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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 153-160
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Vascular release of plasminogen activator inhibitor-1 impairs
fibrinolysis during acute arterial thrombosis in mice
Tomihisa Kawasaki,
Mieke Dewerchin,
Henri R. Lijnen,
Jos Vermylen, and
Marc F. Hoylaerts
From the Center for Molecular and Vascular Biology, University of
Leuven, and Center for Transgene Technology and Gene Therapy, Flanders
Interuniversity Institute for Biotechnology, Leuven, Belgium
 |
Abstract |
The role of plasminogen activator inhibitor-1 (PAI-1) in the plasma,
blood platelets, and vessel wall during acute arterial thrombus
formation was investigated in gene-deficient mice. Photochemically induced thrombosis in the carotid artery was analyzed via
transillumination. In comparison to thrombosis in C57BL/6J wild-type
(wt) mice (113 ± 19 × 106 arbitrary light units
[AU] n = 15, mean ± SEM), thrombosis in PAI-1 / mice (40 ± 10 × 106 AU,
n = 13) was inhibited (P < .01), indicating that PAI-1
controls fibrinolysis during thrombus formation. Systemic
administration of murine PAI-1 into PAI-1/ mice led
to a full recovery of thrombotic response. Occurrence of fibrinolytic
activity was confirmed in  2-antiplasmin
(2-AP)-deficient mice. The sizes of thrombi developing
in wt mice, in 2-AP+/ and
2-AP/ mice were 102 ± 35,
65 ± 8.1, and 13 ± 6.1 × 106 AU,
respectively (n = 6 each) (P < .05), compatible with
functional plasmin inhibition by 2-AP. In contrast,
thrombi in wt mice, t-PA/ and u-PA/
mice were comparable, substantiating efficient inhibition of fibrinolysis by the combined PAI-1/2-AP action. Platelet
depletion and reconstitution confirmed a normal thrombotic response in
wt mice, reconstituted with PAI-1/ platelets, but
weak thrombosis in PAI-1/ mice reconstituted with wt
platelets. Accordingly, murine (wt) PAI-1 levels in platelet lysates
and releasates were 0.43 ± 0.09 ng/109 platelets and
plasma concentrations equaled 0.73 ± 0.13 ng/mL. After
photochemical injury, plasma PAI-1 rose to 2.9 ± 0.7 ng/mL (n = 9, P < .01). The plasma rise was prevented by
ligating the carotid artery. Hence, during acute thrombosis,
fibrinolysis is efficiently prevented by plasma 2-AP,
but also by vascular PAI-1, locally released into the circulation after
endothelial injury.
(Blood. 2000;96:153-160)
© 2000 by The American Society of Hematology.
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Introduction |
Thrombi dissolve as a consequence of activation of the
fibrinolytic system. The physiologic plasminogen activators tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) are serine proteinases activating the proenzyme plasminogen to
the broad specificity enzyme plasmin.1 The
serpin plasminogen activator inhibitor-1 (PAI-1) is the
main inhibitor of both t-PA and u-PA and constitutes a critical
regulator of plasminogen activation.2 Several clinical
studies have associated elevations in plasma PAI-1 with increased risk
for thrombosis,3,4 whereas a drop in plasma PAI-1 levels
may be a cause of recurrent bleeding.5,6 Animal studies
substantiated that increased circulating PAI-1 levels are associated
with a prothrombotic tendency,7 whereas inhibition of PAI-1
with monoclonal anti-PAI-1 antibodies8,9 or low-molecular
weight compounds10 has antithrombotic consequences. Plasmin
is very rapidly and specifically inhibited by
alpha2-antiplasmin ( 2-AP), which circulates
in plasma at a high concentration of 1 µmol/L.11 The
physiologic importance of 2-AP is illustrated by the
significant bleeding tendency in patients with homozygous 2-AP deficiency; heterozygotes have no or only mild
bleeding complications.12
Whereas 2-AP is synthetized in the liver and released
into the circulation, the origin of active PAI-1 in the circulation is
less well-defined. PAI-1 is produced by several cell types, including
endothelial cells, smooth muscle cells, fibroblasts, and
hepatocytes.13 Two distinct pools of PAI-1 exist in the circulation, 1 in platelets and 1 in plasma. Human platelets are a
major reservoir of PAI-1, with up to 90% of the circulating human
PAI-1 contained within platelet -granules,14 yielding up
to 700 ng PAI-1 per 109 platelets.15 However,
platelet PAI-1 exists predominantly in a latent or inactive
form,15 suggesting its effect on fibrinolysis to be rather
limited. Nevertheless, the inhibitory effect of platelets on clot
lysis16,17 was proposed to be mediated partly by platelet PAI-1, a conclusion supported by a differential clot lysis efficiency in the presence of normal platelets or platelets derived from PAI-1-deficient patients.18
Different experimental conditions in various in vitro studies may
explain discordant results on the role of platelet PAI-1 in
fibrinolysis.16,17,19 In addition, important species
differences were reported with respect to the relative concentrations
of PAI-1 in plasma, platelets, inside thrombi, and in
tissues.20-23 Furthermore, species-dependent differences in
the proportion of active versus latent PAI-1 have been
observed.23 The study of mouse gene knock-outs has made
clear that a combined t-PA and u-PA gene deficiency leads to extensive
spontaneous fibrin deposition,24 but also that in
PAI-1/ mice, lysis of pulmonary plasma clots
is significantly enhanced.25
In a murine model of ferric chloride-induced arterial thrombosis,
significantly smaller residual thrombosis was shown 24 hours after
injury induction in PAI-1/ mice than in
wild-type (wt) mice.26 In addition, from the shorter reperfusion times observed during thrombolytic therapy of platelet-rich arterial thrombi in PAI-1/ mice, Zhu et
al27 concluded that PAI-1 is a major determinant of the
resistance to thrombolysis by pharmacologic t-PA concentrations. In
more acute studies of photochemically induced arterial thrombosis, a
significant prolongation of the time required to occlude the vessels
with thrombi was observed in PAI-1/ mice,
whereas u-PA and t-PA deficiencies had no effect on closure time,28 highlighting the importance of PAI-1 during
thrombus formation.
In this study, we have investigated the role of PAI-1 for the control
of fibrinolysis during acute thrombosis, by focusing on PAI-1 present
in different compartments. By performing PAI-1 reconstitution
experiments in plasma and platelets, this study provides the first
evidence that PAI-1 released from the vasculature participates in the
control of endogenous fibrinolysis in mice, at sites of arterial injury
initiating acute thrombosis.
| |
Materials and methods |
Reagents
Recombinant t-PA (Actilyse) was from Boehringer Ingelheim
(Ingelheim, Germany). The chromogenic plasmin substrate S-2403 was purchased from Chromogenix (Antwerp, Belgium) and equine tendon collagen from Nycomed (München, Germany). Busulfan was acquired from Sigma (St Louis, MO). Recombinant murine and human
PAI-1 were prepared as described elsewhere.25 Other
reagents were obtained commercially.
Animals
2-AP, PAI-1, t-PA, or u-PA gene-deficient mice were
generated via homologous recombination in embryonic stem cells, as
described.24,25,29,30 To avoid potential effects of strain
differences, consecutive generations of mice carrying the
null gene allele were backcrossed repeatedly to C57BL/6J mice. Only
mice generated after 5 or more backcrosses were used in this study.
Genotyping of mice was performed by Southern blot analysis of tail tip
DNA.29
Experimental thrombosis model
All animal experiments were reviewed and approved by the
Institutional Review Board of the University of Leuven and were
performed in accordance with protocols approved by the Institutional
Animal Care and Research Advisory Committee. Thrombus was induced as recently described.31 Mice (10 to 12 weeks old) of both
sexes weighing 19 to 31 g were anesthetized by intraperitoneal
injection of sodium pentobarbital (60 mg/kg) and fixed on a heated
operating table. Atropine sulfate was also injected and endotracheal
intubation carried out. A 2F venous cathether was inserted into the
right jugular vein for injection of reagents and rose-bengal. The left carotid artery was carefully exposed from the surrounding tissue and
mounted on a transilluminator. The exposed artery was irradiated with
the green light (wavelength 540 nm) of a Xenon lamp (L4887, Hamamatsu
Photonics, Hamamatsu, Japan), equipped with a heat-absorbing filter and
a green filter. Irradiation was directed via a 3-mm diameter optic
fiber attached to a manipulator. Just after the injection of
rose-bengal (20 mg/kg) via the intravenous catheter, irradiation was
started for various time intervals (1-4 minutes).
The analytical procedures for the quantitation of mural thrombi in the
mouse carotid artery have been described.32 The total light
intensity versus time curve was established over 40 minutes, and
thrombus formation was measured by comparing the area under the curve
and expressed in arbitrary light units (AU).
Administration of recombinant murine PAI-1
Immediately after a 3-minute photochemical injury, recombinant
murine PAI-1, diluted with saline just before use, was injected into
PAI-1/ mice (n = 6) as a single bolus (35 ng), followed by a continuous infusion (76 ng/h) for 40 minutes via a
microinfusion pump (Perfusor, Braun, Melsungen, Germany)
and an intravenous catheter. Blood samples for platelet counting and
plasma PAI-1 measurements were collected at the end of the experiment
via the vena cava into a syringe containing 3.8% sodium citrate as an
anticoagulant (1/10 volume). After centrifugation at 3000 rpm for 10 minutes, the citrated plasma was stored at  70°C.
Platelet reconstitution experiments
Thombocytopenia was induced in wt mice and
PAI-1/ mice via a triple intraperitoneal
injection of busulfan at 20 mg/kg,33 dissolved in
polyethylene glycol 400 diluted with saline to 25% just before the
injection, on day 0, 3, and 9. On day 21, circulating platelets sampled
via tail bleeding were counted on a Cell Dyn 1300 (Abbott,
Ottignies/Louvain-la-Neuve, Belgium), and mice were subdivided in
groups to test thrombus induction without and after reconstitution with
murine washed platelets. Thrombocytopenic animals were anesthetized as
outlined above and reconstituted with 5 × 108
platelets, prepared from wt or PAI-1/ mice
and injected as a bolus in a volume of 200 µL into the jugular vein.
For this purpose, platelets were prepared after sampling blood
from the vena cava of donor mice on acid-citrate dextrose (ACD).
Platelet-rich plasma (PRP) was prepared via 2 subsequent centrifugations at 800 rpm for 5 minutes. Pooled PRP was then mixed
with an equal volume of ACD and centrifuged at 2000 rpm for 10 minutes
to pellet platelets. The platelet pellet was resuspended in saline at
2.5 × 106 platelets/µL. Platelets were counted at
the end of the thrombosis experiment both in wt mice and in
platelet-reconstituted thrombocytopenic mice.
Amidolytic assay
Blood samples were collected from PAI-1+/+ and
PAI-1/ mice (n = 2 each) as described
above, and washed platelets (prepared as above) were lysed with 1%
Triton X-100 and 5 minutes later 10-fold diluted with
phosphate-buffered saline (PBS). Active PAI-1 was evaluated by first
adding human t-PA (23 pmol/L) to the platelet lysate (or dilutions) for
10 minutes at room temperature, and then by measuring residual t-PA
activity via the activation of plasminogen. Therefore, human
Lys-plasminogen (0.2 µmol/L) was incubated with the treated human
t-PA sample at 37°C in the presence of CNBr-digested human
fibrinogen fragments (0.1 µmol/L) as described.34 Generated plasmin was measured by the simultaneous addition of S-2403
(0.5 mmol/L) and A(405 nm) was measured up to 40 minutes with a
multiscan spectrophotometer ELx808 (Bio-Tek Instruments, Highland Park,
Winooski, VT). t-PA activities were derived from plots of
A(405 nm) versus (time),2 which were proportional to the
t-PA activity. Initial activation rates of plasminogen by t-PA in the
absence or presence of platelet lysates/releasates were monitored in
the same way. Measurements were performed in duplicate.
Preparation of lysates and releasates from murine platelets for
PAI-1 dosage
Washed platelets prepared from vena cava blood of C57BL/6J mice
(n = 6) were resuspended up to 5 × 108
platelets/mL in PBS containing 0.002% Tween 80, 5 mmol/L EDTA, and
0.1% bovine serum albumin (BSA). After centrifugation, platelets in
the pellet were lysed with 1% Triton X-100 in 100 µL and, after 5 minutes of incubation, 10-fold diluted with buffer. Alternatively, platelets were incubated for 10 minutes at 37°C with equine tendon collagen (20 µg/mL; this concentration induced murine platelet aggregation during classical aggregometry). After centrifugation, platelet lysates and releasates were immediately analyzed via enzyme-linked immunosorbent assay (ELISA) (see below).
Tissue and plasma sampling after photochemical injury
Blood samples were collected from the vena cava from C57BL/6J
controls and from mice 40 minutes after the 3-minute photochemical injury in wt mice (n = 18), respectively, in thrombocytopenic mice,
reconstituted with wt platelets or PAI-1/
platelets (3 groups of n = 6). To delineate the origin of the plasma
PAI-1 elevation after injury, blood sample controls were prepared after
injection of rose-bengal, but without injuring light irradiation
(n = 6) or after carotid artery ligation, with (n = 6) or without
(n = 6) vessel wall injury. Citrated plasma was stored at
70°C. Carotid arteries of both injured and noninjured sites
were carefully dissected and rinsed with saline. Tissues (pooled from 3 arteries to raise the sensitivity) were homogenized with 100 µL of
PBS containing 1% Triton X-100, diluted with 900 µL PBS, and stored
at 70°C. Tissue protein content was determined using a
dye-binding assay kit (Bio-Rad, Hercules, CA) with BSA as a standard.
Measurements were performed in duplicate.
PAI-1 antigen and activity determinations
Murine PAI-1 antigen levels in tissue and platelet extracts and in
plasma withdrawn before/after photochemical injury were measured by a
sensitive combined monoclonal-polyclonal antibody assay, calibrated
with recombinant murine PAI-1.35 For this purpose, the
murine antimurine PAI-1 monoclonal antibody H34G6 (4 µg/mL) was
coated on microtiter plates for 48 hours at 4°C. Bound PAI-1 in the
samples was revealed using a biotinylated and diluted (1:250) rabbit
polyclonal antiserum raised in the laboratory against murine PAI-1,
incubated for 1 hour at 37°C, and then by a peroxidase-labeled
avidin-biotin complex, according to standard protocols. In this assay,
a linear relationship exists between A(492 nm) and antigen
concentrations between 0.03 ng/mL and 3 ng/mL PAI-1. Recovery
experiments validating the precision of the PAI-1 antigen determination
in lysed platelets consisted of the addition of known amounts of murine
PAI-1 (0-3 ng/mL) to the platelets before sample preparation via lysis.
For PAI-1 activity determinations, the monoclonal antihuman PAI-1
antibody 16F11 (4 µg/mL), which cross-reacts with murine PAI-1, was
coated on microtiter plates, for 48 hours at 4°C.29 Samples containing t-PA-PAI-1 complexes were incubated as described above for the antigen-ELISA, followed by addition of horseradish peroxidase (HRP)-conjugated monoclonal antibody 62E8 (directed against
human t-PA; dilution 1:8000) for 2 hours at room temperature. For
calibration, purified active recombinant human PAI-1 was
used,36 after preincubation with an excess of t-PA. All
activity data are expressed in nanograms per milliliter (ng/mL) complex
and all ELISA measurements were performed in duplicate. With the use of
human t-PA-PAI-complexes, a linear dose-response was observed between
0.03 and 10 ng/mL of complex.
Complex formation between t-PA and PAI-1 extracted or released both
from human and murine platelets was performed as follows. t-PA (30 ng/mL) was added to washed platelets
(0-5 × 108 platelets/mL) and extraction was
performed with 1% Triton X-100 for 5 minutes, followed by a 10-fold
dilution to reduce the Triton X-100 concentration, avoiding
detergent-induced conformational changes in the extracted
PAI-1.37 Separate control experiments in which recombinant
murine PAI-1 was preincubated with 1% Triton X-100 for 5 minutes
confirmed that this treatment was without effect on the PAI-1 activity.
The addition of t-PA to the platelets before initiation of lysis with
Triton X-100 was likewise performed to shorten the exposure time of
released PAI-1 to the detergent. Recovery experiments validating the
precision of the PAI-1 activity determination were performed by adding
known amounts of murine PAI-1 to the platelets (0-3 ng/mL) before
addition of t-PA, sample preparation via lysis and ELISA.
Plasma samples were measured 10-fold diluted, at which dilution no
plasma matrix-dependent effects were observed. Platelet lysates,
platelet releasates, and tissue extracts were measured 2-fold diluted,
both in the antigen and in the activity ELISA.
Statistical analysis
Data are presented as mean ± SEM. Comparison between
groups was performed using the Dunnett multiple range test,
Student t test, Mann-Whitney test, Dunn's multiple
comparison test, or Tukey-Kramer multiple comparison test, as described
in the text. P values of < .05 are considered significant.
| |
Results |
Thrombus generation in PAI-1+/+,
PAI-1+/, and PAI-1/ mice
As shown in Figure 1, the absence of
PAI-1 was associated with an apparent antithrombotic effect. The
average thrombus size in wt mice after a 3-minute photochemical injury
equaled 113 ± 19 × 106 AU (n = 15).
Thrombus formation in PAI-1/ mice was
significantly decreased (40 ± 10 × 106 AU,
n = 13, P < .01 by Dunnett multiple range test). The
average thrombus size in heterozygotes
(124 ± 33 × 106 AU, n = 6) did not differ
from that in wt mice (P < .05 by Dunnett multiple range
test, versus the thrombus size in PAI-1/
mice). Injection into PAI-1/ mice of a bolus
of recombinant murine PAI-1 of 35 ng, followed by a continuous infusion
at 76 ng/h until the end of the 40-minute experiment, normalized
thrombus formation (Figure 1). The average thrombus size
(137 ± 30 × 106 AU, n = 6,
P < .01 by Student t test versus thrombus size in nontreated PAI-1/ mice) was comparable to
that in PAI-1+/+ mice. Plasma PAI-1 antigen levels at the
end of the infusion were 10 ± 2.5 ng/mL (n = 6).
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| Fig 1.
Thrombus generation in PAI-1+/+,
PAI-1+/, and PAI-1/ mice.
Cumulative thrombus formation over 40 minutes after a 3-minute carotid
artery photochemical injury, expressed as total light intensity;
+/+: wild-type mice; +/: PAI-1
heterozygotes;/: PAI-1-deficient
homozygotes; thrombus formation in PAI-1/
mice after an intravenous bolus (35 ng) with recombinant murine PAI-1,
administered immediately after photochemical injury, followed by
continuous infusion (76 ng/h) for 40 minutes via an intravenous
catheter (n = 6). Data are the mean ± SEM for the number of mice
indicated in parentheses. *P < .01 by Dunnett multiple
comparison test and  P < .05.
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Figure 2 shows representative tracings of
the light intensity versus time curves during thrombus formation in wt
mice, in PAI-1/ mice, and recombinant murine
PAI-1-treated gene-deficient PAI-1/ mice. In
wt mice, a cyclic flow pattern of massive thrombus formation was
induced immediately after the photochemical injury, leading to a slowly
decreasing thrombus (Figure 2A). In contrast, in
PAI-1/ mice, thrombi only developed just
after the photochemical injury and no stable thrombus was observed at
later time points (Figure 2B). The injection of recombinant murine
PAI-1 restored injury induced thrombus formation in the later phase of
the observation period (Figure 2C).
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| Fig 2.
Thrombus progression after photochemical injury.
Typical patterns of light intensity recorded during 40 minutes after
photochemical injury of the carotid artery of PAI-1+/+ mice
(A), PAI-1/ mice (B), and recombinant murine
PAI-1 treated PAI-1/ mice (C).
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Thrombus generation in 2-AP+/+,
2-AP+/, and
2-AP/ mice
As shown in Figure 3, the absence of
plasma 2-AP was also associated with an apparent
antithrombotic effect. The average thrombus size in wt mice after a
3-minute photochemical injury (102 ± 35 × 106
AU, n = 6) did not differ from that in wt controls in Figure 1.
The thrombus size, however, was gene dose-dependent. In
2-AP/ mice, it was greatly and
significantly decreased (13 ± 6.1 × 106 AU,
n = 6, P < .05 by Dunnett multiple range test). In
heterozygote 2-AP+/ mice, an
intermediate intensity of thrombus generation
(65 ± 8.1 × 106 AU, n = 6) was observed, in
agreement with the expression of intermediate plasma levels of
2-AP.38
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| Fig 3.
Thrombus generation in
2-AP+/+,
2-AP+/, and
2-AP/ mice.
Cumulative thrombus formation over 40 minutes after a 3-minute carotid
artery photochemical injury, expressed as total light intensity;
+/+: wild-type mice; +/:
2-AP heterozygotes; /:
2-AP-deficient homozygotes. Data are the mean ± SEM
for the number of mice indicated in parentheses (*P < .05
by Dunnett multiple comparison test in comparison with the
2-AP+/+ group).
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Thrombus generation in t-PA/ and
u-PA/ mice
Thrombogenicity in these gene-deficient mice was investigated after
induction of a milder photochemical injury, consisting of a 2-minute
irradiation, because of the potential increase in thrombogenicity. As
shown in Figure 4,
t-PA/ mice were inclined to more intense
thrombosis (32 ± 8.6 × 106 AU, n = 8) than
wt mice (15 ± 4 × 106 AU, n = 9,
P = .078). In contrast, the average thrombus size in
u-PA/ mice
(27 ± 12 × 106 AU, n = 7) was almost
identical to that in wt mice (27 ± 6.6 × 106
AU, n = 6). Comparison of all data revealed no significant
differences between wt mice and t-PA/ or
u-PA/ mice.
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| Fig 4.
Thrombus generation in t-PA+/+ and
t-PA/ mice versus u-PA+/+ and
u-PA/ mice.
Cumulative thrombus formation over 40 minutes after a mild 2-minute
carotid artery photochemical injury, expressed as total light
intensity; +/+: wild-type mice;/:
t-PA- or u-PA-deficient homozygotes. Data are the mean ± SEM for
the number of mice indicated in parentheses.
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Effect of PAI-1 on plasminogen activation by t-PA
To investigate whether physiologic t-PA concentrations are inhibited
by murine platelet PAI-1, a sensitive kinetic and coupled assay of
plasminogen activation was carried out, necessitating only
picomolar concentrations of t-PA. In the presence of
CNBr-fibrinogen fragments, Lys-plasminogen (0.2 µmol/L) was activated
in a time-dependent manner by t-PA (final concentration, 20 pmol/L),
leading to an exponential increase with time of A(405 nm), after
hydrolysis of the plasmin substrate S-2403 (not shown). Control
incubations of t-PA with recombinant human PAI-1 at 30 pmol/L inhibited
subsequent plasminogen activation completely (not shown). In the
presence of platelet lysates (derived from 7.5-30 × 104
platelets/µL), plasminogen activation was inhibited in a
dose-dependent manner, but to a comparable degree for lysates from
PAI-1+/+ and PAI-1/ mice (not
shown). Because under these conditions t-PA activity was influenced by
other factors present in the platelet lysate than the presence of
PAI-1, this approach was abandoned and further analyses were
exclusively made by ELISA.
Murine PAI-1 antigen and activity levels in plasma and platelets
With the use of a sensitive sandwich ELISA (monoclonal-polyclonal
antibodies), PAI-1 antigen values measured in plasma obtained from 9 wt
C57BL/6J mice averaged 0.73 ± 0.13 ng/mL. Recovery experiments, during which murine PAI-1 was added to washed platelets before lysis,
indicated that platelet membrane components did not interfere with the
detection of the PAI-1 antigen in ELISA and that added PAI-1 was fully
detected. In PAI-1/ mice (n = 4), PAI-1
antigen remained undetectable, confirming the specificity of the ELISA,
at least when analyzing 10-fold diluted plasma samples. In plasma from
endotoxin-treated mice (n = 6, pooled), PAI-1 antigen levels rose to
136 ± 20 ng/mL (n = 4), in agreement with intense vascular
release of PAI-1 into the circulation after endotoxin
treatment.21 PAI-1 antigen levels in platelet lysates and
releasates were 0.56 ± 0.19 ng/109 platelets
(n = 6) and 0.34 ± 0.08 ng/109 platelets (n = 9),
respectively, yielding an average value of 0.43 ± 0.09
ng/109 platelets (n = 15).
The feasibility of activity measurements for platelet PAI-1 in platelet
lysates prepared via detergent extraction was first tested with human
platelets. Analysis via the activity-ELISA yielded 22 ± 3.9 ng
t-PA-PAI-1 complex per 109 platelets for lysed platelets
(n = 4) and 11 ± 1.8 ng/109 platelets for the
releasate of collagen stimulated platelets (n = 4), in agreement with
known activity levels for human platelet PAI-1. Further recovery
experiments during which murine PAI-1 was added to the platelets before
the addition of t-PA and lysis with Triton X-100, however, revealed
that only about 30% of the added reactive PAI-1 was detected in
complex with t-PA. Hence, similar to their effect during
the t-PA-induced plasminogen activation, platelet membrane
components interfere slightly with t-PA inhibition by PAI-1.
However, no t-PA-PAI-1 complexes could be detected in t-PA-treated
murine platelet lysates. In view of the total antigen content of
the platelet and the sensitivity of the t-PA-PAI-1 complex assay (0.03 ng/109 platelets to be multiplied with a correction factor
of 3-4 for the presence of platelet membranes), this finding implies
that at best 20% of platelet PAI-1 is active. On the contrary, in
plasma obtained from 7 wt C57BL/6J mice, the ELISA estimated PAI-1
activities equivalent to 0.40 ± 0.10 ng/mL, whereas in
PAI-1/ mice (n = 3), PAI-1 activity
remained undetectable. In plasma obtained from endotoxin-treated mice
(n = 6, pooled), estimated PAI-1 activity values were 175 ± 20
ng/mL (3 separate measurements). These data imply that most of the
circulating plasma PAI-1 is active.
Thrombosis after platelet reconstitution
After treatment with busulfan,33 circulating platelet
numbers dropped progressively as a result of inhibition of
megakaryocyte differentiation and simultaneous clearance of circulating
platelets. Platelet counts in wt mice averaged
8.3 ± 0.4 × 105/µL blood, but 21 days after
initiation of treatment, platelet numbers had dropped to
1.4 ± 0.12 × 105/µL (n = 19) in the
treated wt mice and to 1.7 ± 0.23 × 105/µL
in the PAI-1/ group (n = 7). Figure
5 shows that as a consequence of the
thrombocytopenia, the thrombotic response was greatly decreased in
comparison to the response in untreated wt mice. Furthermore,
reconstituting wt platelets in thrombocytopenic wt mice could restore
thrombus formation completely. However, thrombus formation could be
restored equally well in thrombocytopenic wt mice with
PAI-1/ platelets. In contrast, wt platelets
were not capable of normalizing the thrombotic response in
PAI-1/ mice. The thrombus size in this group
(39% ± 12% of control thrombus) did not differ statistically
from the average thrombus size measured in the
PAI-1/ group in Figure 1 (35% ± 8.8%
of corresponding control group). These experiments confirmed that
platelet PAI-1 is not the determinant of the fibrinolytic control in
vivo. Platelet numbers measured at the end of the thrombosis
experiment averaged 2.6 ± 0.24 × 105/µL
(n = 8) in PAI-1/ mice reconstituted with
wt platelets, and 2.5 ± 0.08 × 105/µL
(n = 7) in PAI-1+/+ mice reconstituted with
PAI-1/ platelets, respectively,
2.4 ± 0.19 × 105/µL (n = 8) in
PAI-1+/+ mice reconstituted with PAI-1+/+
platelets. Platelet numbers in wt controls equaled
4.3 ± 0.34 × 105/µL (n = 8) at the end of
the thrombosis experiment. Therefore, reconstituted platelet numbers
equal about 60% of the normal count (about
5 × 105/µL initially), in agreement with the
injected amount of 5 × 108 platelets per mouse.
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| Fig 5.
Thrombus generation in platelet reconstituted
thrombocytopenic PAI-1+/+ and PAI-1/
mice.
Cumulative thrombus formation over 40 minutes after a 3-minute carotid
artery photochemical injury, expressed as total light intensity;
+/+: wild-type mice;/:
PAI-1-deficient homozygotes. Thrombocytopenia was induced with
busulfan in the groups indicated; mice were reconstituted with
5 × 108 PAI-1+/+ or
PAI-1/ platelets and thrombosis was
quantitated after injury induction. Data are the mean ± SEM for the
number of mice indicated in parentheses. *P < .01 by
Tukey-Kramer multiple comparison test in comparison with the groups
indicated by the horizontal brackets; P < 05.
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PAI-1 antigen levels in tissue and plasma after
photochemical injury
PAI-1 antigen levels in plasma of wt mice 40 minutes after the
photochemical injury were 2.9 ± 0.7 ng/mL, compared with
0.57 ± 0.09 ng/mL in the control group, exposed to the dye
without injury (n = 9 each, P < .01 by Dunn's multiple
comparison test) (Figure 6). Because this
increase could not be explained by release of the very low PAI-1
content in platelets (less than 1 ng per mouse), we investigated
whether PAI-1 would be released by the injured carotid artery. Figure 6
shows that a double ligation (without photochemical injury) around the
carotid artery hardly triggers any increase of plasma PAI-1 by itself.
However, preventing contact between the injured area and the
circulation, by ligating the injured vessel area, almost completely
abolishes the observed rise of plasma PAI-1 after injury. Hence, these
results substantiate that significant amounts of PAI-1 are released
from the vessel wall in the vicinity of the developing thrombus.
Likewise, PAI-1 levels in the injured carotid artery, removed 40 minutes after the photochemical reaction, were 1.3 ± 0.42
ng/mg protein extracted, compared with 4.9 ± 1.5 ng/mg for
control carotid arteries (n = 6 for samples consisting of 3 pooled
arteries, P < .05 by Student t test).
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| Fig 6.
Release of PAI from the vessel wall.
PAI-1 antigen levels in plasma 40 minutes after a 3-minute carotid
artery photochemical injury, compared with control values (rose-bengal
injection without injury); effect of ligation without injury (second
bar) and of ligation in combination with injury (fourth bar) on PAI-1
plasma concentrations is also shown. Data are mean ± SEM for the
number of animals indicated between parenthesis. *P < .01
by Dunn's multiple comparison test in comparison with the groups
indicated by the horizontal brackets; P < .05;
 P < .05 by alternative t test for the direct
comparison between the groups indicated; these groups had unequal SD
distribution.
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Separate measurements of plasma PAI-1 in thrombocytopenic mice at the
end of the thrombosis induction confirmed that the rise of PAI-1 did
not depend on the presence of platelets. In comparison to plasma PAI-1
in nonthrombocytopenic and noninjured wt mice injected with rose-bengal
(0.35 ± 0.04 ng/mL; n = 6), plasma PAI-1 rose to
1.76 ± 0.1 ng/mL after injury in thrombocytopenic wt mice (n = 6) and to 1.79 ± 0.12 ng/mL in thrombocytopenic wt mice
reconstituted with wt platelets (n = 6), respectively, 2 ± 0.23
ng/mL in thrombocytopenic wt mice reconstituted with
PAI-1/ platelets (n = 6). Although these
numbers are slightly lower than those in Figure 6, these experiments
confirm that the rise of plasma PAI-1 after vessel wall injury is not
influenced by the PAI-1 content of the circulating platelets.
| |
Discussion |
Several models in mice have been reported, in which thrombosis is
induced via a vascular injury, inflicting damage to endothelial cells,
elastic lamina, and smooth muscle cells.39,40 The model used in this study causes only mild damage to endothelial cells; thrombi are platelet-rich and resemble clinical thrombi by electron microscopic analysis.41 By combining transillumination and
photochemical vessel wall injury in the mouse, it has become possible
to link the degree of vessel wall injury to the intensity of
thrombosis, which develops as a consequence of endothelial cell
destruction.31 This methodology was used in this study to
investigate the consequences for thrombus generation of gene
inactivation of inhibitors of fibrinolysis, and more in particular to
study the contribution of PAI-1 present in plasma, endothelial cells
and platelets.
The role of PAI-1 in controlling fibrinolysis in vivo has already been
investigated extensively. Thus, Fay et al42 recently reported a significant decrease in residual thrombi 24 hours after vessel wall injury with FeCl3 in
PAI-1/ mice, compared with wt mice. However,
they reported similar initial thrombus formation in
PAI-1/ and PAI-1+/+
mice.26,42 In contrast, Matsuno et al28
recently identified a delay in acute thrombus formation in
PAI-1/ mice, suggesting that PAI-1 controls
fibrinolysis also in the early phase of thrombus development. However,
this interpretation is at variance with a normal tail bleeding and
blood loss after amputation of the cecum in
PAI-1/ mice.25 The spontaneous
lysis of 125I-fibrin-labeled pulmonary emboli in
PAI-1+/ mice was not different from that in
PAI-1+/+ mice after 4 hours, but significantly weaker
fibrinolysis was found in PAI-1+/ mice after 8 hours.25 During recent t-PA-induced thrombolysis studies
in mice, reperfusion occurred in all mice that received t-PA, but
reperfusion times were significantly shorter in
PAI-1/ mice.27
Our current results indicate that both 2-AP and PAI-1
play a determinant role in controlling acute arterial thrombus
formation. Inactivation of 2-AP indeed leads to a potent
and gene dose-dependent antithrombotic effect. Gene inactivation of
PAI-1 is associated with a somewhat weaker antithrombotic effect, which
was not gene dose-dependent; ie, heterozygous
PAI-1+/ mice showed normal arterial thrombus
formation. It has recently been shown that 2-AP
cross-links to fibrin.43 Therefore, theoretically, the
reduced thrombus formation observed in PAI-1 and 2-AP
gene-deficient mice could be postulated to result from a reduced
contribution to thrombus formation of PAI-1, respectively
2-AP. However, the efficiency of spontaneous
thrombolysis seems to be controlled primarily by circulating
2-AP and not by the amount of 2-AP cross-linked to fibrin38 and 2-AP
cross-linking does not seem to affect the lysability of fibrin clots by
the murine fibrinolytic system. These data support our interpretation
that 2-AP plays a relevant role in regulating thrombus
formation, in relation to its high plasma content, via controlling
fibrinolysis, but not via influencing fibrin clot architecture. Because
of the lack of a strict gene dose-dependent antithrombotic effect for
PAI-1, its role in acute thrombus formation appeared to be more
complex, which findings were the basis for this study, that
investigates contributions of PAI-1 present in different compartments
to the control of thrombosis. Our finding that thrombus generation in t-PA/ and u-PA/
mice was equal to that in wt mice agrees with results of Matsuno et
al.28 It therefore seems that during an acute thrombotic response, t-PA- and u-PA-induced plasmin formation in wt mice is
efficiently inhibited by the combined action of PAI-1 and
2-AP.
The intravenous injection of recombinant murine PAI-1, in combination
with a continuous PAI-1 infusion, fully restored thrombus development
in PAI-1/ mice, at steady-state plasma PAI-1
levels (at the end) equal to 10 ± 2.5 ng/mL (equivalent to
15-fold the wt plasma concentration). These findings confirm that
soluble plasma PAI-1 is responsible for t-PA inhibition during thrombus
formation. In contrast to work performed by others, studying the
effects of exogenous PAI-1 in thrombosis models at high doses of
PAI-144,45 because of the short half-life in vivo,46
this study shows that low plasma concentrations of active PAI-1
suffice to restore inhibition of fibrinolysis. To investigate whether
platelet PAI-1 would be involved in t-PA inhibition during the
continuous deposition of platelets in a growing thrombus, crossover
design experiments were performed, in which
PAI-1/ mice were reconstituted with
PAI-1+/+ platelets and PAI-1+/+ mice with
PAI-1/ platelets. These experiments confirmed
that thrombocytopenia could be induced in mice using busulfan and that
thrombocytopenia resulted in loss of a thrombotic response to vascular
injury. Furthermore, reconstitution with wt platelets in wt mice led to a full recovery of thrombus development, whereas reconstitution of
PAI-1/ mice with wt platelets could not
restore thrombus development. Because a normal thrombotic response was
measured in wt mice reconstituted with PAI-1-deficient platelets, it
became clear that thrombosis was not controlled via platelet PAI-1.
Histologic and immunohistochemical studies confirmed endothelial cell
destruction by the photochemical injury, in agreement with earlier
findings. Because large amounts of granulocytes were found in the
vicinity of the injured site (not shown), we conclude that PAI-1 is
released into the circulation after injury and the accompanying
inflammatory reaction, generating high concentrations of PAI-1 in the
vicinity of the developing thrombus. This conclusion is supported by
the 4- to 5-fold increase in plasma PAI-1 after injury in wt mice but
also in thrombocytopenic mice. Accordingly, vascular tissue PAI-1
antigen concentrations were reduced after injury, presumably as a
consequence of the release from the vessel into the circulation.
Furthermore, ligating the injured area could completely abrogate the
rise of plasma PAI-1. A mechanism for the regulation of thrombolysis in
vivo has been proposed in which PAI-1 released from endothelial cells
is incorporated into developing fibrin clots.47 Our own
immunofluorescent PAI-1 staining of carotid artery cross sections was
too diffuse to conclude decisively whether PAI-1 would predominantly be
released from endothelial or from smooth muscle cells. Several studies
indicate, however, that mouse endothelial cells do not contain PAI-1
messenger RNA (mRNA)48,49 and even take up PAI-1 from the
circulation. Because these authors found faint PAI-1 expression by
resting murine medial smooth muscle cells, those findings are
indicative of PAI-1 release from the media in this study. Nevertheless,
we can conclude that at sites of vascular injury, thrombi develop in an
antifibrinolytic microenvironment, due to local secretion of PAI-1. The
potent inhibition of plasmin by circulating 2-AP
completes this antifibrinolytic action.
To understand the lack of effect on thrombosis of platelet PAI-1, we
adapted existing ELISA assays to accurately measure PAI-1 antigen and
activity levels in mouse platelets. The low murine PAI-1 antigen
concentrations measured in plasma (below 1 ng/mL) are consistent with
data described elsewhere.29,35 These levels are more than
10-fold lower than in human plasma15,50 but comparable to
rat plasma.22 At least half of this concentration was
estimated to represent active PAI-1, as also observed in man.15,50
Murine platelet PAI-1 antigen levels measured in lysates and
releasates were around 0.5 ng/109 cells; ie, about 50-fold
and 500-fold lower than in rat and human platelets,
respectively.15,22,50,51 We could easily detect active
PAI-1 in human platelets, but not in murine platelets. From the lack of
reactivity with human t-PA, and correcting for interferences during the
detection, we estimate that at best 20% of the PAI-1 antigen in murine
platelets represents active PAI-1, as in human
platelets,40-42 corresponding to an active PAI-1 pool circulating in platelets equivalent to 100 pg only. These numbers explain in our cross-over thrombosis studies that platelet PAI-1 antigen levels and activity are too low to exert any effect during platelet accumulation in developing thrombi. They also support our
findings that it is the local release of vascular PAI-1 that controls
fibrinolysis in the developing thrombus. They further explain the lack
of a gene-dose effect in the PAI-1+/ mice, because
when released from the vessel wall, locally high PAI-1 concentrations
can be reached, even in heterozygous animals.
The currently described results do not detract from conclusions in
other animal models in which small amounts of active PAI-1 in platelets
played a role in regulating fibrinolysis. The 500-fold lower levels of
PAI-1 in mouse platelets compared with human platelets rather provided
a unique opportunity to study the role of PAI-1 from other sources in
regulating fibrinolysis. Rather than diminish the role of platelet
PAI-1 for human biology, this study raises the possibility that
vascular release of PAI-1 may play a similar role in humans.
In conclusion, this study confirms that, during acute thrombosis,
fibrinolysis is efficiently prevented by the combined action of plasma
2-AP and PAI-1. PAI-1 is released locally into the circulation from the vessel wall, as a consequence of the injury, and
participates in the control of thrombus formation. Thus, our findings
show a role for the vessel wall in controlling the fibrinolytic system
during thrombosis in vivo.
| |
Acknowledgments |
We thank Dr P. Declerck (Laboratory for Pharmaceutical Biology and
Phytopharmacology, Faculty of Pharmaceutical Sciences, KU Leuven) for
generously providing some of the antibodies and the recombinant murine
PAI-1 used in this study. The help of I. Vreys (immunohistochemical
studies) and H. Moreau (ELISAs |
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Abstract
Introduction