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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3442-3450
Structure-Function Analyses of Thrombomodulin by Gene-Targeting in
Mice: The Cytoplasmic Domain Is Not Required for Normal Fetal
Development
By
Edward M. Conway,
Saskia Pollefeyt,
Jan Cornelissen,
Inky DeBaere,
Marta Steiner-Mosonyi,
Jeffrey I. Weitz,
Hartmut Weiler-Guettler,
Peter Carmeliet, and
Désiré Collen
From the Center for Transgene Technology and Gene Therapy, Flanders
Interuniversity Institute for Biotechnology, University of Leuven,
Leuven, Belgium; The Blood Center for Southeastern Wisconsin,
Milwaukee, WI; the Hamilton Civic Hospital Research Center, Hamilton,
Ontario, Canada; and The Toronto Hospital and Department of Medicine,
University of Toronto, Toronto, Ontario, Canada.
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ABSTRACT |
Thrombomodulin (TM) is a widely expressed glycoprotein receptor that
plays a physiologically important role in maintaining normal hemostatic
balance postnatally. Inactivation of the TM gene in mice
results in embryonic lethality without thrombosis, suggesting that
structures of TM not recognized to be involved in coagulation might be
critical for normal fetal development. Therefore, the in vivo role of
the cytoplasmic domain of TM was studied by using homologous
recombination in ES cells to create mice that lack this region of TM
(TMcyt/cyt). Cross-breeding of F1 TMwt/cyt mice
(1 wild-type and 1 mutant allele) resulted in more than 300 healthy
offspring with a normal Mendelian inheritance pattern of 25.7%
TMwt/wt, 46.6% TMwt/cyt, and 27.7%
TMcyt/cyt mice, indicating that the tail of TM is not
necessary for normal fetal development. Phenotypic analyses showed that
the TMcyt/cyt mice responded identically to their wild-type
littermates after procoagulant, proinflammatory, and skin wound
challenges. Plasma levels of plasminogen, plasminogen activator
inhibitor 1 (PAI-1), and  2-antiplasmin were
unaltered, but plasmin:2-antiplasmin (PAP) levels were
significantly lower in TMcyt/cyt mice than in
TMwt/wt mice (0.46 ± 0.2 and 1.99 ± 0.1 ng/mL,
respectively; P < .001). Tissue levels of TM antigen were
also unaffected. However, functional levels of plasma TM in the
TMcyt/cyt mice, as measured by thrombin-dependent
activation of protein C, were significantly increased (P < .001). This supported the hypothesis that suppression in PAP levels may
be due to augmented activation of thrombin-activatable fibrinolysis
inhibitor (TAFI), with resultant inhibition of plasmin generation. In
conclusion, these studies exclude the cytoplasmic domain of TM from
playing a role in the early embryonic lethality of TM-null mice
and support its function in regulating plasmin generation in plasma.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THROMBOMODULIN (TM) is a transmembrane
receptor for thrombin that is widely expressed in a variety of tissues
in adults and during development.1-3 The function of TM is
best characterized with respect to its role in hemostasis, in which it
acts as a cofactor in the activation of protein C, thereby providing
anticoagulant properties,4,5 the physiological importance
of which is exemplified by the prethrombotic state of those who are
functionally deficient in protein C6 or who have factor
VLeiden.7 Mutations in TM have also been reported to be associated with a thrombotic tendency.8
Recently, TM was found to be a cofactor in the activation of plasma
procarboxypeptidase B (thrombin-activatable fibrinolysis inhibitor
[TAFI]),9 which interferes in the transformation of
plasminogen to plasmin by altering the cofactor, fibrin. Thus, TM is
presumed to have both antifibrinolytic and anticoagulant properties.
A critical role of TM during development, unrelated to coagulation, is
evidenced by the embryonic lethality before the development of a
vascular tree in mice with TM gene inactivation.10
Furthermore, mice expressing a TM variant with markedly reduced
thrombin-dependent protein C activation survived to
adulthood,11,12 additional evidence that TM has
noncoagulation roles during critical stages of murine development.
The lethal phenotype of TM / embryos implies that
normal development beyond 8.5 to 9.5 days post coitum (dpc) depends on expression of intact TM at a specific site at that developmental stage
or, alternatively, that a structural domain of TM, not involved in
coagulation, is essential for normal development. Although there is
considerable information as to the role of epidermal growth factor-like
repeats,3-6 little is known about the function of the other
domains of TM.13-15 The N-terminal extracellular region with weak homology to lectins16-18 appears to be required
for recycling of the molecule in vitro19 and may have a yet
to be identified ligand. The juxtamembranous serine-threonine-rich
region contains sites for N- and O-linked glycosylation and addition of
a chondroitin sulfate moiety, the latter which enhances the
anticoagulant function of TM. The cytoplasmic tail is not required for
constitutive recycling of TM in vitro,20,21 although it may
be important for multimerization. Although the cytoplasmic tail of TM
contains serine, threonine, and tyrosine, consensus sequences for
phosphorylation have not been identified. However, most intriguing is
the recent observation that both the N-terminal lectin-like domain
and the cytoplasmic domain of TM may be important in modulating the
growth of tumor cells.22
To determine the physiologic role of the cytoplasmic tail of TM, we
have used gene targeting in embryonic stem (ES) cells to generate mice
lacking this domain. Mice expressing tail-less TM (ie, lacking the
cytoplasmic domain) are both viable and fertile, with a normal response
to skin wounds, and to procoagulant and proinflammatory stimuli. The
cytoplasmic domain of TM regulates its functional level in plasma, in
turn regulating the fibrinolytic pathway possibly via activation of TAFI.
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MATERIALS AND METHODS |
Isolation of the murine TM gene.
Oligonucleotide primers TM.3330s (5'-TCTCCGCACTAGCCAAGCTGCAG) and
TM.3331as (5'-CTGCGGGAGCTGTAAACCGATCC), based on the published murine TM cDNA sequence,23 were used to screen a murine
129Sv genomic PAC library (Genome Systems, South Bend, IN). Three
clones were provided, each demonstrated by Southern blotting to contain the murine TM gene. As previously reported, an intracisternal A-particle (IAP) provirus was found in the 5' end of the
TM gene in this strain of mice.24 A 12-kb
Kpn I fragment, containing the entire coding region, was
subcloned into pBS (Stratagene Inc, Mississauga, Ontario, Canada),
resulting in Kpn12/BS.
Construction of a targeting vector to delete the cytoplasmic domain
of TM.
To replace the wild-type coding region of TM with one that
encodes a tail-less TM (ie, lacking the cytoplasmic domain of TM), polymerase chain reaction (PCR)-based mutagenesis with complementary oligonucleotides TM.S1951i (sense
5'-CTCTGTCACCTGCGCAAGTGAGGGATTTGCTCCAGA) and TM.AS1850i
(antisense 5'-TCTGGAGCAAATCCCTCACTTGCGCAGGTGACAGAG) was used. Two
PCRs were performed. In the first, oligonucleotide primer TMS.1951i was
paired with antisense primer TM.AS2613EO (5'-TGGACTAGTTAATTAAGATCTTCCTCGAGGCGCGCCGTTCAGCTGAAATATTTTAGC), resulting in a 643-bp fragment. In the second, antisense
oligonucleotide primer TM.AS1850i was paired with sense primer TM.S-240
(5'-TTCTGTGGTGGCGCCTGCAGGCCACGCCCG), yielding a 2,050-bp
fragment. These products were purified and used for recombinant PCR
with oligonucleotide primers TM.S-240 and TM.AS2613EO. The recombined
2,740-bp amplicon was subcloned into the TA-cloning vector pCR2.1
(Invitrogen, San Diego, CA), and DNA sequencing
confirmed the presence of the desired deletion. This DNA fragment
extends from a Nar I restriction enzyme site 230 bp upstream of
the transcriptional start site, through the coding region of the gene,
and 643 bp into the 3' untranslated region (3'-UTR).
Oligonucleotide primer TM.AS2613EO resulted in the addition of
Asc I, Xho I, Bgl II, Pac I, and
Spe I restriction sites at the 3' end of the recombined
product to be used for subcloning and ES cell DNA screening. The final
translated protein product represents the intact TM protein, lacking
the COOH-terminal 34 amino acid residues of the cytoplasmic tail
(NH2-KQGAARAELEYKCASSAKEVVLQHVRTDRTLQKF), yet retaining the
native in-frame stop codon.
A targeting vector was constructed (Fig 1)
by replacing the above mutated TM DNA into Nar I-Spe
I-digested Kpn12/BS, generating Kpn12mut/BS. The 3.5-kb Spe
I-Spe I fragment of 3' homology was excised from
Kpn12mut/BS, and the remaining vector was religated. A 1.5-kb
Kpn I/Bgl I fragment was removed from the most 5'
end of the gene and, after mung bean nuclease digestion, the ends of
the vector were also religated, resulting in approximately 3.4 kb of
5' homology. After digestion of the latter construct with
Xho I and Bgl II, a loxP-flanked neomycin
phosphotransferase-thymidine kinase (neo-tk) gene cassette
was subsequently inserted within the 3'-UTR. The resultant vector
was cut with Pac I, and the previously purified 3.5-kb
Spe I-Spe I fragment representing 3'-homology was
inserted in the correct orientation. Finally, for negative selection,
the gene encoding cytosine deaminase (cda) was inserted at the
3' end of the targeting vector between the Sal I and
Not I sites.
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| Fig 1.
Strategy to introduce TM lacking the cytoplasmic domain
into ES cells via homologous recombination. The wild-type allele for
the TM gene, which is intronless, encodes a lectin-like domain
(LLD), 6 epidermal growth-factor like repeats (EGF), a serine-threonine
rich region (STR), a single transmembrane domain (TM), and a
cytoplasmic tail (CT). DNA probes used for Southern blotting are shown.
After homologous recombination of the targeting vector, the targeted ES
cells were exposed to cre-recombinase for excision of the
neo-TK genes.
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Targeting of mutated TM gene into ES cells.
Targeting vector DNA (20 µg) was linearized with Not I and
introduced into R1 ES cells by electroporation, after which the cells
were plated onto confluent layers of neomycin-resistant embryonic
fibroblasts in the presence of G418 and 5-fluorocytosine (5-FC). DNA
from surviving colonies was screened for homologous recombination by
Southern blotting using a 3' external probe E (Fig 2A). Random integrations were excluded
by Southern blotting with a neomycin DNA probe and internal probes A
and B (Fig 1). Using DNA from the homologously recombined ES cell
clones, the expected deletion was confirmed by PCR with primer pair
TM.s1303 (5'-AGAGTGCGTGGAGCTTCTGGATC) and TM.as2250
(5'-TGCCTTCAAGCCTACAGATCAG), followed by DNA sequencing of
the PCR product.
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| Fig 2.
Southern blots of DNA derived from targeted ES cells and
tails of gene-targeted mice. (A) Southern blot of genomic DNA from ES
cells before electroporation (TMwt/wt), with homologous
recombination of the targeting vector (TMwt/cyt)), and
after successful cre-recombinase excision of the neo-tk genes
from the targeted allele (TMwt/cyt-cre). (B) Southern blot
of tail DNA from gene-targeted mice. A single 5.4-kb band is detected
in the TMcyt/cyt mice, reflecting homologous recombination
of both TM alleles with the mutant form. In (A) and (B), DNA was
digested with EcoRI/Xho I and bands were detected with
probe E (see Fig 1).
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In vitro excision of lox-P flanked neo-TK from targeted ES cell
clones.
Targeted ES cell clones were expanded and the cre-recombinase
gene25 was transiently introduced by electroporation.
Transfected ES cells were plated at low density on feeders and exposed
to gancyclovir, and surviving colonies were selected. Excision of the
neo-TK gene was confirmed by Southern blotting and PCR analysis.
Introduction of mutated TM into mice.
Targeted ES cells were aggregated26 with morula-stage
embryos derived from C57Bl6/J mice and introduced into pseudopregnant female National Institutes of Health (NIH) Swiss white mice. Two chimeric male offspring resulted in the establishment of germline transmission of the mutant TM allele. Large numbers of F1 and F2 offspring were intercrossed, avoiding brother-sister matings. Genotyping was performed on tail DNA both by Southern blotting and by
PCR. The chimeric males were also backcrossed with C57Bl/6 and 129sv/ev
mouse pedigrees for comparative purposes.
Expression of recombinant mutated murine TM in mammalian cells.
The cDNA encoding wild-type and tail-less TM were subcloned into the
expression vector pcDNA3.1 (Invitrogen) for transfection into COS
cells. Serial dilution of the cells under continuous selection with
G418 resulted in isolated clones of TM-expressing cells. A vector-alone
control COS cell line was also generated. Expression of cell surface TM
was confirmed by indirect immunofluorescence27 using highly
specific rabbit antirat TM antisera (gift of Dr Robert Jackman, Boston,
MA). The cofactor activity of cell-surface expressed recombinant TM was
evaluated by activation of purified bovine protein C with exogenously
added bovine thrombin.28
RNA isolation and reverse transcriptase-PCR (RT-PCR).
Total RNA was isolated from tissue by the method of Chomczynski and
Sacchi.29 For RT-PCR, cDNA was synthesized from total RNA
by reverse transcription using murine leukemia virus (M-MLV) reverse
transcriptase and a cDNA synthesis kit (NV Life Technologies, Merelbeke, Belgium). First-strand synthesis was primed
using random hexanucleotides. To confirm the deletion of the
cytoplasmic domain of TM in the gene-targeted mice by RT-PCR,
oligonucleotide primers that flank the deleted region, TM.s1303 and
TM.as2250, were used, and the amplicon was sequenced.
Quantitation of tissue levels of TM.
Relative amounts of TM in lung tissue and plasma were quantitated using
a previously reported sandwich radio-immunoassay30 with
monoclonal antibodies 201B and 34A (kindly provided by Dr S. Kennel,
Oak Ridge, TN).
Embryo sections and immunohistochemical staining.
For embryonic samples, mice were mated, conception was assessed by the
presence of a coital plug (with the morning of coital plug being scored
as 0.5 dpc), pregnant females were killed at specific developmental
time points, and embryos were carefully removed by dissection. Whole
embryos were fixed and embedded in paraffin, and 10-µm sagittal
sections were made and either stained with hematoxylin and eosin or
incubated with rabbit anti-TM antisera (1:1,000) for subsequent
detection by immunoperoxidase staining.
Plasma levels of components of the fibrinolytic system.
Previous reported double antibody enzyme-linked immunoassays were
performed to detect murine plasma levels of
 2-antiplasmin, plasminogen activator inhibitor 1 (PAI-1), plasminogen, and plasmin-2-antiplasmin (PAP)
complex.31,32
Quantitation of plasma levels of fibrinopeptide A (FPA).
Antibodies directed against the carboxy-terminus of FPA were raised
against a homologue of murine FPA that was synthesized by solid-phase
methods33 (QCB, Inc, Hopkinton, MA). An amino-terminal cysteine residue of the FPA homologue was cross-linked to Keyhole Limpet hemocyanin (Sigma, St Louis, MO) with
m-maleimidobenzoic acid N-hydroxysuccinimide ester (Pierce Chemical Co,
Rockford, IL) according to the manufacturer's instructions, and
Ellman's reagent (Pierce Chemical Co) was used to compare the
sulfhydryl concentration of the conjugate with that of the starting
material. Murine FPA (112 mol) was covalently coupled to each mole of
Keyhole Limpet hemocyanin. Immunization of sheep by subcutaneous
injection of the coupled murine FPA suspended in complete Freund's
adjuvant, followed by incomplete Freund's for booster immunizations,
was performed by Affinity Biologicals (Hamilton, Ontario, Canada). IgG
fractions were isolated from the sheep sera, dialyzed against HEPES-buffered saline, and then dialyzed against an equal volume of
glycerol for storage in aliquots at 20°C.
Murine blood from the inferior vena cava was collected into a plastic
syringe preloaded with 1/10 vol of an anticoagulant solution consisting
of 100 kallikrein inhibitor units (KIU)/mL bovine lung aprotinin
(American Diagnostica, Montreal, Quebec, Canada), 1,300 U/mL porcine
intestinal mucosa heparin (Sigma H9399), 10 mmol/L adenosine
(Calbiochem, Mississauga, Ontario, Canada), 20 mmol/L theophylline, and
0.1 mmol/L MeO-Suc-Ala-Ala-Pro-ValCh2Cl (CK-20; Enzyme
Systems Products, Livermore, CA) in HEPES-buffered saline,
pH 7.4. Plasma fractions obtained by 5 minutes of centrifugation at
15,000g were stored at 70°C for subsequent assay.
Murine FPA homologues, with or without an amino-terminal tyrosine
residue, were synthesized (QCB, Inc). The tyrosinated peptide was
radiolabeled with 125I-Na with a specific activity of at
least 0.32 µCi/pmol. Known concentrations of murine FPA or plasma
sample (500 µL) diluted in 50 mmol/L Tris-HCl, pH 8.5, 100 mmol/L
NaCl, 0.1% ovalbumin, 0.02% sodium azide (TBS) were incubated
overnight at 4°C with tracer (50 µL; ~15,000 cpm) and sheep
antimouse FPA IgG (100 µL; at a dilution to bind ~35% of total
cpm). Unbound tracer was precipitated by addition of 1 ml of 1.25%
(wt/vol) suspension of activated charcoal in TBS and incubation for 20 minutes at 4°C, followed by centrifugation at 3,000g for 20 minutes at 4°C. Supernatants were decanted and counted for 1 minute
(LKB Instruments, Gaithersburg, MD). Based on standard curves generated
with the FPA homologue, the assay has a limit of detection of 0.01 nmol/L mouse FPA and interassay and intra-assay coefficients of
variation of 4.7% and 2.8%, respectively.
Functional level of plasma TM.
After anesthesia of mice, blood was drawn via cardiac puncture into
preloaded syringes containing 1/10 vol 3.8% sodium citrate. Ten
microliters of bovine thrombin (15 nmol/L) was added to 120 µL
HEPES-buffered saline, 5 µL plasma, and 20 µL bovine protein C
(2,372 nmol/L), and the reaction was incubated at 37°C for 45 minutes, after which a molar excess of PPACK, with respect to thrombin,
was added. The TM-dependent conversion of protein C to activated
protein C was measured by adding the reaction mixture to 500 µL of
0.4 mmol/L chromogenic substrate S2238 (Helena Laboratories, Beaumont,
TX) and quantitating the rate of change in absorbance at 405 nm.
Preincubation of plasma with PPACK, hirudin, or specific polyclonal
anti-TM antibodies known to interfere with TM cofactor activity
resulted in the absence of conversion of protein C to its active form,
as measured by the chromogenic assay.
Protein electrophoresis and immunoblot of plasma TM.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
was performed using an 8% gel according to the method of
Laemmli34 under nonreducing conditions and as previously described.35 Citrated plasma obtained from 8-week-old mice
was loaded in each lane in Laemmli buffer, and after electrophoretic separation and transfer to nitrocellulose, the filter was incubated overnight with monoclonal anti-TM antibodies 201B and 34A (Dr S. Kennel) in Blotto at a concentration of 0.5 µg/mL.
Detection was accomplished using the ECL method (Amersham, Gent, Belgium).
Thrombogenic stresses.
Mice were exposed to 5.5% oxygen for 16 to 18 hours in a normobaric
chamber, as previously described,36 after which they were
immediately anesthetized. The sternum was split for cardiac puncture to
withdraw blood into appropriate anticoagulants for subsequent assays.
The vasculature was perfused via the heart with phosphate-buffered
saline (PBS). Tissues were quickly dissected and either fixed for
histological analysis or placed into liquid nitrogen for protein or RNA
studies. Tissue levels of fibrin were determined as
reported.11 Transverse sections of the lungs were cut for
immunoperoxidase staining (without counter-stain) with polyclonal goat
antimouse fibrin/ogen antibody (1:400) (Nordic, Trilburg,
The Netherlands).
Induction of disseminated intravascular coagulation in mice.
Lipopolysaccharide (LPS) from Escherichia coli serotype
0111:B4 (Sigma) was injected intraperitoneally into 10- to 12-week-old mice. Animals were closely monitored each day until either recovery or
cessation of breathing.
Wound-healing in mice.
Under anesthesia, two linear, parallel, vertical 3-cm incisions were
made on the back, through the skin to the depth of the dermis. Mice
were then housed in separate cages to prevent scratching. Wounds were
inspected daily. After 8 days, the animals were killed and the
surrounding tissue including each wound was excised and fixed for
histological analysis.
Animal care.
All animal experiments were approved by the Institutional Animal Care
and Use Committee of the University of Leuven.
Statistical analyses.
Statistical analyses of data using standard methods were conducted with
the StatView computer program (Abacus Concepts Inc, Berkeley, CA). The
means are provided with associated standard errors (SD). P
values were determined using the unpaired t-test.
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RESULTS |
Deletion of the cytoplasmic domain of TM and expression in COS cells.
To evaluate the role of the cytoplasmic domain of murine TM, we deleted
the entire domain using recombinant PCR while retaining the native
in-frame stop codon. The two basic amino acid residues within the
cytoplasmic domain and immediately adjacent to the membrane, arginine
and lysine, were maintained to minimize the possibility of shedding of
the mutated molecule from the cell surface. COS cells were transfected
with murine TM cDNA encoding both wild-type and tail-less TM. Northern
analysis of RNA derived from the TM-expressing cells and control cells
transfected with the expression vector (pcDNA3.1) alone confirmed the
specificity and expected TM mRNA processing (not shown). Indirect
immunofluorescence using a specific rabbit anti-TM antibody showed that
wild-type and tail-less TM could be transported through the cell for
stable surface expression. Furthermore, protein C activation by
thrombin was markedly accelerated on the surface of COS cells that
expressed either wild-type or tail-less murine TM. The rate of change
in absorbance of the chromogenic substrate S2238 at 405 nm used to determine TM-cofactor function in thrombin-dependent activation of
protein C was 0.01 U/min in vector-alone transfected COS cells. However, it was 0.14 ± 0.02 (n = 3) U/min and 0.16 ± 0.03 (n = 3) U/min for those COS cells transfected with wild-type
or tail-less TM, respectively, indicating that both forms of TM were
functional, as would be expected given that the entire extracellular
domains of the protein were intact.
Generation of mice lacking the cytoplasmic domain of TM.
A targeting vector was constructed (Fig 1) in which the wild-type
coding region of the murine TM gene (TM is intronless)
was replaced with one that encoded a truncated form of TM, ie, lacking the most COOH-terminal 34 amino acid residues of the cytoplasmic domain, while retaining an in-frame stop codon. After electroporation of R1 ES cells, more than 300 clones were picked, 2 of which were determined to have homologously recombined the replacement vector in a
single copy, as evaluated by Southern blotting (Fig 2A). PCR and DNA
sequencing were used to confirm that the entire coding region of the
mutated allele with the appropriate deletion was intact. After
expansion of the two positive ES cell clones, excision of the lox-P
flanked neo-TK cassette was accomplished by transient introduction of the cre-recombinase gene. Successful excision was demonstrated in numerous cre-recombinase exposed,
gancyclovir-resistant ES cell clones. Several of these were aggregated
for generation of chimeric mice, two of which transmitted to germline.
The reported results are not likely to reflect a strain-specific
artifact, because in limited studies, back-crossing onto 129sv/se and
Bl6 backgrounds resulted in similar phenotypes.
Viability of gene-targeted mice.
Cross-breeding of F1, TMwt/cyt (1 mutant and 1 wild-type
allele) mice has resulted in more than 300 offspring. Genotyping of
tail DNA was performed by PCR analysis and occasionally confirmed by Southern blotting (Fig 2). The genotypes of F2 progeny were distributed in a Mendelian inheritance pattern of 25.7%
(TMwt/wt), 46.6% (TMwt/cyt), and
27.7% (TMcyt/cyt) at birth, indicating that intrauterine
death was not occurring. Furthermore, there was an equal distribution
of male and female births, and there were no detectable differences in
weight, appearance, or growth and development up to 10 months of age.
Females and males were fertile, producing offspring of the different
genotypes in the expected distribution.
Expression of TM by TMwt/wt, Tmwt/cyt, and
TMcyt/cyt mice.
Deletion of the cytoplasmic domain of TM in vivo did not affect
cellular distribution, quantitative synthesis, cell membrane expression, or protein C cofactor activity of the molecule.
Immunoperoxidase staining of sagittal sections of 14.5 dpc embryos
showed TM in all tissues as previously reported, including within the
lung, brain, spleen, liver, kidneys, and heart. The total amount of TM
in lung tissue was quantitated using a double antibody sandwich radioimmunoassay. From four mice of each genotype, 3 measurements were
performed on each lung sample. TMwt/wt,
TMwt/cyt, and TMcyt/cyt mice had similar TM
lung tissue levels of 340 ± 21, 350 ± 10, and 280 ± 23 cpm/µg of total protein, respectively. Similarly, there were no
differences in TM antigen levels in the brain and kidney of the same
mice (not shown).
Plasma levels of fibrinolytic components and functional TM.
In view of the recently described role of TM in mediating an
antifibrinolytic effect via TAFIa, PAP complex in plasma was measured
by using an enzyme-linked immunosorbent assay (ELISA) in which one
antibody detects murine plasmin/ogen while the second antibody detects
2-antiplasmin. In five separate experiments, performed
with 3 to 5 mice from each genotype, there was a significantly lower
plasma level of PAP in the mice expressing tail-less TM (P < .005; Table 1). Plasma levels of
2-antiplasmin and plasminogen were essentially identical
(P > .1) in each group (TMwt/wt v
TMcyt/cyt) by using sensitive and specific ELISAs
(Table 2). Thus, mice expressing tail-less
TM have significantly lower plasmin generation than their wild-type
counterparts.
PAI-1 levels were not significantly different in the plasma of those
mice expressing tail-less TM as compared with their wild-type littermates (Table 2) or the TMwt/cyt mice, indicating that
the cytoplasmic domain of TM does not mediate regulation of release of
PAI-1. An increase in soluble forms of TM in the plasma might enhance
the activation of TAFI, thereby diminishing plasmin generation. Using a
chromogenic substrate assay to measure thrombin-dependent protein C
activation as a function of plasma TM,28 it was determined
that mice expressing tail-less TM had significantly higher functional
levels of soluble plasma TM (Table 1). Protein C activation was
entirely abrogated by preincubation of plasma samples with anti-TM
antisera or with PPACK, indicating the specificity of the protein C
activation by the thrombin-TM complex and supporting our hypothesis
that excess functional TM in the plasma might result in suppression of
plasmin generation via the activation of TAFI. In 4 mice of each
genotype, plasma TM antigen levels were not significantly different
(P > .1; 58 ± 7 cpm/µg and 63 ± 9 cpm/µg in
wild-type and mutant mice, respectively). Furthermore, Western
immunoblotting showed that the pattern of plasma TM fragments was
similar in the TMcyt/cyt mice and their wild-type
littermates (Fig 3).
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| Fig 3.
Western immunoblot detection of plasma TM. Citrated
murine plasma from TMwt/wt and TMcyt/cyt mice
(lanes A and B, respectively) in Laemmli buffer was separated
electrophoretically by SDS-PAGE under nonreducing conditions, and the
gel was transferred to a nitrocellulose filter for immuno-detection of
TM with specific monoclonal anti-TM antibodies, as detailed in
Materials and Methods. Molecular weight markers are on the left.
Several molecular forms of TM were identified, the most prominent of
which are noted with arrows on the right. No differences in patterns
between the two lanes could be discerned.
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Thrombogenic stresses.
Lethal doses of LPS (40 µg/g of body weight) resulted in
similar survival times of 33 ± 13 hours and 28 ± 11 hours (n = 7, P = .22) for TMwt/wt and TMcyt/cyt
mice, respectively. Human protein C has been shown in some animal models to protect against the lethality of Escherichia coli
infusions.37 Pretreatment of LPS-exposed mice with
bovine protein C (10 µg) had no effect on survival. Sublethal doses
of LPS (5 µg/g) resulted in blindness, altered hair
appearance, and irregular behaviour within 20 to 30 hours. There were
no obvious differences in time of onset, severity of symptoms, or time
to full recovery (~72 hours after LPS administration) between
genotypes, indicating that the cytoplasmic domain does not play a
direct role in inflammation or coagulation. Pretreatment with bovine
protein C again had no effect on the response to sublethal doses of
LPS.
Exposure of mice to hypoxia for 16 to 18 hours has been shown to result
in the deposition of fibrin and platelet thrombi within the lung
vasculature, with increases in thrombogenicity observed in mice
heterozygous for the TM gene12 and in mice
expressing TM that has approximately 0.5% protein C cofactor
activity.11 The thrombogenic response to hypoxia was
studied by quantitation of lung tissue fibrin,11
measurement of FPA, and computer-aided quantitation of fibrin/ogen
immunostaining in lung tissue sections (Table 3). Baseline levels of these
parameters in the TMwt/wt, TMwt/cyt, and
TMcyt/cyt mice were similar, and hypoxia did not
significantly affect these markers (P > .1). Previous in
vitro and in vivo studies have indicated that TM mRNA levels may be
augmented in response to stress.38 Quantitation of lung
tissue TM antigen in mice expressing wild-type and tail-less TM before
and after hypoxia exposure showed no significant response to the
stress. Overall, the results of these experiments would suggest that
the cytoplasmic domain of TM has no role in altering the coagulation
system in response to this stress.
Wound healing.
TM expression by suprabasal keratinocytes has been demonstrated to be
upregulated both during epidermal differentiation and after injury,
particularly at the migrating edge of a healing skin
wound.39 However, the presence or absence of the
cytoplasmic tail of TM had no effect on the rate of wound healing, the
apparent quality of the wounds, or the histologic appearance of
sections of the wounds (data not shown).
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DISCUSSION |
TM is known to be expressed by a wide variety of tissues, including
several tumor cells, syncytiotrophoblasts, vascular endothelium, neutrophils, monocytes, synovial lining cells, platelets, smooth muscle
cells, keratinocytes, and meningeal cells. Referred to as fetomodulin,
TM was recognized as a marker protein of fetal development.2,3 The multidomain structure of this
glycoprotein suggests that it may have several functions in addition to
its role as a cofactor in activating protein C and TAFI. The
observation that inactivation of the TM gene in mice leads to
embryonic lethality without evidence of thrombosis,10
warranted a search for alternative functions for this protein, as well
as for the etiology of the intrauterine death of TM null embryos.
Recent in vitro studies have elucidated several of the
structure-function correlates of TM. For example, EGF-like repeats 3 through 6 are critically involved in both the activation of protein
C5 and TAFI.40,41 Attachment of the chondroitin
sulfate moiety in the juxtamembranous serine-threonine region provides
optimization of anticoagulant function.42 The
NH2-terminal lectin-like domain may interact with other
proteins and thereby mediate regulation of intracellular
routing,19 whereas the cytoplasmic tail of TM is probably
required for multimerization of the receptor and may contribute to
clathrin-coated pit-mediated endocytosis.20 Finally, both
the lectin-like and the cytoplasmic domains have been implicated in
regulating a thrombin-independent antiproliferative effect.22
To directly evaluate the in vivo role of the cytoplasmic domain of TM,
mice that are lacking this structure were generated by homologous
recombination in ES cells. The targeting vector strategy was designed
to maintain the integrity of the gene encoding TM while selectively
deleting the DNA sequence encoding the cytoplasmic domain. The
Cre-lox P system was used to excise the selection marker genes,
neomycin phosphotransferase and thymidine kinase, from
the 3'-untranslated region of the targeted gene, excluding the
possibility of these affecting the phenotype of the resultant gene-targeted mice. Although a single loxP site was left within the 3'-UTR, this insertion did not appear to affect TM protein levels in the mutant mice, indicating that the effect of the mutant alleles was restricted to a structural defect and did not alter overall
quantitative expression of TM.
TM has well-defined anticoagulant properties, mediating the activation
of protein C, the latter which also possesses an anti-inflammatory role. In view of the link via TM between coagulation and inflammation, the response of mice expressing tail-less TM to both procoagulant and
proinflammatory stimuli was evaluated. The effects of hypoxic injury to
induce fibrin deposition in the lungs, and of lethal and sublethal
injections of LPS, were identical irrespective of the integrity
of the cytoplasmic domain of TM. These results support the biochemical
and cell culture data indicating that the anticoagulant properties of
TM reside within the EGF-like repeats and that regulation of expression
of cell-surface functional TM does not depend on the cytoplasmic domain.
Preinfusion of the mice with bovine protein C before LPS
exposure had no apparent anti-inflammatory effect. Although purified bovine protein C may be activated by human or bovine thrombin in the
presence of murine TM as a cofactor, it is not known whether bovine
activated protein C has anti-inflammatory activity in vivo in mice.
Thus, these results should be cautiously interpreted until the
experiments are repeated exclusively with murine-derived proteins.
Links between coagulation and fibrinolysis have been further documented
by the observation that generation of the fibrinolysis inhibitor,
TAFIa, by thrombin requires TM as a cofactor.43 It has long
been recognized that keratinocytes can produce tissue type plasminogen
activator (t-PA) and urokinase type plasminogen activator
(u-PA) both in vitro and in vivo.44 Direct
evidence for fibrinolysis playing a critical role in epithelialization was provided by studies in which the gene encoding plasminogen was
inactivated in mice, resulting in profoundly impaired wound healing.45,46 TM also is believed to be involved in this
process, but its specific role is unknown. TM expression by suprabasal keratinocytes is dramatically enhanced at the migrating edge of a
healing wound. It was therefore reasonable to assume that TM may serve
to regulate fibrin deposition, fibrin dissolution, and inflammation at
the site of a wound, by virtue of its functions via protein C and TAFI.
Raife et al47 have previously shown that
overexpression of TM in transgenic mice does not affect wound healing,
although collagen deposition was altered. Although not a quantitative
study, we could detect no obvious difference in either the formation or
healing of skin wounds in mice lacking the cytoplasmic domain of TM,
suggesting that signals critical for normal wound healing do not appear
to be mediated by this domain. To definitively identify the role of TM
in keratinocytes and skin wound healing, cell-specific inactivation of
TM will be necessary. These studies are in progress.
In view of the activation of TAFI being dependent on TM, we quantitated
several fibrinolytic parameters in the mutant mice. Those mice
expressing tail-less TM had significantly lower PAP levels than their
wild-type counterparts (P < .001). However, both plasminogen
and 2-antiplasmin levels were similar in both wild-type
and mutant mice, supporting the conclusion that plasmin generation was
diminished in those mice with tail-less TM. We would not have expected,
based on this observation, that the TMcyt/cyt mice would
exhibit a predisposition to thrombosis, given the fact that
inactivation of the genes for t-PA and u-PA in mice did not result in
significant spontaneous fibrin deposition.48 Nonetheless,
the possibility that the cytoplasmic domain of TM might modulate
plasmin generation was intriguing in that new insights into
fibrinolysis regulation may be provided. There are several potential
explanations for the surprising decrease in PAP levels in the
TMcyt/cyt mice. Plasmin generation is regulated by the
interplay between plasminogen activators and inhibitors. Furthermore,
thrombin has been shown in vitro to regulate the production of PAI-1
and t-PA in human umbilical vein endothelial cells
(HUVEC), augmenting both, presumably via activation of
protein kinase C.49 It has not been determined whether one
of the thrombin receptors or TM mediated these effects. We therefore
considered the possibility that the cytoplasmic domain of TM might
mediate signals that regulate the release of functional t-PA, u-PA, or
PAI-1, in which case suppression of plasminogen activators or increases
in PAI-1 would be expected to decrease the generation of plasmin and
thus explain the lower PAP levels in the TMcyt/cyt mice. We
excluded the possibility that the cytoplasmic domain of TM mediates,
either directly or indirectly, an increase in PAI-1 levels, but it
remains to be established whether the tail of TM plays a role in
regulating release of plasminogen activators. Detailed signaling,
protein, and mRNA expression studies on cells isolated from the
TMcyt/cyt mice will help to elucidate the role of the
cytoplasmic domain of TM in the fibrinolytic pathway and the functional
relationship(s) between thrombin receptors and TM.
PAP levels may also be diminished in the TMcyt/cyt mice as
a result of TM-mediated activation of TAFI. Tail-less TM might be more effective than wild-type TM as a cofactor in activation of TAFI, resulting in reduced plasmin generation. Alternatively, the
TMcyt/cyt mice may have increased shedding of TM, resulting
in augmented activation of TAFI. However, the inclusion of two basic
amino acids of the cytosolic tail of TM adjacent to the membrane was designed to minimize shedding of tail-less TM from the cell surface. TM
antigen levels were indeed very similar in the lung, kidney, and brains
of mice expressing wild-type and tail-less TM, and there was no
evidence of increased antigenic TM in the plasma of the
TMcyt/cyt mice. In contrast, functional levels of TM,
measured by thrombin-dependent activation of protein C, were
significantly elevated in the TMcyt/cyt mice. Subtle
increases in TM shedded into the plasma might account for this
increase. Alternatively, this apparent discrepancy suggests that the
cytoplasmic domain of TM plays a role in regulating the pattern of
proteolysis as it is released into plasma. Two monoclonal antibodies
were used in the quantitative sandwich radioimmunoassay for TM, and
because multiple proteolytic forms of TM are known to exist in plasma,
all of the forms are not necessarily detected with equivalent
sensitivity. Consequently, a relative increase in the TAFI-activatable
forms of TM might not have been detected by the radioimmunoassay or
discerned by Western immunoblot. In summary, increased plasma levels of
functional TM probably contribute to suppression of plasma PAP levels
via TAFI activation.
The most notable observation from these studies is that the cytoplasmic
domain of TM is not required for normal fetal development. The pattern
of expression of TM during development was unaltered by the absence of
the cytoplasmic tail. Furthermore, postnatal growth and development up
to 1 year of age was similar in mice expressing tail-less or
full-length TM. In view of the suggestion by Zhang et al22
that the cytoplasmic tail may modulate cell proliferation, mice
expressing tail-less TM were examined for spontaneous development of
tumors, none of which were evident during the period of observation up
to 1 year. However, this does not necessarily exclude a role for the
cytoplasmic domain in tumor/cell proliferation. Genetic crosses with
mice predisposed to developing tumors are being set up to more directly
evaluate the function of the cytoplasmic domain of TM in this process.
Finally, to evaluate the role of other structural domains of TM during
embryonic development, further in vivo gene targeting studies are underway.
| |
FOOTNOTES |
Submitted November 2, 1998; accepted January 12, 1999.
Supported in part by the Heart and Stroke Foundation of Ontario. E.M.C.
and J.I.W. are Career Investigators of the Heart and Stroke Foundation
of Canada.
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 Edward M. Conway, MD, Center for Transgene
Technology and Gene Therapy, KU Leuven, Gasthuisberg O&N, 9th Floor,
Herestraat 49, B-3000 Leuven, Belgium; e-mail:
ed.conway{at}med.kuleuven.ac.be.
| |
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ABSTRACT
INTRODUCTION