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From the W. M. Keck Center for Transgene Research and theDepartment of Chemistry and Biochemistry, University of NotreDame, Notre Dame, Indiana
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Abstract |
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chain, which results in a total Fg deficiency state
(FG-/-), aborted the fetuses
at the equivalent gestational stage seen in humans. Results obtained
from timed matings of FG-/- mice
showed that vaginal bleeding was initiated as early as embryonic day
(E)6 to 7, a critical stage for maternal-fetal vascular
development. The condition of afibrinogenemia retarded embryo-placental
development, and consistently led to abortion and maternal
death at E9.75. Lack of Fg did not alter the extent or distribution
pattern of other putative factors of embryo-placental
attachment, including laminin, fibronectin, and
Factor XIII, indicating that the presence of
fibrin(ogen) is required to confer sufficient stability at
the placental-decidual interface. The results of these studies
demonstrate that maternal Fg plays a critical role in maintenance of
pregnancy in mice, both by supporting proper
development of fetal-maternal vascular communication and
stabilization of embryo implantation.
/Bß/)2. This protein is the precursor
of fibrin, formed during the hemostatic response. Three separate genes,
FGA, FGB, and FGG, located on a 50-kb
region of chromosome 4q28-q31, are translated in a coordinated fashion
to code for the A-, Bß-, and -chains, respectively, of
Fg.1
For its conversion into fibrin, thrombin first
catalyzes release of fibrinopeptides A and B from the A and Bß
chains, thus leading to exposure of polymerization sites that organize
the initially formed fibrin monomers into a polymeric network of fibers
that form the major protein component of the blood clot. This clot is
ultimately covalently stabilized by crosslinking of specific
-NH2 groups of Lys and -COOH groups of Glu
residues of fibrin monomer, a process catalyzed by a transglutaminase,
namely, activated Factor XIII (FXIIIa).2 In addition to their function in clot formation, Fg and fibrin play roles in other pathophysiological processes, such as infection,3 wound healing,4,5 and clot retraction.6 Further, both Fg and fibrin have been implicated in the progression of certain types of tumors,7 and in the severity of atherosclerosis.8
Congenital afibrinogenemia (MIM no. 202400), an autosomal recessive disorder, was first described in 1920,9 and since that time more than 150 cases have been reported.10 The underlying causes of afibrinogenemia have been determined to be because of a large number of genetic abnormalities in FGA (donor splice site, frameshift, and nonsense mutations);11,12 FGB (missense mutations);13 and FGG (deletion, splice site, and missense mutations).14-16 Despite excessively prolonged plasma clot times, the phenotypes manifested by these patients involve hemorrhagic diatheses no more severe than hemophilia A or B. A common event after birth is an uncontrolled bleeding from the umbilical cord. Later in life, spontaneous intracerebral bleeding and splenic rupture may occur.17
Fg deficiencies also result in spontaneous miscarriage in the early gestational period.18-20 Although reasons for fetal loss in pregnant females homozygous for the FGG deletion, which results in a total deficiency of Fg (FG-/-), are primarily uncertain, protection against premature bleeding, offered in part by Fg, is most likely involved in allowing pregnancy to advance. It has also been proposed that there is a need for maternal Fg for proper anchoring of the cytotrophoblasts after they invade the endometrium. This entire process may additionally involve fibronectin (FN) and FXIIIa.21 To address issues involved in the maintenance of pregnancy of afibrinogenemic females, and the role of Fg in this process, we have developed a murine model of congenital afibrinogenemia by targeted deletion of the entire FGG gene that results in FG-/- state.22 Embryos removed from timed matings of FG-/- females were then analyzed for localization of these specific proteins during implantation and development of pregnancy, up to the time of formation of the chorioallantoic placenta, at which point spontaneous miscarriage occurs. This article provides a report of these studies.
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Materials and Methods |
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Mice with a targeted deletion of the entire coding sequence of the FGG chain were used.22 The strain was the F1 offspring of the mating of 129 SvJ and C57BL/6J mice. Either wild-type (WT) or FG-/-male mice were bred with female mice heterozygous for the FGG deletion (FG+/-) to obtain all combinations of genotypes, which were identified as previously described.22
For timed matings, Gtrosa-26 male mice that express ß-galactosidase (ß-Gal), purchased from Jackson Laboratories (Bar Harbor, ME), were paired with either normal WT or FG-/- females. This mating strategy allowed the distinction of embryonic from maternal tissue in early placentas.
Timed matings of mice, which were paired in the evenings, were used to obtain embryos at various gestational ages. The noontime after the morning of the observation of the vaginal plug was designated as embryonic day (E) 0.5. At the desired gestational stages, entire uteri were removed surgically from the mouse and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) (10 mmol/L phosphate/140 mmol/L NaCl, pH 7.2) for 2 hours. The tissue was then processed for histological examinations. After fixation, individual embryos, along with the uterine walls, were excised.
Housing and surgical procedures involving experimental animals were approved by the Institutional Animal Care and Research Advisory Committee of the University of Notre Dame.
Histochemistry and Immunohistochemistry
Preparation of Embryos
The embryos were embedded in paraffin and sectioned at a thickness of 4 µm. Representative slides were routinely stained with hematoxylin II and eosin Y (H&E) (Richard Allen Scientific, Kalamazoo, MI) for morphological analysis. For immunoperoxidase techniques, all slides were incubated in Peroxo-block (Zymed Laboratories, San Francisco, CA) to inhibit endogenous peroxidase activity, and with avidin- and biotin-blocking solutions (Zymed Laboratories) to eliminate endogenous avidin- or biotin-like substances.
In some cases, whole mount embryos were initially stained for ß-Gal expression before sectioning. When this was desired, entire uteri were cross-sectioned between embryos. The embryos were then transferred to 70% ethanol before processing. The tissues were rinsed several times in X-Gal rinse buffer (0.02% Nonidet P-40/0.01% sodium deoxycholate/2 mmol/L MgCl2, in PBS), and stained overnight in X-Gal staining solution (5 mmol/L potassium ferrocyanide/5 mmol/L potassium ferricyanide/1 mg/ml X-Gal, from a 40-mg/ml stock in dimethylformamide) (Research Products International, Mount Prospect, IL). After several rinses in PBS, the tissues were dehydrated in graded alcohols, cleared in xylene, infiltrated and embedded in paraffin at 60°C, and finally serially sectioned at 4 µm. The slides were then used for other histochemical and immunohistochemical analyses.
Anti-Von Willebrand Factor (VWF) and Anti-Laminin Double-Labeling
Slides were incubated with trypsin for antigen unmasking, then blocked with normal rabbit serum and placed in DAKO-EPOS rabbit-anti-human-VWF antibody conjugated to horseradish peroxidase (HRP) (DAKO, Carpinteria, CA). The chromogen 3,3'-diaminobenzidine was used for color development. These same sections were then washed, blocked in normal swine serum, and incubated with a rabbit anti-rat laminin polyclonal antibody (DAKO) solution. This was followed by addition of a swine anti-rabbit biotin F(ab)2-IgG solution (DAKO), and then by a streptavidin-HRP conjugate (Biogenex, San Ramon, CA). SG chromogen (Vector Laboratories, Burlingame, CA) was applied for color development. Sections were counterstained with Nuclear Fast Red (Vector Laboratories).
Anti-Fibronectin (FN) Immunostaining
Heat-induced antigen retrieval was accomplished with 0.1 mol/L citrate buffer, pH 6.0. Sections were then blocked with normal swine serum, followed by incubation with a rabbit anti-human FN polyclonal antibody (Labvision, Fremont, CA). Next, swine-anti-rabbit biotin IgG-F(ab)2 complex (DAKO) and streptavidin-HRP conjugate solutions were added in succession. SG chromogen, along with a Nuclear Fast Red counterstain, or, 3-amino-9-ethylcarbazole (AEC), followed with a hematoxylin counterstain (Biomeda, Foster City, CA), were used.
Anti-Fibrin(ogen) Immunostaining
After heat-induced antigen retrieval, as above, a preimmune rabbit serum block was applied. The sections were then incubated in a goat anti-mouse fibrin(ogen) antibody (Nordic Immunology, Tillburg, The Netherlands), followed by rabbit anti-goat IgG in 10% normal mouse serum. A complex of HRP, conjugated to a goat anti-HRP-IgG solution (DAKO) was added. The slides were developed with AEC and followed with a hematoxylin counterstain.
Anti-FXIII Immunostaining
Heat-induced antigen retrieval was performed on representative slides, as above, followed by a block with normal swine serum. Sections were incubated with a rabbit anti-human FXIII subunit A-polyclonal antibody (Biogenex), followed in succession by biotinylated swine anti-rabbit IgG-F(ab)2 and streptavidin-HRP solutions. AEC chromogen was used for color development. Sections were counterstained with hematoxylin.
Rescue of Pregnancies of FG-/- Female Mice by Fg Supplementation
FG-/- males were paired with FG-/- females and times of vaginal plugs noted. At various times after plug formation, 10 mg of human Fg (Enzyme Research Laboratories, South Bend, IN), dissolved in 0.25 ml of isotonic saline, was administered into the tail veins of the pregnant dams. A volume of 0.1 ml of blood was periodically removed from the eyes of the pregnant females in heparinized capillary tubes, and used for determinations of plasma levels of Fg, using the Fibri-Prest Automate Kit (Diagnostica Stago, Asnieres-Sur-Seine, France).
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Histochemical and Immunohistochemical Characterization of Pregnancy in FG-/- Mice
The development of the murine placenta in WT and FG-/- females was studied in mice at various stages of pregnancy. Several strategies were used in the timed matings of FG-/- females with both WT and FG-/- males. FG-/- males breed normally and were used in most cases so that none of embryos would contain Fg. WT males were used for special reasons. In these cases, male mice were used that produced ß-Gal, the presence of which could then be detected with the chromogenic stain, X-Gal. This strategy was useful in cases in which it became necessary to clearly distinguish maternal from embryonic tissue and cells, particularly in later stages of the abnormal events that occurred in uteri with the pregnant FG-/- females.
Embryonic Day 6
Figure 1
illustrates H&E staining of E6.0 embryos from these animals. The
embryos of both WT and FG-/-
mice appear to be at similar stages of development, with giant
trophoblasts invading the epithelium and a well-formed ectoplacental
cone present (Figure 1, A and B)
. It is already evident that excessive
maternal blood is present in the uterus, in the vicinity of the
implantation site, in the FG-/- mouse.
The ovaries of both animals examined at this stage exhibit normal
architecture and displayed stroma and oocyte development that did not
appear to differ in FG-/- and
WT mice (Figure 1, C and D)
.
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Figure 2
illustrates the results of
a variety of stains that characterize the implantation site of the E7
embryo. The H&E stains show that the ectoplacental cone in the
WT mouse exhibits signs of the presence of blood (Figure 2A)
, likely a normal event because of rupturing of capillaries as the
cone interdigitates between cells of the decidua and endometrium. This
bleeding, which is more pronounced in the
FG-/- mouse, likely cannot be
effectively controlled in the absence of Fg (Figure 2B)
. Figure 2C
shows the presence of sites of anti-fibrin(ogen) staining that
originate from maternal vessels, which are beginning to form at E7.
This protein, which serves to limit this bleeding, is present in the
ectoplacental cone, in the surrounding decidua and endometrium, and in
embryonic distal regions. The presence of Fg and/or fibrin is also
noted at the implantation site (Figure 2C)
. Obviously, no
anti-fibrin(ogen) staining is observed in the
FG-/- control animal (Figure 2D)
.
Intense anti-FN immunostaining is found in both abembryonic and
distal embryonic locations, as well as at the implantation sites in
both genotypes (Figure 2, E and F)
. Profuse interstitial staining for
FN is seen in the nondecidualized endometrium, with more focal areas in
the decidua. Embryonic endoderm and mesoderm membranes also show the
presence of FN. In both genotypes, the VWF immunostains (Figure 2, G and H)
show profuse endothelial cell-lined vessels in the ectoplacental
cone and implantation site, likely the result of maternal-fetal
communication. The distal areas of the embryo also show strong
vascularity. Both genotypes also display similar anti-laminin
immunostaining throughout the decidua and endometrium, as well as in
the embryo itself. Figure 2, I and J
, shows that FXIII is co-localized
at the implantation site with Fg and FN in the WT animal,
and with FN in the FG-/- mouse, making
this protein available for cross-linking reactions at this site.
Similarly, FXIII is also co-localized with these same proteins in the
distal embryonic region.
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The E8.0 embryos presented in Figure 3
, derived from a mating of a Gtrosa male
with a FG-/- female, were first whole-mount
stained for ß-Gal expression. Additional H&E staining of the E8.0
WT placenta (Figure 3A)
shows a defined implantation of the
ectoplacental cone. The fact that embryonic material can be
distinguished from maternal tissue in this complex biological
environment using this novel approach, because of ß-Gal staining
solely by embryonic tissue, greatly assists in deriving conclusions
regarding the development of the embryonic and maternal placentas.
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. The
implantation site in the WT animal stained positively for
Fg/fibrin (Figure 3C)
, FN (Figure 3E)
, and FXIII (Figure 3I)
. Although
the fibrinoid layer is formed in the
FG-/- E8.0 placenta, it obviously does
not contain Fg/fibrin (Figure 3D)
. However, FN (Figure 3F)
and FXIII
(Figure 3J)
are present in this altered fibrinoid layer. In both cases,
anti-VWF immunostaining reveals a heavily vascularized implantation
region, with endothelium-derived vessels and laminin present throughout
the decidua and embryo (Figure 3, G and H)
. Embryonic Day 9.0
At E9, continued development of the labyrinthine layer occurs in
the WT placenta, as seen in all panels of Figure 4
, and the placenta appears smaller in
the FG-/- mouse. A distinct fibrinoid
layer is present in the WT mouse, surrounding the
trophoblast giant cells, and contains Fg/fibrin (Figure 4C)
, FN (Figure 4E)
, and FXIII (Figure 4I)
. Similarly the fibrinoid layer formed in the
FG-/- placenta contains FN (Figure 4F)
and FXIII (Figure 4J)
, but not Fg/fibrin (Figure 4D)
. Signs of
separation of the fibrinoid layer are appearing in the
FG-/- mouse. As is the case at the
earlier gestational ages, the implantation site in both genotypes is
heavily vascularized (Figure 4, G and H)
with the vessels containing
blood and associated blood seepage (Figure 4, A and B)
.
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At E9.75, H&E staining shows that the
WT chorioallantoic placenta, now fully formed, possesses a
clearly defined labyrinth, with an abundance of large blood-filled
sinuses where maternal/fetal exchange is occurring, and a
spongiotrophoblast layer, separated from the decidua by giant
trophoblasts (Figure 5A)
. Nucleated fetal
red blood cells within the labyrinth and nonnucleated maternal red
blood cells in the spongiotrophoblast layer and decidua are clearly
evident in Figure 5A
. Figure 5B
shows the presence of a Fg/fibrin-rich
fibrinoid layer, with Fg/fibrin surrounding giant cells at the
implantation site. The decidua is also heavily stained for interstitial
Fg/fibrin. The fibrinoid layer also contains FN (Figure 5C)
. Anti-VWF
staining occurs mainly in the endothelium-derived vessels of the
decidua and fibrinoid layer, but not in the labyrinth layer, suggesting
that these latter sinuses are not endothelium-derived (Figure 5D)
.
Laminin is present throughout the entire placenta (Figure 5D)
. Lastly,
FXIII is also co-localized at the fibrinoid layer with Fg/fibrin and FN
in the WT E9.75 placenta (Figure 5E)
.
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, with a variety of
histochemical and immunohistochemical stainings. In the case of the
WT female (Figure 6A)
, a normally developed fetus is
present, with the yolk sac-containing embryo-vitelline vessels and
blood lakes lining the cylinder. The implantation site is also evident.
On the other hand, Figure 6B
shows the occurrence of a massive bleeding
event in the egg cylinder of a fetus from a
FG-/- female. In addition, a much
smaller yolk sac and fetus is noted. Examination of the thickened upper
wall of the yolk sac cylinder shows the presence of spongiotrophoblasts
that most likely originated from a detachment at the implantation site.
With use of the Gtrosa male, and the subsequent ability to identify the
locations of embryonic tissue, speculation on the position of the
original implantation site of this embryo could be made. As can be seen
in Figure 6C
, embryonic cells in the decidual region of the egg
cylinder (top) can be observed (black arrow), and a magnified view of
this area (Figure 6D)
shows the existence of embryonic giant cells
within this region (red arrows). This indicates that detachment of the
yolk sac likely occurred from this placental region. Similarly, this
latter site also contained overexpression of FN (Figure 6E
, black
arrows), along with giant cells (Figure 6F
, red arrows). VWF (Figure 6
,
G and H, black arrow), is also present at that site, probably
originating from both endothelium-derived vasculature and from
platelets present at the site of bleeding. FXIII, along with giant
trophoblasts, are also evident at this implantation region,
co-localized with FN (Figure 6
, I and J).
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Using the Fg dosage schedule described in Figure 7
, we found that
none of the treated female FG-/- mice
showed obvious genital bleeding during pregnancy. These mice delivered
normally and appropriately managed their offspring. Matings
(n = 5) of FG-/-
males with FG-/- females provided
full-term FG-/- litters, ranging from 6
to 11 FG-/- offspring, with both genders
represented in nearly equal numbers. Only two of the pups presented
fatal neonatal bleeding. The remainder of the pups survived weaning (3
weeks postnatal), and beyond. After weaning the pups, plasma levels of
the mothers (n = 4) and representative weanlings
(n = 6) were obtained and did not show
detectable levels of plasma Fg. One of the
FG-/- mothers experienced three
pregnancies after weaning each litter, and, with the same course of Fg
treatment, experienced delivery of a total of 18
FG-/- pups.
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183
± 32 mg/dl (Figure 7A)
. The following
day (E6.5), without additional treatment, Fg levels were
77 ± 22 mg/dl (normal levels in WT nonpregnant
female mice are, 185 mg/dl), and the protein was below the level of
detection (20 mg/dl) 3 days after injection (Figure 7A)
. Injections of
10 mg of Fg were subsequently made at E8.5, and every 2 days afterward,
up to E18.5. In all cases, Fg levels of 70 mg/dl to 140 mg/dl were
found on the day after injection, with the lower ranges obtained in
late pregnancy, possibly indicating increased consumption at
times approaching delivery. In all cases, at times of 2 days
after Fg treatment, very low levels of Fg (20 mg/dl to 30 mg/dl) were
found in plasma of the pregnant FG-/-
mice.
A time course of Fg determinations on WT
(n = 8) and FG+/-
(n = 14 to 16) pregnant females, without any
additional Fg administration, demonstrated that Fg levels rose
slightly from their normal values after E8.5 during the course of
pregnancy (Figure 7B)
, whereas the value for
FG+/- pregnant females remained somewhat
constant. In this study, we find that initial levels of Fg as low as 90
mg/dl could support normal pregnancy without intervention (Figure 7B)
.
This is also the case in the few humans that have been studied in this
regard.24,25
Of course, it is possible that even lower
concentrations of this protein would suffice.
As a result of this information, a number of Fg administration protocols were attempted, and we found that a minimal dosage schedule of 10 mg of Fg at E8.5 and E17.5 would result in a successful vaginal deliveries in FG-/- females.
Characteristics of the E9.75 Chorioallantoic Placenta of a FG-/- Pregnant Mouse Administered Fg
Figure 8
presents representations of
the embryos and implantation sites of an E.9.75
FG-/- pregnant female (mated with a
Gtrosa male) who had been administered Fg. The final dose of Fg was
provided at E8.5, and the plasma level of Fg of this mouse at sacrifice
(E9.75) was 102 mg/dl (see Figure 7A
). Photographs of the embryo egg
cylinders in utero are shown in Figure 8
for E9.75
FG-/- (Figure 8A)
and WT
(Figure 8B)
mice, as well as a FG-/-
gravida administered Fg (Figure 8C)
. The severe intrauterine bleeding
observed in the FG-/- animal (Figure 8A)
was not observed in the rescued FG-/-
mouse (Figure 8C)
, which had the same gross features as the
WT mouse uterus at this same gestational stage (Figure 8B)
.
The microscopic features of the implantation of one of the embryos in
panel C also appear to be normal. Specifically, H&E stainings of an
embryo attached to the uterus of panel C (Figure 8D)
show well-defined
labyrinth and spongiotrophoblast layers, one or both containing large
sinuses, lacunae, and arteries, with fetal and maternal blood present.
The spongiotrophoblast layer is clearly separated from the decidua by a
layer of fetal trophoblast giant cells. A clear distinction of
maternal- and embryonic-derived tissue can be made from the ß-Gal
stainings (blue) of serial sections of an embryo of panel C, shown in
panels E, F, H, and I (Figure 8)
. The only possible source of the Fg
immunostaining present throughout the maternal decidua (Figure 8E)
is
from that injected into the plasma of the mother. This maternal Fg is
also observed in random vessels of the spongiotrophoblast layer (Figure 8E)
, showing that vascular communication between the mother and fetus
has been established. A heavier layer of Fg/fibrin, along with FN
(Figure 8F)
and FXIII (Figure 8H)
, appears at the interfacial
implantation site that composes the fibrinoid layer anchoring the
trophoblast giant cells. FN and FXIII also appear similarly within
spongiotrophoblast-located vessels, indicating these two proteins may
have interacted in the circulation. The strong immunostaining for FN in
interstitial regions of the labyrinth, chorionic plate, yolk sac,
decidua, and the fact that Fg and FXIII are not co-localized with
interstitial FN in the embryonic labyrinth and chorionic plate,
suggests that this form of FN may be fetally derived, whereas Fg/fibrin
and FXIII at the fibrinoid layer may need to originate from maternal
circulation. The labyrinth, spongiotrophoblast, yolk sac, and decidua
of this same placenta are heavily vascularized with embryonic and
maternal endothelium-derived cells, as evidenced by the positive VWF
immunostaining of Figure 8G
. Finally, a x40 view of the ß-Gal/FN
doubly-stained embryo attached to the uterine wall (a lower resolution
view of Figure 8F
) is presented in Figure 8I
. Contrasting this rescued
pregnancy at E.9.75 with the unrescued pregnancy of Figure 6B
, shows
the clear benefits that result from Fg administration. The bleeding and
placental detachment observed in the unrescued animal are not observed
in the mouse treated with Fg, and the rescued mouse seems to maintain
the same egg cylinder-uterine contact as is present in the same stage
WT mouse (Figure 6A)
.
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Discussion |
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This murine model of congenital afibrinogenemia thus seems to be very suitable to investigate mechanistic features of spontaneous miscarriage in humans with this genetic defect, and, even more important, to define the role of Fg in supporting a successful outcome of pregnancy.
The histochemical and immunohistochemical assessment of embryonic, placental, and decidual tissues in timed pregnancies allowed precise determination of the course of fetal and maternal demise in Fg-deficient pregnant mice. The first subtle event, visualized in the uteri of pregnant animals on E6, is an exacerbation of the normal bleeding that occurs in regions surrounding the ectoplacental cone. Lack of Fg allows for continued bleeding, leading to lacunae filled with blood at E8 to E9 of the gestational period. Blood seepage from the sinuses of the developing labyrinths of the placenta are more extensive in FG-/- mice. The bleeding continues throughout the placental development, so that by E9 large areas of bleeding initiate the disruption of the placenta and yolk sac from the endometrium, causing complete placental abruption on E9.75. As a result, the pregnant mice die from profuse bleeding. The Fg deficiency does not alter embryonic development, but formation of the placenta and yolk sac is significantly compromised. Both the placentas and yolk sacs in afibrinogenemic animals are smaller than those found in corresponding WT animals. A most important site of Fg/fibrin deposition is the fibrinoid layer that starts to peel from the decidua in the absence of Fg/fibrin. This event is associated with disruption of sinusoids of the labyrinths. This leads to severe hemorrhage along the planes of placental-decidual attachment. This adherence is enforced by other proteins including laminin, FN, and FXIII, which co-localize with Fg in WT mice. A lack of Fg does not change this typical distribution pattern, but its presence is required to give sufficient stability to the placental-decidual binding.
Platelet adhesion and aggregation at the site of vascular compromise
are the first key events that serve to arrest
bleeding.28,29
By virtue of bridging GPIIb/IIIa on
adjacent platelets, Fg leads to platelet aggregation, which ultimately
forms a platelet thrombus. VWF contributes to the activation of the
integrin, IIbß3, on the platelets through an inside-out signaling
pathway.30
The lack of Fg does not seem to alter the
distribution of VWF on the extensive vascular network, which develops
both within the embryo and placenta as well as the surrounding decidua.
In the absence of Fg, it is thought that VWF contributes to platelet
adhesion and aggregation.31
Even in the absence of both Fg
and VWF, platelet thrombi can form in mice that lack both VWF and Fg,
although adhesion and aggregation is delayed.31
This
finding shows that the abnormal bleeding in the uteri of
FG-/- mice is not because of a
compromise in platelet aggregation or formation of platelet thrombus.
Rather, the profuse bleeding in these animals is likely caused by the
coagulation defect that results from lack of Fg. This protein is
thought to be critical to coagulation because the insoluble fibrin
matrix stabilizes the thrombus.2
In
FG-/- mice, the thrombi that are formed
are quite unstable and lead to vessel embolization and
occlusion.31
The weakest point of thrombi in
FG-/- mice seems to lie at the
thrombus-vessel wall interface.31
This event is likely to
play a role in exacerbation of the placental abruption in the
FG-/- mice. The ability to easily form
thrombi is thought to be shared by humans with a Fg deficiency, because
blood from these patients also forms thrombi on subendothelium in
perfusion chambers.32
This thrombotic tendency in
afibrinogenemic patients seems to be an additional factor in the
development of complications of pregnancy.
Hypercoagulable responses resulting from a number of mutations that lead to thrombosis of the uteroplacental vessels, spiral artery vasculopathies, and impaired placental perfusion are all associated with abnormal development of the fetus, pregnancy losses, and obstetrical complications. Notable among these are genetic predispositions toward thrombophilia, such as the FV-Leiden mutation,33 leading to thrombosis because of anti-coagulant protein C resistance; the homozygous methylenetetrahydrofolate mutation,33 resulting in hyperhomocysteinemia and a consequent thrombotic condition; the prothrombin G20210A mutation,34 resulting in elevated plasma prothrombin concentrations and the associated risks of coagulopathies; and the vascular thrombotic dispositions of an anti-phospholipid syndrome.35 Many of the early associations between thrombophilia and pregnancy loss were studied through retrospective analyses with small numbers of patients, thus tempering the conclusion as to whether causal relationships exist between complications of pregnancy and thrombosis. However, more recent case-controlled prospective studies support the view that genetic markers of thrombophilia may predispose women to a number of severe complications during pregnancy.34,36,37
In the E8 mouse, the chorioallantoic placenta is developing, and abnormalities appear in the pregnancy of the FG-/- female. The spongiotrophoblast layer is beginning to separate and shows signs of hypoplasia. This leads to further separation, which is evident in the E9.0 embryos. In this case, a smaller placenta in the FG-/- mouse is seen, with a spongiotrophoblast layer that has begun to detach from the fibrinoid layer. At E9.75, terminal events in the pregnancy have occurred in the FG-/- mouse. Massive bleeding is seen in the egg cylinder cavity and an apparently forceful detachment of the spongiotrophoblast layer from the fibrinoid layer is observed, with portions of the giant cell-associated fibrinoid layer remaining at the implantation site and cells from the spongiotrophoblast layer present in the detached yolk sac. These events are sufficient to terminate the pregnancy, in that clear placental abruption has occurred.
The results of this investigation reveal very important elements of pregnancies in normal mice and divulge a possible encompassing role for Fg in this process. It seems as though the course of pregnancy and development of the placenta in mice possess some similar features to that of humans. These include formation of a fibrinoid layer that contains Fg, FN, laminin, and FXIII. Fg is a critical component of the fibrinoid layer and stable attachment of the spongiotrophoblast to this layer does not occur in afibrinogenemic mothers. Placental abruption in these animals thus involves separation of the fibrinoid layer from the spongiotrophoblast layer, and this is another novel finding that resulted from this study.
Thus, the role of Fg in supporting a successful pregnancy certainly
involves its function as a hemostatic agent, by assuring that normal
bleeding during development of immature vessels is effectively
controlled. In addition, Fg is required in a less appreciated role, by
assisting in formation of a stable fibrinoid layer that is essential
for anchoring the spongiotrophoblast layer to the decidua. This process
may involve the interaction of stromal FN, itself bound to collagen at
the surface of decidual cells at the implantation site,38
with its integrin receptors, eg,
5ß1, on invading
spongiotrophoblast cells.39
Stromal Fg, supplied by the
vasculature of the mother, may also bind to FN and/or the
5ß1
integrin,40
stabilizing the fibrinoid layer. FXIII, which
is supplied by maternal plasma and by macrophage-derived mononuclear
cells in the decidua, is capable of further stabilizing the fibrinoid
layer via crosslinking of FN to Fg41,42
and of FN to
decidua-derived collagen.43
In conclusion, the loss of embryo in afibrinogenemic mice is because of an abortive process that is initiated as an exacerbation of the hemorrhage that normally occurs around E6 during the critical stage of maternal-fetal vascular development and at a time when the embryo is invading the maternal decidua. This event gives rise to a robust bleeding that does not cease during days 8 and 9 of gestation. Ultimately, the profuse bleeding causes extensive placental disruption that results in the loss of embryo and death of the mother. This entire course of events can be normalized by elevation of the maternal plasma Fg levels and successful vaginal deliveries experienced. This shows that embryonically derived Fg is not required for a completion of a successful pregnancy and that all Fg needed in this regard is derived from the mother.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health (grants HL-13423 to F. J. C. and HL-63682 to V. A. P.), a grant from the W. M. Keck Foundation (to F. J. C.), by the Kleiderer-Pezold Family Endowed Professorship (to F. J. C.), by a grant from the Leda Sears Medical Trust, as well as through collaboration with the Walther Cancer Foundation.
Current Address of T. K.: Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
Accepted for publication December 7, 2001.
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gene account for the majority of cases of congenital afibrinogenemia. Blood 2000, 96:149-152This article has been cited by other articles:
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