater response to PE than rings from WT mice
with EC50 of 11.35 and 33.67 nM, respectively (n ⫽ 5; P ⬍ .0001). To
examine the effect of endogenous tPA on blood pressure, we compared
the blood pressure of tPA⫺/⫺ mice to that of WT. No significant
differences between the blood pressure of tPA⫺/⫺ and WT mice were
found; MABP was 75.33 and 74.5 mm Hg, respectively (Figure 8).
In contrast to the similar MABP seen in WT and tPA⫺/⫺ mice,
Figure 8 shows that after the intravenous injection of PE (0.1 ␮g),
tPA⫺/⫺ mice had a greater response than WT mice—MABP was
144.5 ⫾ 4.76 compared with 123.17 ⫾ 3.19.
Discussion
This study was designed to examine the effects of tPA and PAI-1 on
blood vessel vasoactivity. Our data show that these 2 components
of the fibrinolytic system do indeed regulate blood vessel tone. Ex
vivo data in isolated aorta rings strongly suggest that tPA contains 2
distinct epitopes with opposite effects. One epitope inhibits the
contraction induced by PE, whereas the other enhances it. The
finding that TNK-tPA had no inhibitory effect but maintained the
capacity to stimulate PE-induced vasoconstriction supports the
contention that we are dealing with 2 independent epitopes.
Figure 8. Effect of endogenous tPA on blood vessel vasoactivity. Mean blood
pressure was monitored in WT and tPA⫺/⫺ mice (n ⫽ 6 per group) at baseline and after
intravenous injection of 0.1 ␮g PE. (Mean ⫾ SD, *P ⬍ .0001 vs WT mice, 2-way ANOVA.)
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902
BLOOD, 1 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 3
NASSAR et al
Another interesting finding is the observation that PAI-1 abolished
the effect of tPA on blood vessel tone. The involvement of PAI-1 in
tPA-mediated vasoactivity clarified the molecular basis of plasminogen
activator vasoactivity. Binding of PAI-1 to its docking site, situated
outside the catalytic site (aa 296-299), seems to induce conformational
changes in tPA that affect the exposure of epitopes that regulate blood
vessel tone. This assertion is supported by the ability of the PAI-1–
derived peptide (EEIIMD) to inhibit tPA vasoactivity without affecting
its catalytic activity and, to some extent, by the inability of TNK-tPA
(mutated at the docking site) to cause vasorelaxation.
It is clear that the effect of tPA is endothelial cell dependent and,
more specifically, NO synthase dependent. The fact that only the
provasoconstrictive effect of tPA was inhibited by LRP antagonists
suggests that the binding of tPA to LRP leads to the inhibition of NO
synthase. On the other hand, tPA seems to bind to another receptor
present in endothelial cells that mediates the stimulation of NO synthase
activity and thus inhibits the vasoconstrictive effect of PE.
The capacity of PAI-1 to inhibit the LRP-mediated vasoactivity of
tPA is in line with its well-known effect on the tPA–LRP interaction and
suggests that the binding of tPA to the (putative) receptor that mediates
the provasodilatory effect is also regulated by PAI-1.
The normal blood pressure of tPA⫺/⫺ mice, despite the increased vasoactivity of isolated aorta rings from the same animals,
suggests that the physiologic effect of tPA on blood vessel tone is
strictly regulated and may become evident only under special
physiologic or pathologic conditions; This conclusion is supported
by the increased response of blood pressure to PE in tPA⫺/⫺ mice.
The finding that 2 mg/kg tPA induced vasodilatation is consistent
with the ex vivo data that show the inhibitory effect of tPA on the
contraction of isolated aorta rings. On the other hand, the inability of
high-dose WT-tPA to induce clear vasoconstriction in vivo remains an
open question. One reason for the apparent difference between the in
vivo and ex vivo data could be that tPA exerts its opposite effects on
blood vessel tone through 2 different receptors, each of which can
function differently in vivo or in vitro. A change in the activity of either
of the 2 receptors could be attributed to the availability of alternative
ligands or regulators under in vivo conditions.
The finding that TNK-tPA induces vasoconstriction suggests
that in vivo the signal transduction pathway that mediates the
vasoconstrictive effect of tPA is functional and that its activity may
become active under certain physiologic or pathologic conditions.
Furthermore, the vasoconstrictive effect of TNK-tPA may explain
the discrepancy between its performance as a thrombolytic agent in
vitro and in patients. In other words, the inability of TNK-tPA to
induce vasodilatation (and possibly to induce more vasoconstriction) could limit its delivery to the clot that has to be cleaved.
In conclusion, our findings describe a novel role for tPA and its
inhibitor, PAI-1, in the regulation of blood vessel tone that could be
of importance in the development of ischemic disorders. Evidently,
additional studies are required to elucidate the detailed mechanisms
by which tPA affects blood vessel vasoactivity.
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2004 103: 897-902
doi:10.1182/blood-2003-05-1685 originally published online
September 25, 2003
In vitro and in vivo effects of tPA and PAI-1 on blood vessel tone
Taher Nassar, Sa'ed Akkawi, Ahuva Shina, Abdullah Haj-Yehia, Khalil Bdeir, Mark Tarshis, Samuel
N. Heyman and Abd Al-Roof Higazi
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