(Circulation. 1995;92:2585-2593.)
© 1995 American Heart Association, Inc.
Articles |
Effects of Disruption of The Plasminogen Gene on Thrombosis, Growth, and Health in Mice
From the Center for Transgene Technology and Gene Therapy (V.A.P., P.C., S.V., I.V.V., L.M., D.C.), Vlaams Interuniversitair Instituut voor Biotechnologie, KU Leuven, Belgium, and the Joseph J. Jacobs Center for Thrombosis and Vascular Biology and the Department of Molecular Cardiology (V.A.P., S.V., E.F.P.), Cleveland Clinic Foundation, Cleveland, Ohio.
Correspondence to D. Collen, Center for Transgene Technology and Gene Therapy, Vlaams Interuniversitair Instituut voor Biotechnologie, Campus Gasthuisberg, O & N, Herestraat 49, B-3000 Leuven, Belgium. E-mail desire.collen@med.kuleuven.ac.be.
 |
Abstract |
|---|
 Top Abstract IntroductionMethods Results Discussion References |
|---|
Background Circumstantial evidence suggests that the plasminogen/plasmin system plays a role in many biological processes, including hemostasis, cell migration, and development.
Methods and Results The in vivo function of the plasminogen/plasmin system was studied by generation of plasminogen-deficient (Plg-/-) mice. Inactivation of the murine plasminogen gene (Plg) was achieved by replacing, via homologous recombination in embryonic stem cells, genomic sequences encoding the exons containing the catalytic site amino acids His605 and Asp648 with a neomycin phosphotransferase expression cassette. Germline transmission of the mutated allele, as determined by Southern blot hybridization and polymerase chain reaction, was obtained via blastocyst injection. Mendelian inheritance of the inactivated plasminogen allele was observed, and homozygous-deficient mice (Plg-/-) displayed normal viability but retarded growth up to at least 12 weeks of age. At 8 weeks of age, body weight was 21.8±1.2 g (n=10) for wild-type (Plg+/+) mice, 21.0±1.1 g (n=16) for heterozygous-deficient (Plg+/-) mice, and 17.4±1.3 g (n=12) for Plg-/- mice; P<.05 versus Plg+/+ or Plg+/-. None of 36 Plg+/+ or 65 Plg+/- mice but 7 of 37 Plg-/- mice (19%) developed rectal prolapse at 7.4±0.6 weeks of age (P=.03 versus Plg+/+ and P=.003 versus Plg+/-); 4 of 37 Plg-/- mice (11%) became runted and apathic at 5.3±0.3 weeks of age (P=.041 versus Plg+/-); and 6 of 37 Plg-/- mice (16%) died prematurely at 8.8±1.7 weeks of age (P=.057 versus Plg+/+ and P=.029 versus Plg+/-). Although male and female Plg-/- mice were able to sire offspring, the fertility of Plg-/- female mice was reduced, possibly owing to their impaired health. Levels of plasminogen-related antigen in plasma, measured by ELISA, were 84±8 µg/mL (n=4) in Plg+/+, 35±2 µg/mL (n=3) in Plg+/-, and 0.076±0.032 µg/mL (n=6) in Plg-/- mice (P<.001 versus Plg+/- and Plg+/+). Plasmin activity generated by urokinase activation was unmeasurable in Plg-/- mice (<5% of Plg+/+ mice). Plasminogen-specific immunoreactivity was observed in hepatocytes from Plg+/+ mice but not from Plg-/- mice (<10% of Plg+/+ mice). Neither native nor variant plasminogen mRNA nor translation products could be identified by Northern or Western blot of liver extracts from Plg-/- mice. Spontaneous lysis within 24 hours of a 125I-fibrin–labeled pulmonary plasma clot was 85±5% (n=5) in Plg+/+ mice, 62±7% (n=3) in Plg+/- mice, and -2±1% (n=3) in Plg-/- mice (P<.001 versus Plg+/- and Plg+/+). Delayed clot lysis within 72 hours was 33±1% (n=3) in tPA-/- mice and 26±2% (n=3) in Plg-/- mice (P=.054). Histological examination of several organs revealed fibrin deposition in the liver; lung; and in the stomach, associated with gastric ulcers, in 6- to 12-week-old Plg-/- mice but not in Plg+/+ or Plg+/- littermates.
Conclusions Plasminogen-deficient mice survive embryonic development but develop spontaneous fibrin deposition due to impaired thrombolysis and suffer retarded growth and reduced fertility and survival. The Plg-/- phenotype is reminiscent of the combined tPA-/-:uPA-/- phenotype, which suggests that there is no significant additional pathway for physiological plasminogen activation in mice.
Key Words: genes • fibrinolysis • thrombolysis
|
Introduction |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
The plasminogen/plasmin system has been implicated in playing a pivotal role in a number of normal and pathophysiological processes.1 2 Most notably, its role in maintaining vascular patency by regulating thrombus dissolution has been supported by several studies.2 3 In addition, because of the expression of receptors for plasminogen and its physiological activators UPA and TPA on a number of normal and transformed cells, this system is believed to facilitate directional cell migration by mediating degradation of matrix proteins.4 5 Such plasmin-mediated processes have been implicated in tissue remodeling, angiogenesis, embryogenesis, the inflammatory response, neointimal formation, tumor cell invasion, and metastasis.2 3 4 5 6 The association of high levels of apolipoprotein(a), a structural homologue to plasminogen, and of plasminogen activator inhibitor-1 with atherothrombotic disease suggests that the plasminogen/plasmin system may also play a role in the development or progression of atherosclerotic lesions.7 Although these correlates are numerous, they do not directly document the causal role and/or contribution of plasminogen to these processes.
Phenotypic analysis of mice with disruption of the genes encoding components of the fibrinolytic system, including plasminogen activator inhibitor-1, UPA, and TPA, as well as components of both TPA and UPA3 8 9 and of the UPA receptor,10 has challenged the perceived role of the plasminogen system in embryogenesis, ovulation, and fertilization. Despite a more thrombophilic phenotype and a reduced survival of combined TPA/UPA–deficient (tPA-/-:uPA-/-) mice versus mice with a single plasminogen activator deficiency, it remained to be determined whether alternative plasminogen-dependent or plasminogen-independent mechanisms might be involved. To further assess the physiological relevance of the plasminogen/plasmin system, we disrupted the murine Plg through homologous recombination in ES cells and characterized the effect of null expression on embryonic development, viability, reproduction, thrombosis, and thrombolysis.
|
Methods |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Mice, Blood Collection, and Hematologic Analysis
Housing and procedures involving experimental animals were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee. Unless otherwise stated, the mice were anesthetized by injection of sodium pentobarbital 60 mg/kg IP (Nembutal, Abbott Laboratories), and blood was collected by vena caval puncture with a 24-gauge needle.
For differential cell counts, blood was collected in 0.01 mol/L trisodium citrate and cells were counted by use of an automated analyzer (Cell-Dyn 610U-Hematology Analyzer, Sequoia-Turner Co). Cell counts are expressed per milliliter of whole blood. The activated partial thromboplastin time and thrombin time were determined with standard clinical laboratory procedures. Fibrinogen was determined by a coagulation rate assay as previously described.9
Construction of the Gene-Targeting Vector
The Plg
was isolated from a 
Fix II library
(Stratagene Cloning Systems) containing gDNA from a murine strain 129
liver. A total of 9.3 kb of flanking plasminogen sequence
was included in the parental vector pPNT that contained a
neomycin phosphotransferase and a herpes simplex virus thymidine kinase
expression cassette,11 as represented in Fig 1
. A
2.5-kb Kpn I/Xba I gDNA
fragment upstream of exon 15, which contains the coding sequence for
the active site His605,12 was ligated
into the Xba I site of the parental vector of
pPNT by blunt-end ligation to generate the 5' homologous
flanking sequence of the intermediate vector pPNT.5'-Plg. A
6.8-kb Sph I/Xba I fragment, downstream of exon
17 and spanning exon 19, which contains the coding sequence for the
active site Ser743, was blunt-end ligated into
the Xho I site of pPNT.5'-Plg, generating the 3'
homologous flanking sequence of the targeting vector
pPNT.Plg-7. Correct orientation of the inserts was confirmed
by polymerase chain reaction, restriction-digestion
analysis, and DNA sequencing. Thus, the neomycin
phosphotransferase gene expression cassette in the final targeting
vector replaced the Plg sequences, including exons 15 to 17,
which encode two of the three active site amino
acids:His605 and Asp648 of the
plasminogen proteinase domain (Ser743 is
encoded by exon 19). A similar strategy was previously successfully
used to disrupt the tPA gene.3
|
ES Cell Culture and Transfection
Culture, electroporation,
and selection of D3 ES
cells were performed as previously described.13 Briefly,
ES cells were cultured in DMEM containing 15%
heat-inactivated fetal bovine serum (Hyclone
Laboratories) and 1000 U/mL recombinant leukemia-inhibiting factor
(LIF Esgro, GIBCO BRL). The cells were grown on lethally irradiated
(1500 rad) or mitomycin C–treated (10 µg/mL) primary mouse embryonic
fibroblasts prepared from neomycin-resistant heterozygous
ß2-microglobulin–deficient mice (provided by R. Jaenisch,
Whitehead Institute, Cambridge, Mass).
ES cells (
108)
were electroporated by use of a Bio-Rad
Laboratories electroporator (400 V, 25 µF, room temperature) with 100
µg Not I linearized targeting vector in electroporation
buffer (20 mmol/L HEPES buffer, pH 7.2, containing [in mmol/L]
dextrose 6, Na2HPO4 0.7, KCl 5, NaCl 137, and
ß-mercaptoethanol 0.1).8 The cells were plated
immediately after transfection in 90-mm cell culture dishes containing
inactivated embryonic feeder-layer cells, and selection
was started 24 hours later with 150 to 175 µg/mL G418 (geneticin,
GIBCO/BRL) and 2 µmol/L ganciclovir (Cymevene, Synthex). Five days
later, selection was continued with only G418. After 8 to 10 days in
selection medium, colonies were isolated and transferred to 48-well
culture dishes. At confluence, 80% of the cells were frozen in ES cell
medium containing 10% DMSO and fetal bovine serum (40% final
concentration), and the remaining cells were grown in 24-well culture
dishes for gDNA Southern blot analysis.
Generation of Chimeric and Plasminogen-Deficient
Mice
Targeted clones containing a disrupted Plg were
injected into host blastocysts of strain C57BL/6J mice (Harlan CPB), as
described by Bradley.14 Injected embryos were transferred
into pseudopregnant B6D2F1 (Harlan) foster mothers. Chimeric offspring,
identified by the presence of agouti coat color, were backcrossed to
C57BL/6J mice (Harlan), and germline transmission of ES cell DNA was
scored by agouti coat pigment. Plg+/-
mutants
were identified by Southern blot analysis and polymerase chain
reaction of tail-tip gDNA, as described below. Brother-sister
mating was carried out to generate Plg-/-
progeny.
Southern Blot Analysis of gDNA
DNA was isolated from cultured
ES cells and mouse tail tips and
was digested with the indicated restriction enzymes for Southern blot
analysis as previously described.8 15 Briefly,
cells or tail tips were incubated overnight in lysis buffer (0.2% SDS,
100 or 500 µg/mL, respectively, of proteinase K, 200 mmol/L NaCl, 5
mmol/L EDTA, 100 mmol/L Tris-HCl buffer, pH 8.5), at 37°C (cultured
cells) or 55°C (tail tip), and gDNA was spooled after
precipitation with an equal volume of isopropanol.
To screen for ES
cell colonies harboring a recombined Plg
gene, a Kpn I/Xba I probe (Fig 1
, probe a)
was
used, which encompasses a 700-bp genomic fragment adjacent to the 5'
Kpn I/Xba I fragment of the targeting construct.
This 5' external probe detects a 7.0-kb EcoRI fragment of
the wild-type allele and a 12-kb EcoRI fragment of
the mutated allele, a 9.0-kb Sca I fragment of the
wild-type allele and a 5.1-kb Sca I fragment of the
mutated allele, and a 4.5-kb EcoRV fragment of the
wild-type allele and a 3.6-kb EcoRV fragment of the
mutated allele (cfr, Fig 1).
To document correct
homologous recombination at the 3' end, a 700-bp
Cel II/Bfr I fragment of the 3' Sph
I/Xba I flank in the targeting construct (Fig 1, probe
c)
was used. This probe detected an 20-kb EcoRV fragment in
the wild-type allele and an 17-kb EcoRV fragment
in the mutated allele. An additional internal probe of the 3'
Sph I/Xba I fragment was used in the targeting
construct (Fig 1, probe b) to detect a 9.0-kb EcoRI
fragment
in the wild-type allele and a 12-kb EcoRI fragment
in the mutated allele and verified deletion of the genomic
sequences encoding for His605 and Asp648 of the
catalytic domain.
Northern Blot Analysis
mRNA was isolated from liver of the
Plg+/+ and
Plg-/- mice
with the guanidinium thiocyanate–phenol-chloroform
single-step extraction method16 by use of the RNA
isolation kit supplied by Stratagene Cloning Systems. Denatured total
RNA 20 µg was separated by formaldehyde agarose gel electrophoresis
and bound to a nylon membrane (Hybond-N) by capillary transfer by use
of standard procedures.15 Blots were prehybridized for 6
to 12 hours and hybridized for 48 hours at 42°C in a hybridization
solution consisting of 1.0 mol/L sodium phosphate buffer, pH 7.2,
containing 0.25 mol/L NaCl, 7% (wt/vol) SDS, 1 mmol/L EDTA, 50%
formamide, 5% (wt/vol) dextran sulfate, and 0.01 mg/mL denatured
herring sperm DNA. After hybridization, blots were washed twice in 2x
SSC containing 0.1% SDS for 2x5 minutes at room temperature, followed
by two 30-minute washes with 0.5xSSC containing 0.1% SDS, all at
65°C. A 266-bp BamHI/EcoRI fragment from
plasmid pmVPlg-4 (nucleotides 945 to 1921 of the
murine Plg cDNA; notation as in Reference 12) and a 1.1-kb
BamHI/EcoRI fragment from plasmid
pmVPlg-5 (nucleotides 1 to 1069 of the murine
Plg cDNA; notation as in Reference 12) were used as
probes.
Histopathological Examination
Plg+/+ (n=4),
Plg+/- (n=4), and
Plg-/- (n=11) mice of either
sex, aged 6 to 12
weeks, were anesthetized by injections of sodium pentobarbital
60 mg/kg IP (Abbott Laboratories) and perfused via 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
5-µm sections of all tissues were examined after staining with
hematoxylin and eosin. The tissue sections included 5 cross sections of
brain; 3 cross sections of heart, thymus, lung, liver, spleen, kidney,
small and large intestine, stomach, cecum, leg muscle, and
reproductive organs (vas deferens, testis, and
epididymis or uterus and ovaries); and 1 cross section of lymph node,
adrenal gland, and pancreas.
Immunohistochemistry
Immunostaining for fibrinogen was
performed on
all paraffin-embedded tissue sections. The sections were incubated
with goat antiserum against murine fibrinogen (Nordic, 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 anti-goat IgG (Dako, prosan, dilution 1:400) and with
peroxidase-labeled avidin-biotin complex (Dako).
Immunostaining for plasminogen was
performed on liver sections with a polyclonal rabbit antiserum against
purified murine plasminogen that was purified by
immunoaffinity chromatography on insolubilized human
plasminogen.17 Peroxidase-labeled swine
anti-rabbit IgG (Dako, dilution 1:100) was used as second
antiserum. Antibody binding was visualized with diaminobenzidine,
resulting in brown staining of the immunoreactive sites. All sections
were briefly counterstained with Harris' hematoxylin, dehydrated, and
mounted with DePeX. The specificity of the primary antibodies was
tested by adsorption of the antisera with their homologous antigen.
Method specificity was performed by substitution of preimmune
goat or rabbit antiserum for the primary antibodies.
Determination of Plasminogen Antigen and
Activity
SDS-PAGE without reduction was performed on 10% to 15%
gradient gels using the PhastSystem (Pharmacia). After proteins were
transferred to nitrocellulose sheets, immunoblotting
was performed according to the method of Towbin et al18
with the purified polyclonal rabbit antibodies to murine
plasminogen described above. Quantitative determination of
plasminogen antigen in murine plasma was performed by ELISA
with the purified rabbit polyclonal antibodies. In addition,
plasminogen antigen levels in liver extracts obtained from
Plg+/+,
Plg+/-, and
Plg-/- mice were monitored by
immunoblotting and were quantified by ELISA (in µg/g
protein).
Plasminogen activity in murine plasma was determined by activation with human UPA after acidification and neutralization to inactivate protease inhibitors19 and quantification of generated plasmin with the chromogenic substrate L-pyroglutamyl-L-phenylalanyl-L-lysine-p-nitroanilide hydrochloride (S-2403) (Chromogenix).
125I-Fibrin–Labeled Plasma Clot Lysis In
Vivo
Lysis of 125I-fibrin–labeled murine plasma
clots injected via a jugular vein and embolized into the
pulmonary arteries was determined essentially as previously
described.20 Briefly, a 25-µL
125I-fibrin–labeled plasma clot, containing 70 000
cpm human 125I-fibrinogen (corresponding to 0.07 µCi
125I), was prepared from a plasma pool of
Plg+/+ mice and injected into the
jugular vein.
Clot lysis was evaluated by measurement of the residual radioactivity
in the heart and lungs ex vivo at various time points and was defined
as the amount of radioactivity that had disappeared, expressed as a
percent of the total amount of radioactivity injected.
Statistical Analysis
Data are represented as mean±SEM.
The statistical
significance of differences between groups was determined by
2 analysis or by ANOVA, followed by
Student's t test with the Bonferroni correction. Body
weights were analyzed by piecewise linear mixed-effects
models, with break point at 5 weeks and with random intercepts and
random slopes for time. F tests were used to test for group
differences. All calculations were done with the SAS procedure
PROC MIXED.
|
Results |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Targeting of the Plgsion/
To target the murine Plg, a replacement-type vector was constructed, in which the neomycin phosphotransferase gene (neo) replaced the genomic sequences containing exons 15 to 17, which comprise two of the three active site amino acids, ie, His605 and Asp648, of the catalytic domain, as schematically outlined in Fig 1
. Correct homologous
recombination thus would result in deletion of these catalytically
essential domains and inactivation of the Plg. After
electroporation of the Not I linearized targeting vector,
711 clones surviving double selection in geneticin and ganciclovir were
analyzed. Homologous integration was observed in 3.2% of
double-resistant (geneticin- and
ganciclovir-resistant) ES cells. As predicted from
restriction-digestion mapping of gDNA, Southern blot
analysis of positive clones with the 5' probe a (Fig 1)
revealed a 4.5-kb fragment for the wild-type allele and a
3.6-kb fragment for the mutated allele after EcoRV
digestion (Fig 2A), a 7.0-kb fragment for the
wild-type allele and a 12.0-kb fragment for the mutated
allele after EcoRI digestion (not shown), and a 9.0-kb
fragment for the wild-type allele and a 5.1-kb fragment for the
mutated allele after Sca I digestion (not shown).
Further expansion of these clones and additional analysis by
Southern blot hybridization with probe c (Fig 1) revealed an
20-kb
fragment for the wild-type allele and a 17-kb fragment for the
mutated allele after EcoRV digestion (not shown). With
probe b (Fig 1), all positive clones contained a 9-kb fragment
for the
wild-type allele and a 12-kb fragment for the mutated
allele after EcoRI digestion (not shown). Finally,
single integration of the targeting construct was confirmed by
EcoRI digestion of genomic DNA and hybridization with the
neo-specific probe d (Fig 1), generating exclusively a
12-kb
fragment (Fig 2B).
|
Germline Transmission of Inactivated
Plg
Four of the targeted clones containing a disrupted
Plg
were injected into C57BL/6J host blastocysts and then transferred into
pseudopregnant B6D2F1 foster mothers. Nineteen offspring exhibiting
>80% chimerism were generated, and two chimeras, one each from clones
Plg7-271 and Plg7-191, were positive for germline
transmission of the mutated allele as verified by Southern blot
analysis of Sca I–digested tail-tip DNA (Fig
3A). Further confirmation was obtained from Southern
blot analysis of EcoRI and EcoRV
digestion patterns (not shown).
|
Viability, Growth, Health, and Fertility of
Plasminogen-Deficient Mice
Agouti offspring (F1 generation)
obtained from the
mating of chimeric males with C57BL/6J females were genotyped
by Southern blot analysis of tail-tip DNA, yielding
restriction patterns similar to those illustrated in Figs 2 and
3.
These heterozygous mice were intercrossed and their F2
offspring genotyped. Fig 3A shows a Southern blot
analysis using probe a with Sca I–digested
tail-tip gDNA from Plg+/+,
Plg+/-, and
Plg-/- littermates. Among 138
F2
littermates analyzed, 36 were Plg+/+
(26%), 65 were Plg+/- (46%), and 37
were
Plg-/- (27%). This distribution is
not
significantly different from the expected mendelian 1:2:1
ratio, indicating equal viability at 3 weeks of age in
Plg+/+,
Plg+/-, and
Plg-/- mice (Table 1).
|
Plasminogen deficiency did, however, affect the growth rate
(Fig 4). Plg-/- mice
developed
normally during the first 4 weeks of life but shortly after weaning
gained less weight than Plg+/- mice
(P<.0001 versus Plg-/-
mice by
F test) or Plg+/+ mice
(P<.0001
versus Plg-/-). At 8 weeks of age,
body weight
values were 21.8±1.2 g (n=10) for
Plg+/+ mice,
21.0±1.1 g (n=16) for
Plg+/- mice, and
17.4±1.3 g (n=12) for
Plg-/- mice. None of 36
Plg+/+ and 65
Plg+/-
mice but 7 of 37 Plg-/- mice (19%)
(all >5
weeks of age) developed rectal prolapse at 7.4±0.6 weeks of age
(P=.035 versus Plg+/+
mice and
P=.003 versus Plg+/-
mice by
2 analysis), 4 of 37
Plg-/- mice (11%) became runted and
apathic
at 5.3±0.3 weeks of age (P=.041 versus
Plg+/- mice), and 6 of 37
Plg-/- mice (16%) died prematurely at
8.8±1.7 weeks of age (P=.05 versus
Plg+/+ mice and P=.029
versus
Plg+/- mice). Compared with
tPA-/-:uPA-/-
mice,
which progressively became runted with age and became severely
cachectic during their preterminal stage beyond the age of 17
weeks,3 Plg-/- mice
appeared to
suffer less severe cachexia syndrome, most likely because of their
younger age at the time of analysis (<12 weeks of age).
Screening of sentinel mice for the presence of pathogens by two
independent laboratories revealed the presence of epidemic disease of
infant mice, Helicobacter, and Pasteurella
pneumotropica but not of Citrobacter freundii 4280, a
pathogen known to cause rectal prolapse in mice.
|
All of 5 male Plg-/- mice (age, 6.2±0.5 weeks), mated for >2 weeks to several female wild-type or Plg+/- mice (age, 7.8±0.8 weeks, n=12), produced 1 to 4 litters each of 7.0±0.5 offspring per litter. These values are not significantly different from the breeding characteristics of Plg+/+ mice, which produced litters of 6.2±0.5 offspring per litter within 2 weeks of mating. Although one Plg-/- female produced a litter of 8 viable offspring, fertility of female Plg-/- mice appeared to be compromised. Indeed, none of 3 male Plg-/- mice (age, 5.7±0.7 weeks; the same mice used for mating to female wild-type or Plg+/- mice) mated for a similar duration to female Plg-/- mice (age, 5.4±0.3 weeks, n=4) produced a litter (P=NS by ANOVA versus female wild-type or Plg+/- mice). In addition, a Plg+/- male (age, 8 weeks) mated for 2 weeks to a Plg-/- female (age, 6 weeks) produced no offspring. Only one of the 5 male but 4 of 5 female Plg-/- mice suffered growth retardation, developed rectal prolapse, or became sick.
Northern Blot Analysis
Northern blot analysis of RNA prepared
from liver of
Plg+/+ and
Plg-/- mice
hybridized with a 5'– or a 3'–end located
Plg-specific
cDNA probe revealed the specific 2.7-kb mRNA in
Plg+/+ mice, whereas no mRNA that would
be
representative of native plasminogen or a
variant thereof could be detected in the
Plg-/- mice (Fig 3B).
Plasminogen Antigen and Activity in
Plasma
The results of plasminogen antigen and activity in
plasma are summarized in Table 2.
Plasminogen-related antigen levels, determined by an
ELISA with affinity-purified polyclonal antibodies and calibration
against murine plasminogen, were 84±8 µg/mL (n=4) in
Plg+/+ mice, 35±2 µg/mL
(n=3) in
Plg+/- mice, and 0.076±0.014
µg/mL (n=6)
in Plg-/- mice (P<.001
versus
Plg+/- and versus
Plg+/+). Thus, plasma from
Plg-/- mice cross-reacted for
0.1% of
that of Plg+/+ mice. Plasminogen
activity was not detectable in Plg-/-
plasma
(detection limit, ±5% that of
Plg+/+ plasma),
whereas the activity was 50% in
Plg+/- mice
(P<.01 by ANOVA versus
Plg+/+)
(Table 2).
|
Macroscopic and Microscopic Examination
The majority of
Plg-/- mice did not
reveal obvious signs of organ dysfunction and appeared healthy during
the first 3 months after birth, although they gained significantly less
weight shortly after weaning. After the age of 7 weeks, a significant
percentage of Plg-/- mice (19%)
developed
rectal prolapse, initially reversible but later persistent. Upon
dissection, macroscopically visible white spots were observed on the
livers of all Plg-/- mice
(n=11; 6 to 12 weeks
of age), similar to those previously observed in age-matched
tPA-/-:uPA-/-
mice.3 In 4 Plg-/- and 1
Plg+/- but none of the
Plg+/+ mice, significant enlargement of
one or
both kidneys was observed, which on microscopic analysis
revealed distension of the pelvis and urine accumulation but no signs
of fibrous adhesions in the peritoneum.
Histological examination of the
white spots in the
liver revealed fibrin deposition in the subcapsular region in all
Plg-/- mice but in none of 4
Plg+/+ or 4
Plg+/-
littermates (6 to 12 weeks of age) (Fig 5A). Similar to
the observation in
tPA-/-:uPA-/-
mice,3 2 of the 11
Plg-/- mice
showed calcification of the fibrin deposits, as revealed by staining
with hematoxylin and eosin (Fig 5C) and alizarin red S (Fig
5D).
Excessive fibrin deposition was also observed at the base of a large
gastric ulceration in a 7-week-old
Plg-/-
mouse, with loss of the intact architecture of the overlying epithelium
and infiltration by inflammatory cells (Fig 5E and
5F). These fibrin
deposits extended into the stroma beneath the unaffected gastric
epithelium, adjacent to the demarcated crater of the ulcer. In another
Plg-/- mouse, a large fibrin deposit
without
apparent epithelial desquamation or ulceration was observed (not
shown). Fibrin deposits were also found in the lung in 3
Plg-/- mice (Fig 5G).
The fibrinous nature of
the liver and stomach deposits was confirmed by
immunostaining of sections with a goat anti-murine
fibrinogen/fibrin–specific serum (Fig 5H). Analysis of
the
kidneys with enlarged pelvis did not reveal any fibrin deposits or
other abnormalities. No extramedullary hematopoiesis, as observed in
older
tPA-/-:uPA-/-
mice (Reference 3 and unpublished data), was observed in the
Plg-/- mice.
|
Hematologic Analysis
tPA-/-:uPA-/-
mice suffer severe anemia beyond the age of 20 weeks, associated with
the development of cachexia (Reference 3 and unpublished data).
Therefore, hematologic parameters of 5- to 9-week-old
Plg-/- mice were examined. As can be
seen in
Table 3, apart from a slightly increased mean
corpuscular value and reduced mean corpuscular hemoglobin concentration
in the Plg-/- mice, no other
significant
differences between Plg+/+ and
Plg-/- mice were observed. The plasma
fibrinogen level determined on a pool of three
Plg-/- mice was 1.98 g/L compared
with 1.02
g/L in a pool of wild-type mice.
|
Immunocytochemical Analysis of
Plasminogen-Deficient Mice
Paraffin sections of the livers of
Plg+/+,
Plg+/-, and
Plg-/- mice were stained with
affinity-purified polyclonal rabbit anti-murine
plasminogen antibodies, which recognized purified murine
plasminogen on a Western blot. Plasminogen
immunoreactivity was observed in the livers of
Plg+/+ and
Plg+/- mice,
when the antibodies were used at a concentration of 
0.25 µg/mL (Fig
6A). Preadsorption of the antibodies with purified
murine plasminogen eliminated the immunopositive staining
(Fig 6C). No immunostaining was observed in
hepatocytes of Plg-/- mice (Fig
6B) when the antibodies were used at a concentration of 
2.5
µg/mL.
However, hepatocytes of Plg-/-
mice also stained positively when the antibodies were used at a
concentration of 2.5 µg/mL or more, but adsorption with excess
purified murine plasminogen did not eliminate this
background staining in either Plg-/-
or
Plg+/+ mice. Plasminogen antigen
levels in liver extracts (expressed in ng/mg protein) were 77±12
(n=4)
in Plg+/+ mice, 41±2
(n=3) in
Plg+/- mice, and 3.6±0.2
(n=6) in
Plg-/- mice (P<.001 by
ANOVA
versus Plg+/+ and versus
Plg+/-).
|
Western blotting of
plasma (Fig 7A) revealed a positive
band with an estimated Mr of 93 kD in plasma of
Plg+/+ mice at a dilution of
1:4000 but not in
plasma of Plg-/- mice at a dilution
of 1:50.
Western blotting of liver extracts (Fig 7B) revealed a similar
band
when 4 µL of a sample with a concentration of 0.25 mg total protein
per milliliter from Plg+/+ mice was
applied. No
bands were observed in liver extracts from
Plg-/- mice at a protein
concentration of 1
mg/mL.
|
125I-fibrin–Labeled Plasma Clot Lysis In
Vivo
As shown in Table 2, spontaneous lysis within 24
hours of a
125I-fibrin–labeled pulmonary plasma clot
produced by injection via the jugular vein was 85±5% (n=5) in
Plg+/+ mice, 62±7% (n=3)
in
Plg+/- mice, and -2±1%
(n=3) in
Plg-/- mice (P<.001 by
ANOVA
versus Plg+/- and
Plg+/+). Comparative results in three
tPA-/- mice were 22±3%, a
value very similar
to that previously obtained with
plasminogen-containing plasma
clots.3 Delayed clot lysis within 72 hours was
26±2% (n=3) in Plg-/-
mice and 33±1% in
tPA-/- mice (P=.054
versus
Plg-/-).
|
Discussion |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Numerous studies have supported a major regulatory role of the plasminogen/plasmin system in a wide range of normal and pathophysiological processes. For example, its involvement in regulating thrombus dissolution and in cell migration via the assembly of its components on cell surfaces is well documented. However, much of this evidence is circumstantial and requires direct confirmation of a causal and/or contributory role of the plasminogen/plasmin system in these events.
To study the role of the plasminogen/plasmin system in
vivo, mice with inactivated plasminogen genes
were generated via homologous recombination in ES cells. Null
expression of plasminogen in mice genotyped as
homozygous-deficient was confirmed by the absence of specific mRNA
in the liver by Northern blotting and of urokinase-inducible
plasmin activity in plasma and by a greatly reduced antigen level in
plasma (<0.1% of wild-type concentration). Cross-reactivity
of liver extracts from Plg-/- mice in
the
ELISA assay (5% of that of Plg+/+
extracts)
was observed, along with some background staining of
hepatocytes from Plg-/- mice with
antiplasminogen antibodies when used at a 10-fold
higher concentration than required for staining of
hepatocytes from Plg+/+ mice.
However, a similar immunostaining persisted in samples
of Plg+/+ mice after adsorption of the
antibodies with excess purified murine plasminogen,
suggesting that these cross-reactivities may be aspecific.
Heterozygous plasminogen deficiency
(Plg+/-) resulted in intermediate
plasminogen antigen and activity levels in plasma,
indicating gene-dosage–dependent expression of the
Plg.
Plasminogen deficiency did not appear to compromise embryonic development and viability of the mice, similar to the tPA-/-:uPA-/- mice, which have previously been shown to produce viable offspring although with reduced life expectancy.3 A previous report of a patient with a markedly reduced plasmin activity in plasma also indicated that the plasminogen system plays a less prominent role in embryo implantation and development than previously assumed.21 Plasminogen deficiency did not appear to affect male fertility in mice, since plasminogen-deficient males were able to sire offspring from wild-type (Plg+/+) and Plg+/- females. This is surprising in view of the presumed role of the plasminogen system in spermatocyte migration and fertilization.22 Although our initial findings suggest that some plasminogen-deficient female mice are less fertile, most of these mice became sick, developed rectal prolapse, and gained less weight than the other mice, suggesting that fertility might have been compromised by their poor general health. Rescue of a possible defect on reproduction secondary to plasminogen deficiency cannot be excluded at the present time and must be examined further. Other proteinase systems, such as the metalloproteinases, however, also may participate in reproduction and development.23
The most prominent phenotype of the Plg-/- mice relates to fibrin homeostasis. Indeed, Plg-/- mice display a greatly reduced spontaneous lysis of pulmonary plasma clots within 24 hours and fibrin deposits in several organs as early as 6 weeks of age. This phenotype is similar but not identical to the thrombophilia observed in patients with hypoplasminogenemia or dysplasminogenemia.21 24 25 26 Significantly, delayed clot lysis within 72 hours occurred to almost the same extent in Plg-/- mice as in tPA-/- mice (present study) and in combined tPA-/-:uPA-/- mice,3 indicating that the residual thrombolytic capacity of Plg-/- or of tPA-/-:uPA-/- mice is mediated via plasminogen-independent mechanisms. In combination with earlier observations in tPA-/- mice,3 the present study establishes that TPA- and UPA-mediated plasminogen activation constitutes the principal physiological mechanism responsible for controlling fibrin deposition. Plasminogen-independent proteinases such as leukocyte-derived cathepsins or elastase27 or the Macglycoprotein–dependent fibrin clearance pathway28 might be involved in delayed clot lysis, although this would probably require incorporation of leukocytes into the thrombus.
Plasminogen-deficient mice were not anemic but displayed retarded postnatal growth and became runted and apathic, although to a lesser extent than the tPA-/-:uPA-/- mice.3 The less severe cachexia syndrome in Plg-/- mice might be related to their younger age. Indeed, the thrombosis and cachexia syndrome in tPA-/-:uPA-/- mice developed gradually over time, with the most severe symptoms in preterminal mice, typically in mice >3 to 4 months of age.3 Whether chronic anemia and reduced food intake (secondary to the inflammation and fibrin deposition in the gingiva), as observed in cachectic and preterminal tPA-/-:uPA-/- mice (Reference 3 and unpublished observations), also occurs in Plg-/- mice and might contribute to cachexia and reduced survival (as suggested by our initial analysis) remains to be determined. Such an effect on health has not been observed in patients with hypoplasminogenemia or dysplasminogenemia21 24 25 26 but may be related to their leaky plasminogen expression, since low levels of plasminogen expression may indeed suffice to rescue increased morbidity and mortality.
A larger proportion of Plg-/- than Plg+/+ mice developed rectal prolapse and gastric ulcerations, similar to the combined tPA-/-:uPA-/- mice.3 Since the genetic background or environmental factors may influence phenotypes, the mice were screened for potential pathogens. Although epidemic disease of infant mice and Helicobacter, which have been claimed to cause rectal inflammation, were documented in our mouse colony (Leuven, Belgium), the presence of Citrobacter freundii 4280, which has been associated with the development of rectal prolapse, was not observed. Since the occurrence of rectal prolapse was, however, clearly genotype-specific, we hypothesize that other mechanisms (possibly intravascular thrombosis) might contribute to the appearance of rectal prolapse, as has been suggested for other inflammatory bowel diseases.29 Helicobacter has, however, been identified as a possible pathogenic agent for the development of gastric ulcerations and might have influenced their occurrence in Plg-/- mice. The increased genotype-specific incidence of gastric ulcerations in Plg-/- mice nevertheless suggests that the plasminogen system plays a role in the prevention and/or healing of tissue damage. Clearly, other aspects of the Plg-/- phenotype, including fertility, postnatal growth, and survival, may be influenced by environmental or genetic factors. In fact, the observation that patients with reduced plasminogen plasma levels display a variable penetrance of thrombophilia supports such a hypothesis.21 24 25 26
An important goal of this study was to examine whether and to what extent alternative plasminogen activation pathways might be involved in biological processes in vivo. The normal embryonic development and viability, the similarly reduced potential to lyse pulmonary plasma clots, the spontaneous fibrin depositions at similar predilection sites and ages, the reduced postnatal growth, the gradual runting, and the apparently reduced survival strongly suggest that TPA and UPA are the only significant plasminogen activators in vivo. The present Plg-/- mice may serve as a model for the study of other physiological processes in which the plasminogen/plasmin system has been implicated, such as vascular injury, neointima formation, atherosclerosis, tumorigenesis, cell invasion, and brain function.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
We are grateful to Ann Bouché, Timothy Burke, Cathérine De Clercq, Sandra Janssens, Lena Kieckens, Berthe Van Hoef, and Sabine Wyns for expert assistance and to Geert Verbeke and Emanuel Lesaffre, PhD, for statistical analyses. This work was supported by grant HL-17964 of the NIH.
Received March 21, 1995; revision received June 2, 1995; accepted June 8, 1995.
|
References |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
1. Astrup T. Fibrinolysis: an overview. In: Davidson JF, Rowan RM, Samama MM, Desnoyers PC, eds. Progress in Chemical Fibrinolysis and Thrombolysis, Vol 3. New York, NY: Raven Press; 1978:1-57.
2.
Collen D, Lijnen HR. Basic and clinical aspects
of fibrinolysis and thrombolysis.
Blood.. 1991;78:3114-3124.
3. Carmeliet P, Schoonjans L, Kieckens L, Ream B, Degen J, Bronson R, De Vos R, van den Oord JJ, Collen D, Mulligan RC. Physiological consequences of loss of plasminogen activator gene function in mice. Nature.. 1994;368:419-424. [Medline] [Order article via Infotrieve]
4. Vassalli JD, Sappino AP, Belin D. The plasminogen activator/plasmin system. J Clin Invest.. 1991;88:1067-1072.
5.
Blasi F, Vassalli JD, Danø K.
Urokinase-type plasminogen
activator: proenzyme, receptor, and
inhibitors. J Cell Biol.. 1987;104:801-804.
6. Reich E. Activation of plasminogen: a general mechanism for producing localized extracellular proteolysis. In: Berlin RD, Heppman L, Lepow IH, Tanzer JM, eds. Molecular Basis of Biological Degradative Processes. New York, NY: Academic Press Inc; 1989:155-169.
7. Liu AC, Lawn RM. Lipoprotein (a) and atherogenesis. Trends Cardiovasc Med.. 1994;4:40-44.
8. Carmeliet P, Kieckens L, Schoonjans L, Ream B, Van Nuffelen A, Prendergast G, Cole M, Bronson R, Collen D, Mulligan RC. Plasminogen activator inhibitor-1 gene-deficient mice, I: generation by homologous recombination and characterization. J Clin Invest.. 1993;92:2746-2755.
9. Carmeliet P, Stassen JM, Schoonjans L, Ream B, van den Oord JJ, De Mol M, Mulligan RC, Collen D. Plasminogen activator inhibitor-1 gene-deficient mice, II: effects on hemostasis, thrombosis and thrombolysis. J Clin Invest.. 1993;92:2756-2760.
10. Dewerchin M, Carmeliet P, Van Nuffelen A, Collen D, Mulligan RC. Inactivation of the mouse urokinase receptor gene. Fibrinolysis. 1994;84(suppl 1):51. Abstract 142.
11. Tybulewicz VL, Crawford CE, Jackson PK, Bronson RT, Mulligan RC. Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell.. 1991;65:1153-1163. [Medline] [Order article via Infotrieve]
12. Degen SJ, Bell SM, Schaefer LA, Elliot RW. Characterization of the cDNA coding for mouse plasminogen and localization of the gene to mouse chromosome 17. Genomics.. 1990;8:49-61. [Medline] [Order article via Infotrieve]
13. Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol.. 1985;87:27-45. [Medline] [Order article via Infotrieve]
14. Bradley A. Production and analysis of chimeric mice. In: Robertson EJ, ed. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford, England: IRL Press; 1987:113-151.
15. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1989.
16. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem.. 1987;162:156-159. [Medline] [Order article via Infotrieve]
17. Lijnen HR, Van Hoef B, Beelen V, Collen D. Characterization of the murine plasma fibrinolytic system. Eur J Biochem.. 1994;224:863-871.[Medline] [Order article via Infotrieve]
18.
Towbin H, Staehelin T, Gordon J. Electrophoretic
transfer of proteins from polyacrylamide gels to nitrocellulose
sheets: procedure and some applications. Proc Natl Acad
Sci U S A.. 1979;76:4350-4354.
19. Mussoni L, Raczka E, Chmielewska J, Donati MB, Lattalo ZS. Plasminogen assay in rabbit, rat and mouse plasma using the chromogenic substrate S-2251. Thromb Res.. 1979;15:341-349. [Medline] [Order article via Infotrieve]
20. Stassen JM, Vanlinthout I, Lijnen HR, Collen D. A hamster pulmonary embolism model for the evaluation of the thrombolytic and pharmacokinetic properties of thrombolytic agents. Fibrinolysis. 1990;4(suppl 2):15-21.
21. Aoki N, Moroi M, Sakata Y, Yoshida N, Matsuda M. Abnormal plasminogen: a hereditary molecular abnormality found in a patient with recurrent thrombosis. J Clin Invest.. 1978;61:1186-1195.
22.
Huarte J, Belin D, Bosco D, Sappino AP, Vassalli JD.
Plasminogen activator and mouse
spermatozoa: urokinase synthesis in the male genital tract and binding
of the enzyme to the germ cell surface. J Cell
Biol.. 1987;104:1281-1289.
23. Lala PK, Graham CH. Mechanisms of trophoblast invasiveness and their control: the role of proteases and protease inhibitors. Cancer Metastasis Rev.. 1990;9:369-379. [Medline] [Order article via Infotrieve]
24. Robbins KC. Dysplasminogenemias. Prog Cardiovasc Dis.. 1992;34:295-308. [Medline] [Order article via Infotrieve]
25.
Azuma H, Uno Y, Shigekiyo T, Sarzo S. Congenital
plasminogen deficiency caused by Ser672 to Pro
mutation. Blood.. 1993;82:475-480.
26. Dolan G, Greaver M, Cooper P, Preston FE. Thrombovascular disease and familial plasminogen deficiency: a report of three kindreds. Br J Haematol.. 1988;70:417-421. [Medline] [Order article via Infotrieve]
27. Plow EF. The major fibrinolytic proteases of human leukocytes. Biochim Biophys Acta.. 1980;630:47-56. [Medline] [Order article via Infotrieve]
28.
Simon DI, Ezratty AM, Francis SA, Rehnke H, Loscalzo J.
Fibrinogen is internalized and degraded by activated
human monocytoid cells via Mac-1 (CD11b/CD18): a nonplasmin
fibrinolytic pathway. Blood.. 1993;82:2414-2422.
29. Wakefield AJ, Dhillon AP, Rowler PM, Sawyer AM, Pittilo RM, Lewis AAM. Pathogenesis of Crohn's disease: multifocal gastrointestinal infarction. Lancet. 1989;1057-1062.
This article has been cited by other articles:
 |
|
 |
|
  S. Netzel-Arnett, T. H. Bugge, R. A. Hess, K. Carnes, B. W. Stringer, A. L. Scarman, J. D. Hooper, I. D. Tonks, G. F. Kay, and T. M. Antalis The Glycosylphosphatidylinositol-Anchored Serine Protease PRSS21 (Testisin) Imparts Murine Epididymal Sperm Cell Maturation and Fertilizing Ability Biol Reprod, November 1, 2009; 81(5): 921 - 932. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Hoover-Plow, E. Hart, Y. Gong, A. Shchurin, and T. Schneeman A Physiological Function for Apolipoprotein(a): A Natural Regulator of the Inflammatory Response Experimental Biology and Medicine, January 1, 2009; 234(1): 28 - 34. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Liu, P. B. Gurpur, and S. J. Kaufman Genetically Determined Proteolytic Cleavage Modulates {alpha}7{beta}1 Integrin Function J. Biol. Chem., December 19, 2008; 283(51): 35668 - 35678. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Park, E. F. Gallo, J. Anrather, G. Wang, E. H. Norris, J. Paul, S. Strickland, and C. Iadecola Key role of tissue plasminogen activator in neurovascular coupling PNAS, January 22, 2008; 105(3): 1073 - 1078. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Swaisgood, M. A. Aronica, S. Swaidani, and E. F. Plow Plasminogen Is an Important Regulator in the Pathogenesis of a Murine Model of Asthma Am. J. Respir. Crit. Care Med., August 15, 2007; 176(4): 333 - 342. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Paul, S. Strickland, and J. P. Melchor Fibrin deposition accelerates neurovascular damage and neuroinflammation in mouse models of Alzheimer's disease J. Exp. Med., August 6, 2007; 204(8): 1999 - 2008. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. P. Fay, N. Garg, and M. Sunkar Vascular Functions of the Plasminogen Activation System Arterioscler Thromb Vasc Biol, June 1, 2007; 27(6): 1231 - 1237. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Tefs, M. Gueorguieva, J. Klammt, C. M. Allen, D. Aktas, F. Y. Anlar, S. D. Aydogdu, D. Brown, E. Ciftci, P. Contarini, et al. Molecular and clinical spectrum of type I plasminogen deficiency: a series of 50 patients Blood, November 1, 2006; 108(9): 3021 - 3026. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Stempien-Otero, A. Plawman, J. Meznarich, T. Dyamenahalli, G. Otsuka, and D. A. Dichek Mechanisms of Cardiac Fibrosis Induced by Urokinase Plasminogen Activator J. Biol. Chem., June 2, 2006; 281(22): 15345 - 15351. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Mackman Tissue-Specific Hemostasis in Mice Arterioscler Thromb Vasc Biol, November 1, 2005; 25(11): 2273 - 2281. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Cheng, Y. Zhao, W. E. Lawson, V. V. Polosukhin, J. E. Johnson, T. S. Blackwell, and D. Gailani The effects of intrinsic pathway protease deficiencies on plasminogen-deficient mice Blood, November 1, 2005; 106(9): 3055 - 3057. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Salmona, R. Capobianco, L. Colombo, A. De Luigi, G. Rossi, M. Mangieri, G. Giaccone, E. Quaglio, R. Chiesa, M. B. Donati, et al. Role of Plasminogen in Propagation of Scrapie J. Virol., September 1, 2005; 79(17): 11225 - 11230. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Li, A. Ny, G. Leonardsson, K. S. Nandakumar, R. Holmdahl, and T. Ny The Plasminogen Activator/Plasmin System Is Essential for Development of the Joint Inflammatory Phase of Collagen Type II-Induced Arthritis Am. J. Pathol., March 1, 2005; 166(3): 783 - 792. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Pawlak, J. P. Melchor, T. Matys, A. E. Skrzypiec, and S. Strickland From The Cover: Ethanol-withdrawal seizures are controlled by tissue plasminogen activator via modulation of NR2B-containing NMDA receptors PNAS, January 11, 2005; 102(2): 443 - 448. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Sabeh, I. Ota, K. Holmbeck, H. Birkedal-Hansen, P. Soloway, M. Balbin, C. Lopez-Otin, S. Shapiro, M. Inada, S. Krane, et al. Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP J. Cell Biol., November 22, 2004; 167(4): 769 - 781. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Sun, U. Ringdahl, J. W. Homeister, W. P. Fay, N. C. Engleberg, A. Y. Yang, L. S. Rozek, X. Wang, U. Sjobring, and D. Ginsburg Plasminogen Is a Critical Host Pathogenicity Factor for Group A Streptococcal Infection Science, August 27, 2004; 305(5688): 1283 - 1286. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Bolon, H.-M. Zhou, Y. Charron, A. Wohlwend, and J.-D. Vassalli Plasminogen Mediates the Pathological Effects of Urokinase-Type Plasminogen Activator Overexpression Am. J. Pathol., June 1, 2004; 164(6): 2299 - 2304. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. S. Heinemann, G. Korza, and J. Ozols A Plasminogen-like Protein Selectively Degrades Stearoyl-CoA Desaturase in Liver Microsomes J. Biol. Chem., October 31, 2003; 278(44): 42966 - 42975. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Solberg, J. Rinkenberger, K. Dano, Z. Werb, and L. R. Lund A functional overlap of plasminogen and MMPs regulates vascularization during placental development Development, September 15, 2003; 130(18): 4439 - 4450. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Chhokar and A. L. Tucker Angiogenesis: Basic Mechanisms and Clinical Applications Seminars in Cardiothoracic and Vascular Anesthesia, September 1, 2003; 7(3): 253 - 280. [Abstract] [PDF]
|
|
|
|
|
|
J. Sato, J. Schorey, V. A. Ploplis, E. Haalboom, L. Krahule, and F. J. Castellino The Fibrinolytic System in Dissemination and Matrix Protein Deposition During a Mycobacterium Infection Am. J. Pathol., August 1, 2003; 163(2): 517 - 531. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. W. Rijneveld, S. Florquin, P. Bresser, M. Levi, V. de Waard, R. Lijnen, J. S. Van der Zee, P. Speelman, P. Carmeliet, and T. van der Poll Plasminogen activator inhibitor type-1 deficiency does not influence the outcome of murine pneumococcal pneumonia Blood, August 1, 2003; 102(3): 934 - 939. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z.-L. Chen, J. A. Indyk, and S. Strickland The Hippocampal Laminin Matrix Is Dynamic and Critical for Neuronal Survival Mol. Biol. Cell, July 1, 2003; 14(7): 2665 - 2676. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Lambert, C. Munaut, P. Carmeliet, R. D. Gerard, P. J. Declerck, A. Gils, C. Claes, J.-M. Foidart, A. Noel, and J.-M. Rakic Dose-Dependent Modulation of Choroidal Neovascularization by Plasminogen Activator Inhibitor Type I: Implications for Clinical Trials Invest. Ophthalmol. Vis. Sci., June 1, 2003; 44(6): 2791 - 2797. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Currier, G. Sabla, S. Locaputo, H. Melin-Aldana, J. L. Degen, and J. A. Bezerra Plasminogen directs the pleiotropic effects of uPA in liver injury and repair Am J Physiol Gastrointest Liver Physiol, March 1, 2003; 284(3): G508 - G515. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. G. Bannach, A. Gutierrez, B. J. Fowler, T. H. Bugge, J. L. Degen, R. J. Parmer, and L. A. Miles Localization of Regulatory Elements Mediating Constitutive and Cytokine-stimulated Plasminogen Gene Expression J. Biol. Chem., October 4, 2002; 277(41): 38579 - 38588. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Burysek, T. Syrovets, and T. Simmet The Serine Protease Plasmin Triggers Expression of MCP-1 and CD40 in Human Primary Monocytes via Activation of p38 MAPK and Janus Kinase (JAK)/STAT Signaling Pathways J. Biol. Chem., August 30, 2002; 277(36): 33509 - 33517. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Guo, P. A. Mathieu, B. Linebaugh, B. F. Sloane, and J. J. Reiners Jr. Phorbol Ester Activation of a Proteolytic Cascade Capable of Activating Latent Transforming Growth Factor-beta . A PROCESS INITIATED BY THE EXOCYTOSIS OF CATHEPSIN B J. Biol. Chem., April 19, 2002; 277(17): 14829 - 14837. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Suelves, R. Lopez-Alemany, F. Lluis, G. Aniorte, E. Serrano, M. Parra, P. Carmeliet, and P. Munoz-Canoves Plasmin activity is required for myogenesis in vitro and skeletal muscle regeneration in vivo Blood, April 15, 2002; 99(8): 2835 - 2844. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. D. Rosen, S. Raymond, A. Zollman, F. Noria, M. Sandoval-Cooper, A. Shulman, J. L. Merz, and F. J. Castellino Laser-Induced Noninvasive Vascular Injury Models in Mice Generate Platelet- and Coagulation-Dependent Thrombi Am. J. Pathol., May 1, 2001; 158(5): 1613 - 1622. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Levi, L. Moons, A. Bouche, S. D. Shapiro, D. Collen, and P. Carmeliet Deficiency of Urokinase-Type Plasminogen Activator-Mediated Plasmin Generation Impairs Vascular Remodeling During Hypoxia-Induced Pulmonary Hypertension in Mice Circulation, April 17, 2001; 103(15): 2014 - 2020. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Lluis, J. Roma, M. Suelves, M. Parra, G. Aniorte, E. Gallardo, I. Illa, L. Rodriguez, S. M. Hughes, P. Carmeliet, et al. Urokinase-dependent plasminogen activation is required for efficient skeletal muscle regeneration in vivo Blood, March 15, 2001; 97(6): 1703 - 1711. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Bezerra, A. R. Currier, H. Melin-Aldana, G. Sabla, T. H. Bugge, K. W. Kombrinck, and J. L. Degen Plasminogen Activators Direct Reorganization of the Liver Lobule after Acute Injury Am. J. Pathol., March 1, 2001; 158(3): 921 - 929. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Swaisgood, E. L. French, C. Noga, R. H. Simon, and V. A. Ploplis The Development of Bleomycin-Induced Pulmonary Fibrosis in Mice Deficient for Components of the Fibrinolytic System Am. J. Pathol., July 1, 2000; 157(1): 177 - 187. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Akassoglou, K. W. Kombrinck, J. L. Degen, and S. Strickland Tissue Plasminogen Activator-Mediated Fibrinolysis Protects against Axonal Degeneration and Demyelination after Sciatic Nerve Injury J. Cell Biol., May 29, 2000; 149(5): 1157 - 1166. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Samis, G. D. Ramsey, J. B. Walker, M. E. Nesheim, and A. R. Giles Proteolytic processing of human coagulation factor IX by plasmin Blood, February 1, 2000; 95(3): 943 - 951. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Bezerra, T. H. Bugge, H. Melin-Aldana, G. Sabla, K. W. Kombrinck, D. P. Witte, and J. L. Degen Plasminogen deficiency leads to impaired remodeling after a toxic injury to the liver PNAS, December 21, 1999; 96(26): 15143 - 15148. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. G. Zhang, M. Chopp, A. Goussev, D. Lu, D. Morris, W. Tsang, C. Powers, and K.-L. Ho Cerebral Microvascular Obstruction by Fibrin is Associated with Upregulation of PAI-1 Acutely after Onset of Focal Embolic Ischemia in Rats J. Neurosci., December 15, 1999; 19(24): 10898 - 10907. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-l. Chen, J. A. Indyk, T. H. Bugge, K. W. Kombrinck, J. L. Degen, and S. Strickland Neuronal Death and Blood-Brain Barrier Breakdown after Excitotoxic Injury Are Independent Processes J. Neurosci., November 15, 1999; 19(22): 9813 - 9820. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Ny, G. Leonardsson, A.-C. Hägglund, P. Hägglöf, V. A. Ploplis, P. Carmeliet, and T. Ny Ovulation in Plasminogen-Deficient Mice Endocrinology, November 1, 1999; 140(11): 5030 - 5035. [Abstract] [Full Text]
|
|
|
|
|
|
D. J. Toft and D. I. H. Linzer Prolactin (PRL)-Like Protein J, a Novel Member of the PRL/Growth Hormone Family, Is Exclusively Expressed in Maternal Decidua Endocrinology, November 1, 1999; 140(11): 5095 - 5101. [Abstract] [Full Text]
|
|
|
|
|
|
V. Schuster, S. Seidenspinner, P. Zeitler, C. Escher, U. Pleyer, W. Bernauer, E. R. Stiehm, S. Isenberg, S. Seregard, T. Olsson, et al. Compound-Heterozygous Mutations in the Plasminogen Gene Predispose to the Development of Ligneous Conjunctivitis Blood, May 15, 1999; 93(10): 3457 - 3466. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Nagai, M. De Mol, H. R. Lijnen, P. Carmeliet, and D. Collen Role of Plasminogen System Components in Focal Cerebral Ischemic Infarction : A Gene Targeting and Gene Transfer Study in Mice Circulation, May 11, 1999; 99(18): 2440 - 2444. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Shi, A. Patel, D. Zhang, H. Wang, P. Carmeliet, G. L. Reed, M.-E. Lee, E. Haber, and N. E. S. Sibinga Plasminogen Is Not Required for Neointima Formation in a Mouse Model of Vein Graft Stenosis Circ. Res., April 30, 1999; 84(8): 883 - 890. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Schott, C.-E. Dempfle, P. Beck, A. Liermann, A. Mohr-Pennert, M. Goldner, P. Mehlem, H. Azuma, V. Schuster, A.-M. Mingers, et al. Therapy with a Purified Plasminogen Concentrate in an Infant with Ligneous Conjunctivitis and Homozygous Plasminogen Deficiency N. Engl. J. Med., December 3, 1998; 339(23): 1679 - 1686. [Full Text] [PDF]
|
|
|
|
|
|
H. R. Lijnen, B. Van Hoef, F. Lupu, L. Moons, P. Carmeliet, and D. Collen Function of the Plasminogen/Plasmin and Matrix Metalloproteinase Systems After Vascular Injury in Mice With Targeted Inactivation of Fibrinolytic System Genes Arterioscler Thromb Vasc Biol, July 1, 1998; 18(7): 1035 - 1045. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Carmeliet, L. Moons, and D. Collen Mouse models of angiogenesis, arterial stenosis, atherosclerosis and hemostasis Cardiovasc Res, July 1, 1998; 39(1): 8 - 33. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. B. Cines, E. S. Pollak, C. A. Buck, J. Loscalzo, G. A. Zimmerman, R. P. McEver, J. S. Pober, T. M. Wick, B. A. Konkle, B. S. Schwartz, et al. Endothelial Cells in Physiology and in the Pathophysiology of Vascular Disorders Blood, May 15, 1998; 91(10): 3527 - 3561. [Full Text] [PDF]
|
|
|
|
|
|
W. Risau Angiogenesis Is Coming of Age Circ. Res., May 4, 1998; 82(8): 926 - 928. [Full Text] [PDF]
|
|
|
|
|
|
P. M. Farrehi, C. K. Ozaki, P. Carmeliet, and W. P. Fay Regulation of Arterial Thrombolysis by Plasminogen Activator Inhibitor-1 in Mice Circulation, March 17, 1998; 97(10): 1002 - 1008. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. A. Ploplis, E. L. French, P. Carmeliet, D. Collen, and E. F. Plow Plasminogen Deficiency Differentially Affects Recruitment of Inflammatory Cell Populations in Mice Blood, March 15, 1998; 91(6): 2005 - 2009. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Ringdahl, M. Svensson, A. C. Wistedt, T. Renne, R. Kellner, W. Muller-Esterl, and U. Sjobring Molecular Co-operation between Protein PAM and Streptokinase for Plasmin Acquisition by Streptococcus pyogenes J. Biol. Chem., March 13, 1998; 273(11): 6424 - 6430. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A.F. Drew, A.H. Kaufman, K.W. Kombrinck, M.J.S. Danton, C.C. Daugherty, J.L. Degen, and T.H. Bugge Ligneous Conjunctivitis in Plasminogen-Deficient Mice Blood, March 1, 1998; 91(5): 1616 - 1624. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Carmeliet and D. Collen Molecular analysis of blood vessel formation and disease Am J Physiol Heart Circ Physiol, November 1, 1997; 273(5): H2091 - H2104. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. E. Tsirka, T. H. Bugge, J. L. Degen, and S. Strickland Neuronal death in the central nervous system demonstrates a non-fibrin substrate for plasmin PNAS, September 2, 1997; 94(18): 9779 - 9781. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Carmeliet, J.-M. Stassen, I. Van Vlaenderen, R. S. Meidell, D. Collen, and R. D. Gerard Adenovirus-Mediated Transfer of Tissue-Type Plasminogen Activator Augments Thrombolysis in Tissue-Type Plasminogen Activator-Deficient and Plasminogen Activator Inhibitor-1-Overexpressing Mice Blood, August 15, 1997; 90(4): 1527 - 1534. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Schuster, A.-M. Mingers, S. Seidenspinner, Z. Nussgens, T. Pukrop, and H. W. Kreth Homozygous Mutations in the Plasminogen Gene of Two Unrelated Girls With Ligneous Conjunctivitis Blood, August 1, 1997; 90(3): 958 - 966. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. P. Fay, A. C. Parker, L. R. Condrey, and A. D. Shapiro Human Plasminogen Activator Inhibitor-1 (PAI-1) Deficiency: Characterization of a Large Kindred With a Null Mutation in the PAI-1 Gene Blood, July 1, 1997; 90(1): 204 - 208. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. T. Sabapathy, M. S. Pepper, F. Kiefer, U. Mohle-Steinlein, F. Tacchini-Cottier, I. Fetka, G. Breier, W. Risau, P. Carmeliet, R. Montesano, et al. Polyoma Middle T-induced Vascular Tumor Formation: The Role of the Plasminogen Activator/Plasmin System J. Cell Biol., May 19, 1997; 137(4): 953 - 963. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Pepper Manipulating Angiogenesis: From Basic Science to the Bedside Arterioscler Thromb Vasc Biol, April 1, 1997; 17(4): 605 - 619. [Abstract] [Full Text]
|
|
|
|
|
|
G. R. Jenkins, D. Seiffert, R. J. Parmer, and L. A. Miles Regulation of Plasminogen Gene Expression by Interleukin-6 Blood, April 1, 1997; 89(7): 2394 - 2403. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. R. Kitching, S. R. Holdsworth, V. A. Ploplis, E. F. Plow, D. Collen, P. Carmeliet, and P. G. Tipping Plasminogen and Plasminogen Activators Protect against Renal Injury in Crescentic Glomerulonephritis J. Exp. Med., March 3, 1997; 185(5): 963 - 968. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. E. Tsirka, A. D. Rogove, T. H. Bugge, J. L. Degen, and S. Strickland An Extracellular Proteolytic Cascade Promotes Neuronal Degeneration in the Mouse Hippocampus J. Neurosci., January 15, 1997; 17(2): 543 - 552. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||