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Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2274-2281
 2-Antiplasmin Gene Deficiency in Mice Is Associated
With Enhanced Fibrinolytic Potential Without Overt Bleeding
By
H.R. Lijnen,
K. Okada,
O. Matsuo,
D. Collen, and
M. Dewerchin
From the Center for Molecular and Vascular Biology, University of
Leuven, and the Center for Transgene Technology and Gene Therapy,
Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium;
and the Department of Physiology, Kinki University School of Medicine,
Osaka, Japan.
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ABSTRACT |
 2-antiplasmin (2-AP) is
the main physiologic plasmin inhibitor in mammalian plasma.
Inactivation of the murine
2-AP gene was
achieved by replacing, through homologous recombination in embryonic
stem cells, a 7-kb genomic sequence encoding the entire murine protein
(exon 2 through part of exon 10, including the stop codon) with the
neomycin resistance expression cassette. Germline transmission
of the mutated allele was confirmed by Southern blot analysis.
Mendelian inheritance of the inactivated
2-AP allele was
observed, and homozygous deficient
(2-AP /) mice displayed normal
fertility, viability, and development. Reverse transcription-polymerase
chain reaction confirmed the absence of
2-AP mRNA in kidney
and liver from 2-AP/ mice, in contrast
to wild-type (2-AP+/+) mice.
Immunologic and functional 2-AP levels were undetectable in plasma of 2-AP/ mice, and were
about half of wild-type in heterozygous littermates (2-AP+/). Other hemostasis
parameters, including plasminogen activator inhibitor-1, plasminogen,
fibrinogen, hemoglobin, hematocrit, and blood cell counts were
comparable for 2-AP+/+,
2-AP+/, and
2-AP/ mice. After amputation of tail
or toe tips, bleeding stopped spontaneously in
2-AP+/+, as well as in
2-AP+/ and
2-AP/ mice. Spontaneous lysis after 4 hours of intravenously injected 125I-fibrin-labeled plasma
clots was significantly higher in
2-AP/ than in
2-AP+/+ mice when injecting clots
prepared from 2-AP+/+ plasma
(78% ± 5% v 46% ± 9%; mean ± SEM, n = 6 to
7; P = .02) or from 2-AP/
plasma (81% ± 5% v 46% ± 5%; mean ± SEM,
n = 5; P = .008). Four to 8 hours after endotoxin
injection, fibrin deposition in the kidneys was significantly reduced
in 2-AP/ mice, as compared with
2-AP+/+ mice (P  .005).
Thus, 2-AP/ mice develop and reproduce
normally; they have an enhanced endogenous fibrinolytic capacity
without overt bleeding.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE MAMMALIAN fibrinolytic system
contains a proenzyme, plasminogen, that is converted to the active
serine proteinase plasmin by tissue-type (t-PA) or urokinase-type
(u-PA) plasminogen activator. Inhibition of the system may occur
through neutralization of the plasminogen activators by plasminogen
activator inhibitors (mainly PAI-1) or through neutralization of
plasmin.1  2-antiplasmin (2-AP) is the main physiologic plasmin inhibitor in
mammalian plasma, whereas excess plasmin may be inhibited by
2-macroglobulin.2-4 2-AP is
synthesized in the liver and is present in plasma at a concentration of
about 1 µmol/L.2-4 Human and murine 2-AP are serpins (serine proteinase inhibitors) with molecular
weight (Mr) 65 to 70 kD,2-5 which inhibit plasmin in a very rapid
reaction resulting in the formation of a stable inactive complex.6 The cDNA and deduced amino acid
sequence,7,8 as well as the gene
organization9,10 of both human and murine 2-AP have been elucidated.
The mouse 2-AP gene encodes
a 491-amino acid protein, with the NH2-terminus Val of the
mature protein corresponding to residue 28,5,10 whereas
mature human 2-AP also consists of 464 residues with Met
as NH2-terminus.11 The reactive site peptide
bond consists of Arg-Met in the inhibitor of both
species.7,8 In human and murine plasma, 2-AP
occurs as a plasminogen-binding (60% to 70%) and as a
non-plasminogen-binding (30% to 40%) form lacking a COOH-terminal
fragment, which contains structures with affinity for the
lysine-binding sites of plasminogen.5,12-14 The
plasminogen-binding form cross-links to fibrin when it is clotted in
the presence of Ca2+ and activated factor
XIII.15 Binding of 2-AP to plasminogen may
prevent subsequent binding of plasminogen to fibrin, and thus result in
an antifibrinolytic effect. Low Mr forms of 2-AP have been detected at low concentration (0.05% of
the plasma concentration) in the -granules of blood platelets; their
function remains unknown.16
Besides in the removal of fibrin, the fibrinolytic system may also play
a role in phenomena such as ovulation, embryogenesis, intima
proliferation, atherosclerosis, tumorigenesis, and
metastasis.17 2-AP may thus exert an
inhibitory effect at different levels on fibrinolysis, as well as on
several other plasmin-mediated biologic processes. Therefore, this
inhibitor appears to be an interesting target to study its biologic
role directly with the use of mice with specific inactivation of the
2-AP gene. This strategy
has successfully been applied to study the biologic function of most
other components of the fibrinolytic system.17 We have
previously characterized the murine
2-AP gene and constructed a
targeting vector for homologous recombination in embryonic stem (ES)
cells.10 In this study, we report the generation of
homozygous 2-AP-deficient mice and evaluate the biologic effects of 2-AP
gene disruption.
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MATERIALS AND METHODS |
Animals, reagents, and assays.
Mice were kept in microisolation cages on a 12-hour day-night cycle and
fed a regular chow. They were anesthetized by intraperitoneal injection
of 60 mg/kg Nembutal (Abbott Laboratories, North Chicago, IL), and
blood was collected by vena cava puncture with a 24-gauge needle.
Platelet-poor plasma was prepared by centrifugation of blood collected
on citrate at 4,000g for 5 minutes.
Murine 2-AP, plasminogen and plasmin, human plasmin, and
two-chain urokinase-type plasminogen activator (tcu-PA) were prepared and characterized as described elsewhere.5 Polyclonal
antibodies against purified murine 2-AP and plasminogen
were raised in rabbits. Before use, the antiserum against murine
2-AP was adsorbed with 10% to 20% (vol/vol) murine
2-AP-deficient plasma. 2-AP and plasminogen antigen levels were quantitated by enzyme-linked
immunosorbent assay (ELISA) using the purified murine proteins for
calibration.5 2-AP activity levels in murine
plasma were determined by addition of human plasmin (final
concentration 5 nmol/L in 500-fold diluted plasma) and measurement of
residual plasmin with the chromogenic substrate S-2403 (final
concentration 0.3 mmol/L) (Chromogenix, Antwerp, Belgium) after
incubation at 37°C for 10 seconds,5 using a calibration
curve constructed with pooled plasma obtained from wild-type C57BL6/J
mice. Plasma PAI-1 antigen levels were determined with a specific ELISA
using monoclonal antibodies produced in gene-inactivated
mice.18 Fibrinogen levels were determined with a clotting
rate assay using human thrombin. Plasma levels of murine
2-macroglobulin were determined by rocket
electroimmunassay, as described,19 using a polyclonal
rabbit antiserum kindly provided by Dr F. Van Leuven (Center for Human
Genetics, University of Leuven, Belgium). Calibration curves were
constructed using pooled plasma from wild-type male or female mice, for
determination of 2-macroglobulin levels in plasma
samples from males or females, respectively. White blood cell, red
blood cell, and platelet counts, hemoglobin and hematocrit levels, mean
corpuscular value, mean corpuscular hemoglobin, and mean corpuscular
hemoglobin concentration were determined on blood collected in
trisodium citrate (final concentration, 10 mmol/L) using an automated analyzer.
Endotoxin (Escherichia coli lipopolysaccharide, serotype 0111:
B4) was purchased from Sigma Chemical Co (St Louis, MO).
Liver or kidney extracts were prepared by homogenization and extraction
with 0.1 mol/L Tris, 0.25% Triton X-100 (Merck,
Darmstadt, Germany), pH 8.0, and protein concentration was determined
using the BCA protein assay (Pierce, Rockford, IL).
SDS-PAGE without reduction was performed on 10% to 15% gradient gels
using the PhastSystem (Pharmacia, Uppsala, Sweden). Immunoblotting, after transfer to nitrocellulose sheets, was performed using antisera against murine 2-AP or plasminogen.
Bleeding times in mice were recorded after amputation of a standardized
fragment of the tail (2 cm) or of a toe. Data are reported as mean ± SEM and statistical analysis was performed using the two-tailed
t-test (nonparametric, Mann Whitney) for comparison between two
groups. Genotype distributions and histopathologic data on fibrin
deposition in kidney sections after endotoxin injection were compared
by Chi-square analysis.
Generation of chimeric and
2-antiplasmin-deficient mice.
The targeting vector
pPNT.2-AP, in which
the neomycin resistance expression cassette replaces a 7-kb
genomic fragment comprising exon 2 through part of exon 10 (including
the stop codon), which represents the entire sequence encoding mature
murine 2-AP, was described previously.10
Electroporation of 129R1 ES cells (obtained from A. Nagy, Samuel
Lunenfeld Research Institute, Toronto, Canada) or of 129/SvJ RW4 ES
cells (Genome Systems Inc, St Louis, MO) with the linearized targeting
vector yielded, respectively, three (out of 127) or eight (out of 93)
correctly targeted clones as confirmed by Southern blot analysis of
purified genomic DNA with appropriate restriction enzymes and
probes.10
Targeted clones were used for aggregation with Swiss morulas (R1 ES
clones) or C57BL6/J morulas (RW4 ES clones). Chimeric offspring,
identified by the presence of agouti (R1 ES cells) or agouti and/or
white (RW4 ES cells) coat pigmentation, were obtained (for both R1 and
RW4 targeted clones), whereas germline transmission of ES-cell DNA was
only obtained with RW4 ES clones (five germline-competent chimeras
originating from three independently targeted clones). Heterozygous
2-AP-deficient germline offspring, identified by
Southern blot analysis of tail-tip genomic DNA, were intercrossed to
generate 2-AP/ progeny (yielding a
genetic background of 50% 129/SvJ and 50% C57BL6/J).
Southern blot analysis of genomic DNA.
DNA was isolated from mouse tail tips, digested with KpnI and
analyzed by Southern blotting using a 3' probe as
described.10
Reverse transcription-polymerase chain reaction (RT-PCR).
Polyadenylated RNA (polyA RNA) was extracted from kidney and liver
using the Quick Prep mRNA purification kit (Pharmacia Biotech Benelux,
Roosendaal, The Netherlands) and was submitted to first strand cDNA
synthesis by oligo(dT) priming using the Ready-to-Go T-primed first
strand kit (Pharmacia). The reaction products (RT-cDNA) were then used
in PCR amplification with primers annealing in the coding part of exon
10 (sense primer: 5'-AATTGTTCCAGGGCCCAGACCTTCGT-3', nucleotides (nt)
1109-1134 of murine 2-AP cDNA, GenBank accession number
Z367748; and antisense primer:
5'-GTCCTCCATGATGAAGAAGAGGAAGGG-3', nt 1302-1276 of murine
2-AP cDNA). The RT-PCR products were analyzed by
separation on a 1% agarose gel.
Histopathologic examination.
2-AP+/+ and
2-AP/ mice (2 males and 2 females each
at 6 and 20 weeks of age) were anesthetized and perfused through
cardiac puncture with 0.9% NaCl followed by 4% formalin in 0.07 mol/L sodium phosphate buffer, pH 7.0.
Organs were removed, postfixed in the same fixative for 20 hours, and
embedded in paraffin. Representative 7-µm sections of all tissues
were examined microscopically after staining with hematoxylin/eosin.
The tissue sections included cross sections of brain, heart, thymus,
lung, liver, spleen, kidney, small and large intestine, stomach, cecum,
leg muscle, reproductive organs (vas deferens, testis and epididymis,
or uterus and ovaries), lymph node, adrenal gland, and pancreas.
Immunostaining for fibrin(ogen) was performed by incubating the
sections with goat antiserum against murine fibrinogen/fibrin (Nordic,
Tilburg, The Netherlands: working dilution 1/500) in 0.01 mol/L Tris,
pH 7.6, containing 0.9% NaCl and 0.1% Triton X-100 for 3 hours at
room temperature. After rinsing, the sections were incubated
consecutively for 60 minutes with biotinylated rabbit antigoat IgG
(Dako, Prosan, Ghent, Belgium; dilution 1/400) and with
peroxidase-labeled avidin-biotin complex (Dako). Antibody binding was
visualized with diaminobenzidine, resulting in a brown staining of the
immunoreactive sites. All sections were briefly counterstained with
Harris' hematoxylin (BDH Laboratory Supplies, Poole,
England), dehydrated, and mounted with DePex (Prosan, Gentbrugge, Belgium). Specificity of the primary antibodies was tested by adsorption of the antisera with murine fibrinogen.
Endogenous thrombolytic potential.
Lysis of 125I-fibrin-labeled murine plasma clots, injected
into age- and weight-matched 2-AP+/+ or
2-AP/ mice through a jugular vein (and
embolized into the pulmonary arteries) was determined essentially as
described previously.20 Therefore, 25 µL
125I-fibrin-labeled plasma clots, containing  70,000 cpm
human 125I-labeled fibrinogen, were prepared from a plasma
pool of 2-AP+/+ or
2-AP/ mice, by addition of thrombin
(final concentration, 1.5 NIH U/mL) and CaCl2 (final
concentration 70 mmol/L). Clot lysis was evaluated by measurement of
the residual radioactivity in the heart and lungs ex vivo at 2 hours
and 4 hours after injection, and was defined as the amount of
radioactivity that had disappeared, expressed as a percent of the total
amount of radioactivity injected.
Endotoxin-induced fibrin deposition.
Mice matched for sex, age (12 to 17 weeks), and weight (25 ± 1 g
and 25 ± 2 g for 2-AP+/+ and
2-AP/ mice; mean ± SEM, n = 8)
were injected intraperitoneally with endotoxin (2 mg/kg, dissolved in
sterile saline). Four or 8 hours after injection, the mice were
sacrificed by injection of Nembutal and immediately perfused for 15 to
30 minutes with saline. For protein extraction, one kidney was removed
and immediately frozen at  80°C. For immunohistochemical analysis,
the other kidney was fixed in 1% paraformaldehyde overnight at 4°C,
washed with phosphate-buffered saline, incubated in 70% ethanol
overnight at 4°C, and embedded in paraffin.
After immunostaining, the extent of fibrin deposition was given a
severity score of 0 to 3.20 Score 0 indicated the absence of fibrin deposits; score 1, the appearance of a few small fibrin deposits, stained very weakly; score 2, the presence of small clearly
stained fibrin deposits; score 3, the presence of large and strongly
stained fibrin deposits.
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RESULTS |
Germline transmission of the inactivated 2-AP
gene.
Inactivation of the murine
2-AP gene was achieved by
replacing, through homologous recombination in ES cells, a 7-kb genomic
fragment comprising the entire coding sequence, with a neomycin
resistance cassette.10 Morula aggregation of
recombinant RW4 cell clones harboring a disrupted
2-AP gene yielded germline-competent chimeras (male), as indicated by the presence of
agouti pups among their offspring after mating with C57BL6/J females.
Heterozygous 2-AP-deficient
(2-AP+/) mice among the agouti
offspring were identified by Southern blot analysis of tail-tip DNA
(not shown), and were intercrossed, yielding
2-AP+/+,
2-AP+/, and
2-AP/ F2 littermates (Fig
1A).
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| Fig 1.
Confirmation at the DNA and RNA level of correct
targeting of the
2-AP gene. (A)
Southern blot analysis of tail-tip genomic DNA of littermates from
intercrosses of heterozygous 2-AP-deficient mice. The
DNA was digested with KpnI and hybridized with a 3' probe
(probe C in ref 10). The 8-kb and 5-kb bands indicate the
presence of the wild-type or mutant allele, respectively. WT,
wild-type; HR, homologously recombined. (B) RT-PCR analysis of polyA
RNA isolated from liver and kidney of
2-AP+/+ and
2-AP/ mice. PCR products were
generated using PCR primers annealing in the coding part of exon 10 of
the murine 2-AP gene
(deleted in the disrupted allele), and were separated on a 1% agarose
gel. PCR with wild type RT-cDNA yielded the expected 193-bp
amplification product (lanes 3 and 5). The absence of signal with
2-AP/ RT-cDNA (lanes 4 and 6)
confirmed the inactivation of the
2-AP gene. Lane 2 (C) represents a negative control PCR reaction performed without DNA
template. The lower band present in all lanes represents dimers of the
primers.
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Deficiency in 2-AP in the
2-AP/ progeny was confirmed at the
mRNA level by the absence of signal in RT-PCR analyses of kidney and
liver polyA RNA using primers annealing in the coding part of exon 10 (Fig 1B).
Viability, fertility, and growth.
Among 161 F2 littermates from heterozygous crosses that
were genotyped at 4 to 5 weeks of age, 23% were
2-AP+/+, 53% were
2-AP+/, and 24% were
2-AP/ (Table
1). This distribution is similar for males
and females, and is not significantly different (by Chi-square
analysis) from the expected Mendelian 1:2:1 ratio, thus indicating
equal viability.
2-AP deficiency did not affect the growth rate of the
mice, as evidenced by weighing the mice at weekly intervals (not
shown). Body weights at 5 weeks of age were (mean ± SEM; n = 4),
17 ± 2 g and 15 ± 1 g for 2-AP+/+ and
2-AP/ mice, respectively, with
corresponding values of 22 ± 2 g and 23 ± 1 g at 10 weeks. Mean
body weights of 2-AP+/ mice
(mean ± SEM; n = 13) were 16 ± 1 g and 22 ± 4 g at 5 and 10 weeks of age, respectively. No macroscopic abnormalities were observed.
2-AP/ mice (F2 and
F3 generations) produced similar sizes of
litters+/ as 2-AP+/+ mice
with similar time intervals between the litters (Table
2).
Hemostasis analysis.
2-AP antigen levels and other hemostatic parameters are
summarized in Table 3.
2-AP antigen levels in plasma were gene-dose-dependent. The functional assay showed an unexpectedly high level of antiplasmin activity in 2-AP/ plasma as compared
with 2-AP+/ and
2-AP+/+ plasma: 22% ± 3%
(mean ± SEM; n = 14) versus 47% ± 5% (n = 10) and 94% ± 5% (n = 13), respectively. The levels in male or female 2-AP/ mice were not significantly
different: 18% ± 4.4% versus 26% ± 4.0% (n = 7;
P = .16). This rapid reacting plasmin inhibitory activity
cannot be due to murine 2-AP activity, because no
complexes corresponding to plasmin-2-AP could be
detected on addition of murine or human plasmin to
2-AP/ plasma (see below).
Plasma levels of 2-macroglobulin antigen were higher in
2-AP/ mice than in
2-AP+/+ mice, for females (128% ± 7%,
n = 10, v 101% ± 5%, n = 7, P = .02), and
for males (134% ± 13%, n = 10, v 100% ± 9%,
n = 6; P = .12). All other measured hemostasis parameters
and cell counts were comparable for wild-type mice and for heterozygous
and homozygous 2-AP-deficient mice.
Bleeding times after amputation of a toe were variable (range, 0.5 to 7 minutes), but were on average (mean ± SEM) not significantly different between 2-AP+/+ (210 ± 64
seconds; n = 5), 2-AP+/ (180 ± 37
seconds; n = 12) and 2-AP/
(210 ± 72 seconds; n = 5) mice. Also after tail amputation,
bleeding stopped spontaneously in all three genotypes. No significant
rebleeding was observed.
Histopathologic examination.
Microscopic analysis of cross-sections through different organs of 6- or 20-week-old 2-AP/ mice, as
described above, did not show any apparent abnormalities or differences
with corresponding sections of 2-AP+/+ mice.
Immunocytochemical analysis.
Western blotting of plasma using polyclonal rabbit anti-murine
2-AP antibodies (Fig 2A),
showed a positive band with an estimated Mr
of 65 kD in plasma of 2-AP+/+ mice but not
in plasma of 2-AP/ mice. In
urokinase-activated plasma (incubation with 50 nmol/L tcu-PA for 1 hour
at 37°C) of 2-AP+/+ mice, but not of
2-AP/ mice, an additional band was
observed with Mr about 140 kD, which
represents plasmin-2-AP complex, as confirmed by blotting with affinity-purified polyclonal rabbit anti-murine plasminogen antibodies (Fig 2C). After addition of purified plasmin, (1 µmol/L) and incubation for 10 seconds at 37°C,
plasmin-2-AP complex was also detected in
2-AP+/+ but not in
2-AP/ plasma (Fig 2B). The faint band
at Mr about 140 kD observed in
2-AP/ plasma (Fig 2B, lane 2 and Fig
2C, lane 4) does not correspond to plasmin-2-AP
complex, as it is not recognized by the antibodies against murine
2-AP (not shown).
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| Fig 2.
Western blot analysis of murine plasma using an
2-AP-specific (A) or a plasminogen-specific (B and C)
antiserum. (A) Lane 1, purified murine 2-AP; lanes 2 and
3, 2-AP+/+ and
2-AP/ plasma. (B) Lanes 1 and 2, 2-AP+/+ and
2-AP/ plasma after incubation with
human plasmin (final concentration, 1 µmol/L) for 10 seconds at
37°C. (C) Lanes 1 and 2, 2-AP+/+ and
2-AP/ plasma; lanes 3 and 4, 2-AP+/+ and
2-AP/ plasma activated with tcu-PA
(final concentration, 50 nmol/L) for 1 hour at 37°C; lane 5, purified
murine plasminogen.
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2-AP antigen levels in liver extracts (expressed in
ng/mg protein) were 37 ± 4 (n = 5) in
2-AP+/+ mice, 24 ± 1 (n = 11) in
2-AP+/ mice, and below the 1 ng/mg
detection level (n = 5) in 2-AP/ mice.
Endogenous thrombolytic potential.
Spontaneous lysis within 2 to 4 hours of a
125I-fibrin-labeled pulmonary plasma clot was always
higher in 2-AP/ than in
2-AP+/+ mice (Fig
3). In
2-AP+/+ mice, lysis of a clot produced from
2-AP+/+ or from
2-AP/ plasma was comparable,
indicating that the 2-AP content of the clot does
not significantly inhibit lysis in plasma with normal inhibitor
concentration. Also in 2-AP/
mice, lysis of both clot types was not significantly different.
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| Fig 3.
Spontaneous lysis of 125I-fibrin-labeled
clots prepared from 2-AP+/+ or
2-AP/ plasma and injected into
2-AP+/+ or
2-AP/ mice. The data are mean ± SEM
of the number of experiments indicated between parentheses. *P < .05 and **, P < .01 versus
2-AP+/+ mice.
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Endotoxin-induced fibrin deposition.
Histopathologic examination and immunostaining of kidney sections after
endotoxin injection in 2-AP+/+ mice showed
the occasional presence of fibrin deposits in the glomeruli of the
outer cortex (Fig 4, IB) and, more
frequently, in the capillaries of the medulla (Fig 4, IIB). In
2-AP+/+ mice without endotoxin injection and
in 2-AP/ mice after endotoxin
injection, significantly less fibrin deposits were detected in the
glomeruli (Fig 4, IA and IC) and in the medulla (Fig 4, IIA and IIC).
Semi-quantitative analysis of fibrin deposition in the glomeruli
indicated that, 8 hours after endotoxin injection, all 15 sections of
2-AP/ mice (4 animals) were free of
fibrin (score 0), whereas only 9 of 16 sections of
2-AP+/+ were devoid of fibrin (Table 4). In
the capillaries of the medulla, fibrin deposits were detected in 4 of
15 sections of 2-AP/ mice, whereas 14 of 16 sections of 2-AP+/+ mice showed fibrin
deposition (Table 4). Chi-square analysis using a two by four contingency table indicated a significant reduction
of fibrin deposits in 2-AP/ mice as
compared with 2-AP+/+ mice
(P = .11 at 4 hours and P < .05 at 8 hours for
the glomeruli; p .005 at 4 hours and at 8 hours for the medulla).
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| Fig 4.
Immunostaining with a specific antiserum against murine
fibrinogen/fibrin of kidney sections taken in the outer cortex (I) or
in the medulla (II) (original magnification ×400) from
2-AP+/+ mice before (A) or 4 hours after
endotoxin injection (B), and from
2-AP/ mice 4 hours after endotoxin
injection (C). The arrows indicate some of the fibrin deposits in the
glomeruli (IB) and in the capillaries (IIB and IIC). The scale bar
corresponds to 50 µm.
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Table 4.
Extent of Fibrin Deposition as Detected by
Immunostaining in Kidney Sections of
2-AP+/+ and
2-AP/ Mice, 4 or 8 Hours After
Injection of 2 mg/kg Endotoxin
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PAI-1 levels (mean ± SEM) in extracts of kidney sections taken at
the end of the experiments were not significantly different for
2-AP+/+ or
2-AP/ mice (2.8 ± 0.44 or
2.3 ± 0.32 ng/mg protein at 4 hours, and 0.96 ± 0.34 or
0.90 ± 0.07 ng/mg protein at 8 hours, as compared with <0.1 ng/mg
protein in both genotypes without endotoxin injection). Plasma PAI-1
levels after endotoxin injection in 2-AP+/+
or 2-AP/ mice were also comparable
(140 ± 26 or 110 ± 14 ng/mL at 4 hours, and 190 ± 93 and
77 ± 14 ng/mL at 8 hours), and were increased more than 50-fold
over baseline levels.
2-AP levels in kidney extracts of
2-AP+/+ mice were not increased after
endotoxin injection (7.5 ± 0.12 and 5.6 ± 3.3 ng/mg protein at
4 hours and 8 hours after injection, as compared with 7.0 ± 0.12
ng/mg protein without endotoxin injection). Plasma 2-AP
levels in 2-AP+/+ mice were also comparable
before (81 ± 5 µg/mL) and 4 hours (73 ± 4 µg/mL) or 8 hours
(56 ± 3 µg/mL) after endotoxin injection.
| |
DISCUSSION |
Congenital homozygous 2-AP deficiency was reported in
patients who presented with hemorrhagic diathesis, whereas several cases of heterozygosity in different families have been described with
no or only mild bleeding symptoms.21-29 In all
heterozygotes described so far, both 2-AP antigen and
activity levels ranged between 40% and 60% of normal, suggesting that
the deficiency is due to decreased synthesis of a normal
2-AP molecule. A single case of dysfunctional
2-AP (2-AP Enschede) associated with
severe bleeding tendency has been reported in two siblings in a Dutch family.30 The ability of this 2-AP to bind
reversibly to plasminogen was not affected, but it was converted from
an inhibitor of plasmin into a substrate, as a result of the insertion
of an extra alanine residue in the reactive center loop.31
The bleeding tendency in patients with 2-AP deficiency
is most likely due to premature lysis of hemostatic plugs, because the
half-life of plasmin molecules generated at the fibrin surface may be
considerably prolonged in the absence of 2-AP. Acquired
2-AP deficiency associated with enhanced fibrinolysis
has been reported in patients with liver disease,32,33
disseminated intravascular coagulation,32 and acute
promyelocytic leukemia.34 Furthermore, 2-AP
levels may be significantly reduced in patients undergoing thrombolytic therapy, especially with nonfibrin-specific agents, as a result of
extensive systemic generation of plasmin.35 After
exhaustion of plasma 2-AP, excess plasmin may degrade
several plasma proteins, including fibrinogen, and eventually lead to
bleeding complications. Pathophysiologic observations in humans thus
support the relevant role of 2-AP in regulating and
controlling plasmin activity. In addition, studies in mice with
deficiency of the main components of the fibrinolytic system indicate
an important role of plasmin in fibrin surveillance and in maintenance
of an intact hemostatic balance.17 To further substantiate
these findings, we have generated mice with homozygous deficiency of
2-AP, the main plasmin inhibitor in mammalian plasma.
2-AP/ mice develop and reproduce
normally. Macroscopic examination and microscopic analysis of
cross-sections of different organs did not show significant hemorrhage
in 6- to 20-week-old 2-AP/ mice.
Furthermore, after amputation of tail or toe tips, bleeding stopped
spontaneously in 2-AP/, as well as in
2-AP+/ and
2-AP+/+ mice. The absence of an overt
bleeding phenotype in 2-AP/ mice
appears somewhat surprising in view of the observations in humans
described above. Several factors may contribute to this apparent
difference between humans and mice. First, although the main components
of the fibrinolytic system are similar in both humans and mice,
important quantitative differences were observed, as a result of which
the fibrinolytic system in mice appeared to be very resistant to
activation.5,36 Second, the spectrum of proteinase
inhibitors in murine plasma, other than 2-AP, may be
more efficient toward plasmin than in human plasma, or additional inhibitory mechanisms may contribute. Inhibition of plasmin has indeed
been observed by several other plasma-proteinase inhibitors, including
2-macroglobulin and 1-antitrypsin. The
residual rapid-reacting plasmin-inhibitory activity in
2-AP-deficient plasma may thus be explained by
alternative inhibitory pathways. We have shown that it is not due to
interaction with 2-AP. Furthermore, the levels of
antiplasmin activity are comparable in male and female 2-AP/ mice and thus do not correlate
with potential sex-related differences in
2-macroglobulin levels. The apparent antiplasmin
activity observed in the functional assay, which persisted in the
presence of higher S-2403 concentrations or with the use of murine
plasmin, may reflect an interaction with plasma proteins rendering the plasmin unavailable for the chromogenic substrate.
The absence of a bleeding phenotype in
2-AP/ mice, as in many heterozygous
patients, probably reflects the fact that the coagulation system
adequately prevents bleeding under circumstances where the fibrinolytic
system is not dramatically challenged. Extensive activation of the
system, in the absence of 2-AP, will, however, result in
efficient fibrin degradation, and may thus cause lysis of hemostatic
plugs and hemorrhagic complications.
In vivo clot lysis experiments confirm that the endogenous thrombolytic
potential is significantly enhanced in
2-AP/ mice, indicating a physiologic
role of 2-AP in fibrin surveillance. Furthermore, the
experiments with cross-linked 2-AP+/+ and
2-AP/ plasma clots in
2-AP+/+ and
2-AP/ mice suggest that the efficiency
of spontaneous thrombolysis is determined primarily by the
2-AP concentration in circulating blood, and not by the
amount that is cross-linked to fibrin. This suggests that cross-linking
of 2-AP to fibrin, which renders a fibrin clot less
susceptible to degradation by plasmin, does not dramatically affect the
lysability of the clot by the murine endogenous fibrinolytic system.
Injection of endotoxin in mice was previously shown to result in
enhanced PAI-1 levels and in significant fibrin deposition in the
kidneys, within 4 to 8 hours after injection.37 Using this
model, fibrin deposition was found to be significantly reduced in
2-AP/ mice as compared with
2-AP+/+ mice. This is most likely not due to
a different degree of fibrin formation in both genotypes; furthermore,
PAI-1 levels in kidney and plasma were enhanced to a similar degree
after endotoxin injection. The observed difference, therefore, would
appear to be due to a higher endogenous fibrinolytic capacity in
2-AP/ mice, again confirming the role
of 2-AP in fibrin clearance.
In conclusion, 2-AP/ mice survive,
develop, and reproduce normally, but show an enhanced endogenous
fibrinolytic capacity without overt bleeding phenotype.
| |
ACKNOWLEDGMENT |
Skillful technical assistance by K. Bijnens, E. Gils, L. Kieckens, T. Vancoetsem, B. Van Hoef, I. Vanlinthout, A. Van Nuffelen, M. Verstreken, and G. Wallays is gratefully acknowledged.
| |
FOOTNOTES |
Submitted September 9, 1998; accepted December 1, 1998.
Supported by a grant from the Belgian National Fund for Scientific
Research (Project 3.0265.95).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to H.R. Lijnen, PhD, Center for
Molecular and Vascular Biology, University of Leuven, Campus
Gasthuisberg O&N, Herestraat 49, B-3000 Leuven, Belgium; e-mail:
roger.lijnen{at}med.kuleuven.ac.be.
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TOP
ABSTRACT
INTRODUCTION