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Regular Article |
From the Department of Chemistry and Biochemistry,*
W. M. Keck Center for Transgene Research, and the Freimann Life
Science Center,
University of Notre Dame,
Notre Dame, Indiana; and the Division of
Hematology/Oncology,
Vanderbilt University,
Nashville, Tennessee
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Other inhibitors regulate enzymes that are needed for coagulation. For example, the serpin, antithrombin III, is one agent that serves this role by direct inhibition of FXa and thrombin. Activated protein C (aPC) is an anticoagulant that functions in a different manner, namely by specific proteolytic inactivation of FV/FVa3 and FVIII/FVIIIa.4 aPC also displays profibrinolytic effects directly by inactivation of fibrinolytic inhibitors, such as plasminogen activator inhibitor-1 (PAI-1),5 and indirectly through attenuation of thrombin formation, which is needed for activation of the zymogen, thrombin-activatable-fibrinolytic-inhibitor.6,7 aPC has also been implicated in host-defense reactions that occur during intravascular inflammation.8
Although a targeted deficiency of PC in mice led to early neonatal death,9 a similar total deficiency of FXI did not result in severe consequences in the unchallenged animal.10 Because of the nature of these phenotypes, an opportunity was present to examine the effect of an additional FXI deficiency on the phenotypes of PC-deficient animals. The issue to be addressed was the extent to which the FXI-dependent pathway contributed to the fibrin deposition observed in PC-/- mice. Thus, creation of PC/FXI double-deficient animals was seen as a means to address this question. This report is a summary of the phenotypic characteristics of these animals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation of mice heterozygous for a null PC allele9 and homozygous for the FXI null allele10 has been described. Briefly, targeted replacement of the PC allele resulted in the deletion of all translatable exons, via substitution of exons 2 to 9 of the PC gene with a neomycin phosphotransferase gene (NEO). The FXI gene mutation resulted in truncation of the protein at exon 5, via disruption of the exon with a NEO cassette. Both transgenic lines were back-crossed to the F4 generation in C57BL/6J mice (ca., 94% C57BL/6J background). Mice heterozygous for PC and homozygous deficient for FXI (PC+/-/FXI-/-) were generated by cross-mating these F4 generation PC+/- and FXI-/- mice. The resulting mice were then subsequently mated to generate PC-/-/FXI-/- progeny. The animals were housed in microisolation cages on a constant 12-hour light/dark cycle with controlled temperature and humidity and given access to food and water ad libitum.
Genotypic Analyses
Ear punches, tails, or portions of embryonic tissue or yolk sac were isolated for the extraction of genomic DNA as previously described.11 PC genotyping was performed by polymerase chain reaction in a 50-µl reaction using three different primers, 1) a common primer (CGT GAT GAG TTT CAG GCA GTG AGA G) for both the wild-type (WT) and null alleles; 2) a specific primer (GAG ATA AGC AGA TCC TGT GGA TTG C) for the WT allele situated in the 5' flank of the PC gene; and 3) a primer (ATT CGC AGC GCA TCG CCT TCT ATC) included in the NEO cassette. Amplicons of 1,000 bp and 600 bp were obtained for the WT and null alleles, respectively. Genotyping for FXI was also performed by polymerase chain reaction using three different primers, 1) an oligonucleotide (TTG CAG CAA AGA TGA GTA CGT GAA C), located at the 5' end of exon 5, served as a common primer for both the NEO and WT alleles; 2) a primer located in the NEO gene, which was the same as that used for PC genotyping; and 3) a primer (ATG GTC GAC ACT GGG AAA ATA CCC) located at the 3' end of exon 5, which was used to amplify the WT allele. Amplicons of 160 bp and 400 bp were obtained for the WT and null alleles, respectively.
Timed Matings
Females were placed with nonsibling males in the late afternoon and the morning of vaginal plug detection was assigned E0.5dpc. Pregnant females were anesthetized with rodent cocktail (9 mg/ml ketamine, 1.8 mg/ml xylazine, 0.3 mg/ml acepromazine in saline), and the embryos harvested at E17.5dpc.
Necropsy
Embryos were dissected free of maternal tissue and their organs isolated and fixed in 10% neutral-buffered formalin overnight. Tails were excised for genotypic analysis. Animals that were sacrificed postnatally were first anesthetized with rodent cocktail. Blood was collected through vena cava puncture into 3.8% citrate at a ratio of 9:1 citrate. For histological analysis, animals were perfused through the left ventricle with saline followed by 10% neutral-buffered formalin. Lungs were additionally inflated with 10% neutral-buffered formalin through the trachea. Organs were then removed and fixed for an additional 3 hours in 10% neutral-buffered formalin.
Histology, Histochemistry, and Immunohistochemistry
After fixation, samples were dehydrated, embedded in paraffin, and sectioned at 2 to 3 µm for hematoxylin and eosin (H&E) staining and 4 µm for all other analyses. Sections were stained with H&E to examine general tissue and cellular morphology, and with Masson’s trichrome for identification of collagen.12 Iron was detected using a periodic acid-Schiff/Mallory method (PAS/Fe).13,14 Blood and thoracic fluid smears were stained with Giemsa solution.15
Fibrin was identified immunohistochemically with a polyclonal goat anti-mouse fibrin(ogen) antibody (Accurate Chemicals, Westbury, NY). Antigen retrieval was performed under high temperature and pressure with citrate buffer (Biogenex, San Ramon, CA), followed by endogenous peroxidase blocking with Peroxoblock (Zymed, South San Francisco, CA). Subsequent to incubation, first with rabbit serum and then with the primary antibody, slides were incubated with the secondary rabbit anti-goat IgG (DAKO, Carpinteria, CA), followed by goat peroxidase anti-peroxidase (DAKO). Peroxidase activity was detected with the substrate 3-amino, 9-ethylcarbazole (Biomeda, Foster City, CA) followed by a hematoxylin counterstain (Biomeda). Macrophages were identified immunohistochemically with a monoclonal rat anti-mouse Mac-3 antibody (Pharmingen, San Diego, CA), followed by a mouse anti-rat biotinylated IgG (Pharmingen) and a streptavidin horseradish peroxidase incubation (Biogenex). Activated platelets were identified via immunostaining for P-selectin using a rabbit anti-human polyclonal antibody (Pharmingen), followed by a swine anti-rabbit biotinylated IgG and streptavidin horseradish peroxidase (Biogenex). Similar antigen retrieval, peroxidase detection, and counterstain were used, as described previously for fibrin detection. Von Willebrand factor (vWF)-positive cell types were identified immunohistochemically using an EPOS anti-human vWF antibody conjugated to horseradish peroxidase (DAKO) and detected with 3-amino, 9-ethylcarbazole. In this case, antigen retrieval was performed with limited trypsin digestion.
Transmission Electron Microscopy
Ultrastructural analyses were performed on the heart of a 45-day FXI-/-/PC-/- mouse. The heart was perfused, fixed (12 hours) with Karnovsky solution, and postfixed (1.5 hours) in 1% osmium tetroxide. Dehydration was accomplished using a graded series of ethanol solutions, and embedded in epoxy resins (Polysciences, Warrington, PA). Ultrathin sections (90 nm) were stained in 2% uranyl acetate and Reynold’s lead stain. Sections were viewed and photographed using a Hitachi H600 transmission electron microscope (Hitachi, Tokyo, Japan) at 75 kV accelerating voltage.
Determination of Soluble Fibrin in Plasma
Plasma fibrin levels were detected using the COATEST soluble fibrin kit, as described by the manufacturer (Chromogenix, Molndal, Sweden). A volume of 2.5 µl of plasma was diluted in 50 µl of 0.1 mol/L Tris-HCl buffer, pH 7.9, and the solution added to a microtiter plate containing plasminogen, recombinant tissue-type plasminogen activator, plasmin inhibitor antibody, and the plasmin-selective chromogenic substrate, S-2403. After a 15-minute incubation, the reaction was quenched with 100 µl of 5% acetic acid. Absorbances, obtained from p-nitroaniline release from substrate S2403, are directly related to the amount of soluble fibrin in the plasma. The absorbance of the batch-specific control provided in the kit was then subtracted from the absorbance of the test samples, and values, expressed in soluble fibrin units, were Tris-buffered saline/Tween and then incubated with 100 µl of plasma sample (1:5 dilution in Tris-buffered saline) or standard (0 to 12.5 ng/ml murine PAI-1 (Molecular Innovations, Southfield, MI ) for 1 hour at room temperature. After a triple wash with Tris-buffered saline/Tween, the plates were incubated with 100 µl of 6 µg/ml anti-murine-PAI-1 polyclonal antibody (Molecular Innovations). The wells were then incubated with 1:1,000 in Tris-buffered saline goat anti-rabbit-IgG-AP (Biorad, Hercules, California) for 1 hour after three washes. Finally, the assay was developed with pNPP (Sigma Chemical Co., St. Louis, MO) for 30 minutes at room temperature and the plates were read in a plate reader at 405 nm.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice carrying the PC+/- genotype were mated with FXI-/- animals to generate PC+/-/FXI+/- progeny. These mice were then crossed with FXI-/- animals to produce PC+/-/FXI+/- mice, which were then bred to provide PC-/-/FXI-/- progeny. Animals possessing the combined PC+/-/FXI+/- and PC+/-/FXI-/- genotypes seemed healthy and bred normally, at least through the age of 6 months.
Postnatal genotypic analysis of litters sired from
PC+/-/FXI-/-
breedings led to identification of eight
PC-/-/FXI-/-
animals who survived the lethal coagulopathy observed in
PC-/- neonates (Table 1)
. The oldest animal (I107, 94 days) was
sacrificed on presentation with severe inflammation, edema, and
gangrene on its extremities such as its nose, tail (Figure 1a)
, and paws (Figure 1b)
. Although none
of the other animals demonstrated this phenotype, the majority were
sedentary and significantly growth retarded (Figure 1c)
as compared to
their littermates. They also demonstrated dyspnea, and either expired
or were sacrificed between the ages of 13 and 94 days.
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. Perfusions
were often difficult to perform, probably because of the presence of
systemic microvascular clots. A total of two of the four animals that
were discovered dead presented with a significant volume (
1 ml) of
lymphatic fluid in the thoracic cavity (Figure 1d)
. The cellular
population was primarily lymphocytic, with occasional erythrocytes,
epithelial cells, monocytes, and neutrophils. Lymph nodes were
significantly enlarged and showed areas of hemorrhage, particularly on
the left side of the body near the salivary glands and kidneys,
adjacent to the carotid bifurcation, and close to the femoral artery
graft (Figure 1e)
. Hearts from sacrificed
PC-/-/FXI-/-
animals were markedly enlarged as compared to littermate controls.
Other anomalies included ventricular infarction (Figure 1f)
, atrial
clotting and fibrosis, and thrombosis near the truncus
pulmonalis. After perfusion and inflation of the lungs,
hemorrhagic lesions were readily apparent (Figure 1g)
. In several
animals, yellow patches of edemic tissue were also evident.
Areas of fibrotic-appearing tissue and the presence of hemorrhagic
cysts were commonly observed on the edges of the liver (Figure 1h)
.
Several animals also demonstrated significant thrombi in larger vessels
such as the thoracic and abdominal aortas (Figure 1i)
. Varying degrees
of hemorrhage were also evident between the brain and the skull plate,
and in other tissues such as the pancreas (Figure 1j)
, preputial gland
(Figure 1k)
, stomach, intestines, bladder (along with mineralization),
adrenal gland, prostate, testicles, epididymus, ovaries, uterus,
thymus, salivary gland, fatty patches, and leg muscles. Analysis of
blood smears indicated varying degrees of leukopenia, neutropenia, and
neutrophil hypersegmentation. In addition, smears demonstrated
reticulocytosis, anisocytosis, and a number of Howell-Jolly bodies.
Because the spleens of these animals appeared normal, the presence of
Howell-Jolly bodies may not be a significant pathological condition in
these mice. Histology
Histological analysis of paraffin-embedded tissues confirmed a wide array of pathologies in the PC-/-/FXI-/- animals. Concurrent with observations made at necropsy, analysis of H&E-stained tissue sections verified the systemic soft tissue hemorrhage. However, the most commonly affected organs were the liver, heart, lungs, and lymph nodes.
The hearts of
PC-/-/FXI-/-
mice were often characterized with multiple small and large
intravascular, epicardial, and lymphatic thrombus formation, as
observed by positive fibrin (Figure 2, A and B)
and vWF immunostaining (not shown), activated platelets trapped
within the thrombi (Figure 2C)
, arterial neointimal formation, acute
hemorrhage, atrial fibrosis and mineralization, and ventricular
fibrosis (Figure 2D)
. Multifocal vacuolization, necrosis, and
myocardial degeneration were also commonly observed. Inflammatory cell
infiltrates (neutrophils, macrophages, mast cells) were seen
surrounding affected vessels and within areas of degeneration and
repair. In a single case, degeneration of the atrioventricular valves
was observed. A transmission electron micrograph of a clot in the right
auricle of a 45-day postnatal animal (Figure 3)
offers confirmation of the
histological and immunohistochemical findings and clearly shows the
presence of activated platelets in a fibrin matrix.
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demonstrated varying degrees of septal thickening,
consolidation, alveolar hemorrhage, and fibroblast infiltration into
the alveolar spaces, as compared to
PC+/+/FXI-/-
controls (Figure 2F)
. Collagen deposition and focal mineralization
(Figure 2G)
, and more rarely, edema, were observed around these
fibrotic lesions. In addition, acute and chronic inflammation
(neutrophils, leukocytes, macrophages, giant cells) (Figure 2H)
,
erythrocytic phagocytosis, and pleuritis were evident. Perivascular
lymphatic vessels were often distended and filled with lymphocytes.
Degeneration of peritracheal muscle and fat were also observed. Small
to moderately sized granulomas were commonly evident.
Livers (Figure 4
; A, B, and C)
consistently demonstrated intravascular thrombosis with multifocal
areas of hyperplasia, mineralization, fibrosis, and hemorrhage. These
areas were often separated from normal tissue with a sharp line of
demarcation. The large blood-filled cysts observed in necropsy were not
surrounded by normal vessel endothelium (via anti-vWF immunostaining,
not shown), but instead appeared to be pockets of blood surrounded by
capsular tissue found adjacent to large mineralized fibrotic areas
(Figure 4D)
. Infiltration of neutrophils, macrophages, and
multinucleated giant cells were also common, particularly within and
surrounding mineralized foci.
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. Analysis of E17.5 Embryos
To determine the effect of FXI deficiency on
PC-/- embryonic coagulopathy, timed
matings between
PC+/-/FXI-/-
crosses were established and embryos harvested at E17.5dpc. Of 35
embryos harvested, eight were identified as
PC-/-/FXI-/-.
A total of two of the eight embryos seemed normal. The remaining six
embryos presented with slightly opaque livers and two of these six
embryos also showed some hemorrhage in their bladders and beneath the
skull plate. H&E and anti-fibrin(ogen) immunostaining demonstrated
varying degrees of hepatic necrosis (Figure 4G)
and fibrin deposition
(Figure 4H)
in all of the embryos, as compared to
PC+/+/FXI-/-
controls (Figure 4I)
. However, the systemic brain, kidney, atrial, and
soft tissue fibrin deposition and hemorrhage observed in
PC-/- embryos at this age were not
seen in the
PC-/-/FXI-/-
embryos.
Soluble Fibrin
Clinically, soluble fibrin is regarded as a molecular marker for
intravascular fibrin formation and, potentially, an elevated level may
be a marker for a hypercoagulative state. Thus, the levels of soluble
fibrin in plasma samples collected from
PC-/-/FXI-/-
animals were examined (Table 2)
. The
assay is based on the ability of fibrin to potentiate tissue-type
plasminogen activator-catalyzed plasmin generation from plasminogen.
Under normal nonhypercoagulative conditions, very low levels of soluble
fibrin should be obtained. Indeed, pooled WT plasma did not
indicate the presence of soluble fibrin. To determine whether the
COATEST assay kit would recognize murine soluble fibrin, WT
mice were injected with a sublethal dose of lipopolysaccharide,
euthanized at different time points (time = 0.5, 1, 2, 5, 10, and
24 hours), and their plasma collected. Only at the last time point (24
hours) was there detectable levels of soluble fibrin (145 soluble
fibrin units). All of the
PC-/-/FXI-/-
plasma samples that were tested (n = 5)
demonstrated measurable levels of soluble fibrin greater than the
WT control supplied by the manufacturers of the assay kit,
and in two cases the soluble fibrin units were greater than that which
was measured in plasma from the 24-hour LPS-injected WT
animals (Table 2)
. Not surprisingly, plasma samples from mice I208
(PC+/-/FXI-/-)
and I209
(PC+/+/FXI-/-)
did not demonstrate significant levels of soluble fibrin greater than
WT levels.
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Because PC has been implicated as a link between coagulation and inflammation, and secondly, because of the significant neutrophilia and recruitment of other inflammatory cell infiltrates into affected organs, enzyme-linked immunosorbent assay and ribonuclease protection assay were performed on plasma and serum samples, and lung and liver RNA, respectively, to assess possible alterations in systemic and tissue-specific cytokine expression levels.
The same five plasma samples tested for soluble fibrin levels were
assayed for the presence of interleukin (IL)-6 (plasma) and tumor
necrosis factor (TNF)-
(serum). Plasma samples from
lipopolysaccharide-injected WT animals were again used as
positive controls for elevated levels of these two cytokines.
PC-/-/FXI-/-
plasma and serum samples were tested in a dilute solution (1:1 in
sample buffer) or tested at an undiluted concentration. In either case,
neither IL-6 nor TNF- was detectable, at least to the pg range. IL-6
and TNF- levels were also undetectable in pools of WT
mouse plasma and serum, respectively.
RPA analysis was performed using total lung and liver RNA collected
from a single
PC-/-/FXI-/-
15-day-old animal (314A), along with its
PC+/- and
PC+/+ littermates. Blood smears
collected from this former animal demonstrated similar leukopenia and
neutropenia, as observed in the other
PC-/-/FXI-/-
animals with significant inflammatory cell infiltration into the lungs
and liver. Total RNA was hybridized with
32P-labeled RNA probes of various cytokines
[TNF-{beta}, lymphotoxin-{beta}, TNF-, interferon (IFN)-
,
IFN-{beta}, transforming growth factor (TGF)-{beta}1, and TGF-{beta}2],
digested with RNase, and the complexes resolved on a denaturing
acrylamide gel. Exposure of the gel to film throughout an extended
period of time (4 to 6 weeks) demonstrated that there were no changes
in the expression levels of any of these cytokines in this
PC-/-/FXI-/-
animal as compared to littermate controls.
PAI-1 Levels
The effects of a hypercoagulative state on fibrinolysis is
dominated by increased levels of PAI-1, leading to insufficient
fibrinolysis. Thus, plasma samples collected from
PC-/-/FXI-/-
animals were analyzed for increased PAI-1 levels (Table 2)
. In
addition, pools of WT mouse plasma from various ages were
also assessed, because PAI-1 levels are thought to be higher in
neonates.16
As is evident in Table 2
, PAI-1 concentrations
(84 ng/ml) in 7-day WT mice are higher than in 24-day
WT mice (15 ng/ml), as compared to 35-day WT mice
(undetectable). Mice I207 and G352-4, two 35-day
PC-/-/FXI-/-
animals, demonstrated higher levels of PAI-1 (7.4 ng/ml and 7.9 ng/ml,
respectively), as compared to an age-matched WT control
(undetectable PAI-1). Control plasma samples from mice I208
(PC+/-/FXI-/-)
and I209
(PC+/+/FXI-/-)
did not exhibit elevated PAI-1 levels. In addition, G462-1 (24-day
PC-/-/FXI-/-)
also possessed a level of PAI-1 (92 ng/ml) greater than its age-matched
WT control (15 ng/ml).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To further understand the consequences of total PC deficiency, our interest was to determine whether the additional loss of FXI, the initiator of the intrinsic maintenance pathway of coagulation, would result in increased survival for PC-/- mice. PC+/-/FXI-/- mice were generated and crossbred to create PC-/-/FXI-/- progeny. At necropsy, E17.5dpc PC-/-/FXI-/- embryos did not demonstrate any significant pathology as compared to PC-/- embryos at this age.9 However, histological analysis did demonstrate varying degrees of focal necrosis and fibrin deposition in the livers of these PC-/-/FXI-/- mice. Thus, the results obtained suggest that levels of embryonic FXI can contribute to coagulopathy in utero, because loss of FXI results in a decrease in severity of the embryonic coagulopathy seen in PC-/- embryos. In addition, a significant number of PC-/-/FXI-/- neonates survived beyond the time period at which the majority of the PC-/- neonates expired (day 0 to 2). Nonetheless, all of these mice eventually demonstrated varying degrees of systemic coagulopathy and inflammatory cell infiltration, resulting in their deaths between the ages of 13 to 94 days. Thus, it is clear that removal of the intrinsic pathway initiator, FXI, can result in at least a partial rescue of the coagulopathy associated with a PC deficiency, but more importantly allowed more mature mice to be studied that possessed a total PC deficiency.
Although it was difficult to assign a pattern of systemic insult correlated to age, the most commonly affected organs of PC-/-/FXI-/- mice were the liver, heart, lungs, and lymph nodes, which consistently demonstrated varying fibrin deposition, fibrosis, mineralization, and inflammatory cell infiltration, as observed through histological analyses. Several PC-/-/FXI-/- animals presented with atherosclerotic lesions and thrombosis in major vessels and in vitro analysis of plasma samples confirmed the presence of increased levels of soluble fibrin and enhanced PAI-1 levels. In addition, hemorrhage, secondary to coagulopathy, was occasionally observed in the stomach, intestines, preputial gland, sex organs, and soft tissues.
Not surprisingly, analysis of blood samples from PC-/-/FXI-/- animals demonstrated a paucity of inflammatory cells that were likely because of heavy recruitment into organs. In addition, the severe systemic hemorrhage resulted in anemia, as evidenced by the presence of higher than expected levels of reticulocytes, Howell-Jolly bodies, and the recruitment of immature erythrocytes in the blood.
Because the occurrence of coagulopathy in these animals is a likely
indicator of up-regulated prothrombin activation, other aspects of
thrombin-related functions were investigated, such as those that would
address the apparent central role of thrombin in the progression of an
inflammation-coagulation autoamplification loop.19
In this
model, it is suggested that in addition to catalyzing fibrin formation,
thrombin also stimulates inflammatory processes. Inflammation, in turn,
can then feedback and promote coagulation, in part through the
resulting damaged endothelium, which becomes prothrombotic. In
addition, the cytokines released during inflammation also influence
coagulation through up-regulation of tissue factor,20
as
well as FVIII and fibrinogen,21
and down-regulation of
thrombomodulin,22
which would attenuate PC activation.
Further contributing to the thrombotic state, the thrombin present can
inhibit fibrinolysis by catalyzing activation of
thrombin-activatable-fibrinolytic-inhibitor. In a setting such as a
PC-/-/FXI-/-
deficiency, it might be expected that this inflammation-coagulation
cross-talk would result in a self-propagating and deleterious feedback
loop, resulting in thrombosis, ischemia, free radical damage, and in
the most severe cases, death. In addition, PC has been suggested to
play a role in the inflammatory response. Although it has been shown
that the aPC/protein S complex up-regulated the proinflammatory
cytokines, IL-6 and IL-8, in human endothelial cells,23
another study24
showed the prevention, by (A)PC/EPCR
binding, of IL-6 and IL-8 up-regulation in Escherichia
coli-challenged animals. It has been further demonstrated that
APC, but not PC, prevented TNF- up-regulation25
and
CD14 down-regulation in macrophages issued inflammatory
challenges,25
and, additionally, that PC functioned in the
blocking of neutrophil activation.26
From another
perspective, the fact that TNF- and IL-1 down-regulate PC production
in mice further suggests that the proinflammatory markers can lead to
an increase in the procoagulant potential.27
From these
studies, it seems that the relationships between PC/aPC and cytokine
production are complex and involve the protease capabilities of aPC in
some cases and/or perhaps different cellular receptors for PC/aPC.
Given the potentially strong relationships between (a)PC and
coagulation/inflammation, it was of interest to assess possible
alterations in systemic and tissue-specific cytokine expression levels
in mice with a total PC deficiency that survived the neonatal
coagulopathy and showed clear signs of inflammation. Interestingly, no
evidence was found of alterations in plasma TNF- and IL-6 levels of
various-aged
PC-/-/FXI-/-
animals. In addition, no changes were observed in TNF-, IL-6,
TNF-{beta}, LT-{beta}, IFN-, IFN-{beta}, TGF-{beta}1, and TGF-{beta}2 in lung and
liver from a 15-day
PC-/-/FXI-/-
animal, as compared to littermate controls. These results were somewhat
surprising, particularly because histological analysis demonstrated
significant neutrophilia and other inflammatory cell infiltration.
Nonetheless, there was no evidence of enhanced cytokine levels in the
animals tested, even with sufficient levels of thrombin to induce
coagulopathy. It is possible that the role of PC in inflammation does
not exclusively rely on the cytokines examined in this report; that the
cytokine responses after clot formation are transient28
and were no longer elevated in the 15-day PC-deficient animal, which
contained clots much earlier in life; and/or the additional absence of
FXI in these animals has established a more complex relationship
between PC and inflammation that does not depend on these particular
cytokines. Unfortunately, it is not possible to test this latter
possibility because adult animals with a total PC deficiency do not
survive without the corresponding total FXI deficiency. However, other
PC-/- rescue strategies are being
probed in this laboratory that may in the future provide the necessary
animals.
The analysis of combined gene deficiencies is a powerful approach to understanding the pathologies of the individual gene deletion phenotypes, thus, further delineating the functions of these proteins. In a similar type of study, an additional FVII deficiency did not compensate for the coagulopathy observed in PC-/- embryos.29 In fact, the coagulopathy in these double-deficient animals was significantly enhanced as compared to the single PC-/- phenotype. This is most likely because of the loss of the anticoagulant function of the tissue factor pathway inhibitor inhibitory complex, which requires FVIIa to function. In contrast, a FVII deficiency has been shown to rescue the coagulopathy observed in embryos deficient in tissue factor pathway inhibitor.30 In this latter case, the effects of lost anticoagulant function of tissue factor pathway inhibitor were not significant in the additional absence of a procoagulant, FVII. Although the significance of the FXI-initiated intrinsic pathway may not have been thoroughly appreciated in the past,31 the results presented in this report clearly demonstrate that alterations in FXI levels, even during embryogenesis, can affect the degree of disseminated intravascular coagulation in PC-deficiency. The coagulopathy observed in the absence of FXI and PC likely results from a continuous activation of FV and FVIII through loss of PC function, and activation of FIX and FX from catalytic levels of thrombin generated from the FVIIa/tissue factor initiated extrinsic pathway. The loss of FXI seems to partially alleviate the neonatal mortality observed in the PC-/- animals; however, the resulting later stage coagulopathy still results in their demise in early adulthood.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grants HL-19982 (to F. J. C.) and HL-63682 (to V. A. P.), a grant from the W. M. Keck Foundation (to F. J. C.), and by the Kleiderer/Pezold Family Endowed Professorship (to F. J. C.).
Accepted for publication October 17, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, or phorbol ester. J Immunol 1994, 153:3664-3672[Abstract]
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