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Blood First Edition Paper, prepublished online January 16, 2013; DOI 10.1182/blood-2012-08-448209
Heparin rescues factor V Leiden-associated placental failure independent of anticoagulation
in a murine high-risk pregnancy model
Running title: Heparin in thrombophilia-associated pregnancy loss
Jianzhong An1, Magarya Waitara1, Michelle Bordas1, Vidhyalakshmi Arumugam1, Raymond G
Hoffmann2, Brian G Petrich3, Uma Sinha4, Paula North1,5,6 and Rashmi Sood1,5
1
Division of Pediatric Pathology, Department of Pathology, Medical College of Wisconsin,
Milwaukee, WI, USA;
2
Quantitative Health Sciences, Department of Pediatrics, Medical College of Wisconsin,
Milwaukee, WI, USA;
3
Department of Medicine, University of California, San Diego, CA, USA;
4
Portola Pharmaceuticals Inc., South San Francisco, CA, USA;
5
Children’s Research Institute, Children’s Hospital and Health System, Milwaukee, WI, USA;
6
Children’s Hospital of Wisconsin, Children’s Hospital and Health System, Milwaukee, WI, USA
Corresponding Author:
Rashmi Sood
Medical College of Wisconsin
8701 Watertown Plank Road
Milwaukee WI 53226
Fax:
414 955 6411
Phone: 414 955 2451
E-mail: [email protected]
Page 1 of 24
Copyright © 2013 American Society of Hematology
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KEY POINTS:
•
Heparin may have beneficial effects on placental health beyond anticoagulation.
•
Thrombin receptor activation on maternal platelets is implicated in placental
developmental failure independent of thrombosis.
ABSTRACT:
Low Molecular Weight Heparin (LMWH) is being tested as an experimental drug for
improving pregnancy outcome in women with inherited thrombophilia and placenta-mediated
pregnancy complications, such as recurrent pregnancy loss. The role of thrombotic processes in
these disorders remains unproven and the issue of antithrombotic prophylaxis is intensely
debated. Using a murine model of factor V Leiden-associated placental failure, we show that
treatment of the mother with LMWH allows placental development to proceed and affords
significant protection from fetal loss. Nonetheless, the therapeutic effect of LMWH is not
replicated by anticoagulation; Fondaparinux and a direct Xa inhibitor, C921-78, achieve
anticoagulation similar to LMWH but produce little or no improvement in pregnancy outcome.
Genetic attenuation of maternal platelet aggregation is similarly ineffective. In contrast, even a
partial loss of thrombin sensitivity of maternal platelets protects pregnancies. Neonates born from
these pregnancies are growth retarded, suggesting that placental function is only partially
restored. The placentae are smaller, but do not reveal any evidence of thrombosis. Our data
demonstrates an anticoagulation-independent role of LMWH in protecting pregnancies and
provides evidence against the involvement of thrombotic processes in thrombophilia-associated
placental failure. Importantly, thrombin-mediated maternal platelet activation remains central in
the mechanism of placental failure.
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INTRODUCTION:
Placental abnormalities have serious consequences for mothers and their babies.
Consequences include fetal growth restriction, early and late fetal death, and maternal
hypertensive disorders, such as preeclampsia. These are disorders of multi-factorial origin that
affect more than 5% of all pregnancies, and are a major cause of preterm deliveries, small birth
weight, and maternal/neonatal morbidity and mortality1. Small for gestation age infants are also at
an increased risk of developmental problems and adult onset cardiovascular and metabolic
disorders2.
Maternal inherited thrombophilia is a risk factor for placenta-mediated pregnancy
complications3. The extent of the risk varies with ethnicity, the type of pregnancy complication
and the thrombophilia mutation under investigation4. The mechanism that connects maternal
thrombophilia with adverse pregnancy outcomes is unknown, and suspected to involve
thrombotic occlusion of the utero-placental circulation. It is hypothesized that the low pressure
placental flow is susceptible to thrombotic complications, much like maternal venous circulation.
Accordingly, interventions aimed at limiting placental thrombosis, such as treatment with Low
Molecular Weight Heparin (LMWH), are being tested to prevent placental dysfunction and the
related sequelae in at-risk pregnancies5-7. While these studies suggest that LMWH may be
beneficial for a subset of women with heritable thrombophilia and recurrent pregnancy loss
(RPL), the risk-to-benefit balance of antithrombotic prophylaxis during pregnancy is a subject of
intense ongoing debate8-11. In addition to study limitations and related methodological concerns,
this is largely due to the uncertain role of thrombotic processes in placental disease12-14 and a lack
of established criteria for identifying high-risk pregnancies that may benefit from the use of
LMWH. Given the multi-factorial and heterogeneous nature of thrombophilia-associated
pregnancy disorders, these issues have proven to be complex, and difficult to resolve based on
epidemiological and clinical data alone15,16. In the last few years, new non-anticoagulant roles of
LMWH have emerged, some of which are directly related to trophoblast function17. Thus, any
beneficial effects of LMWH treatment may not necessarily reflect a causal thrombotic link
between thrombophilia mutations and pregnancy loss.
The mouse is a well studied experimental model system that shares chorioallantoic
placentation with humans; this occurs around the end of first trimester in humans and corresponds
to 9 days post coitum (dpc) in mice18. Both species form a hemochorial placenta where maternal
cells are eroded and zygote-derived trophoblast cells become directly exposed to maternal blood.
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At this feto-maternal interface, hemostasis is coordinately regulated by maternal and fetal
factors19. We have previously provided proof-of-concept evidence that fetal prothrombotic gene
mutations expressed on trophoblast cells are risk modifiers of adverse pregnancy outcome in
women with thrombophilia20. We showed that embryos homozygous for a hypomorphic
Glu387Pro mutation in thrombomodulin (designated as ThbdPro/Pro) die in utero in pregnancies
carried by mothers homozygous for the factor V Leiden mutation (designated as FVQ/Q), but not
in mothers with normal alleles for factor V (FV+/+). In pregnancies of FVQ/Q mothers, placentae of
ThbdPro/Pro embryos are growth retarded and most do not complete chorioallantoic morphogenesis,
resulting in placental insufficiency and fetal death. If the mother is treated with platelet-depleting
antibodies, the chorioallantoic placenta is formed and embryonic development proceeds to term.
Genetic absence of Par4 in the mother (embryo is Par4+/-) also results in the birth of normal
appearing and live ThbdPro/Pro pups20. In the current study we utilize three experimental
approaches to evaluate the role of thrombin and thrombotic processes in this robust model of
thrombophilia-associated fetal loss. In the first approach, we treat the mother with LMWH and
other pharmacological agents that inhibit thrombin activity or generation to examine their effect
on pregnancy outcome. In the second approach, we attenuate thrombin signaling by making the
mother deficient in Protease Activated Receptor 3 (Par3) through genetic breeding experiments.
Thrombin triggers activation of murine platelets via two receptors, Par3 and Par4. Unlike Par4,
thrombin is the only known agonist for Par3. Genetic absence of Par3 in mice increases the
threshold of thrombin-mediated platelet activation21. In the third approach, we utilized genetic
means to inhibit platelet aggregation in the mother by using mutant animals that lack the ability to
activate αIIbβ3 integrin by inside-out signaling22. This experiment is based on the rationale that
Par cleavage on platelets potently activates αIIbβ3 integrin by inside-out signaling leading to
platelet aggregation. The two genetic approaches allow us to investigate the role of thrombinmediated platelet activation and aggregation in the mechanism of placental failure and fetal
demise.
MATERIALS AND METHODS:
Mice: Animal experiments were conducted following standards and procedures approved by the
Animal care and Use Committee of the Medical College of Wisconsin. Thbd Pro (provided by H
Weiler, Blood Center of Wisconsin, Milwaukee, WI), FV Leiden (provided by D. Ginsburg,
University of Michigan, Ann Arbor, MI), Par3-/- (purchased from Jackson Laboratories) Par4-/(provided by S. Coughlin, University of California, San Francisco, CA) and Beta3LA/LA mice have
been described20,22. These were maintained on a C57BL/6 genetic background and bred to each
Page 4 of 24
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other to generate double and triple mutant strains. Correct combinations were identified based on
tail biopsy and PCR-based genotyping.
Analysis of pregnancies: The stage of pregnancies was assessed from days post coitum (dpc),
assuming midday of plug as 0.5 dpc. Embryos were dissected in Phosphate buffered saline and
photographed under a Nikon SMZ-1000 Zoom Stereo Microscope (Nikon, Melville, NY)
equipped with a high resolution 5 Megapixel digital camera and NIS Elements F 3.0 imaging
software. A small sample from each embryo was used for PCR-based genotyping. Nearly all dead
embryos were found at an advanced stage of resorption and were not genotyped. Live embryos
were identified based on the presence of heart beats and movement. Using ImageJ software
(version 1.42q; National Institutes of Health), the head to tail length and lengthwise crosssectional area was measured from digital images of embryos photographed on their sides. The
cross-sectional area of utero-placental discs photographed with maternal side facing the camera
(Supplemental Figure 1) was similarly measured. Formalin fixed, paraffin-embedded placentae
were sectioned and stained with hematoxylin-eosin and independently examined by two
pathologists. Placental sections were photographed using Nanozoomer HT slide scanner equipped
with the NDP view imaging software (Hamamatsu, Japan).
Anticoagulation treatments: Anticoagulant drugs were administered through subcutaneous
micro-osmotic pumps (Model 1002, Durect Corporation, Cupertino, CA) with a release rate of
0.25 μl/hour. These were filled with 12.5 or 25 μg/μl hirudin (lepirudin, brand name Refludan,
Berlex, Wayne, NJ), 100 μg/μl LMWH (enoxaparin, brand name Lovenox, Aventis
Pharmaceuticals Inc, Bridgewater, NJ), 12.5 μg/μl Fondaparinux (brand name Arixtra,
GlaxoSmithKline, Research Triangle Park, NC) or 100 μg/μl C921-78 (Portola Pharmaceuticals,
Inc., South San Francisco, CA)23 prior to implantation. Treatment was initiated at 5.5 dpc and
continued till the day of analysis. Anticoagulation and plasma levels of LMWH, Fondaparinux
and C921-78 were measured in terms of Xa inhibitory activity with Coatest heparin
(Chromogenix, Lexington, MA). Enoxaparin standards were included in these assays to compare
all anticoagulants in terms of enoxaparin equivalent activity. Anticoagulation by hirudin was
assessed by PTT assays done using ST4 instrument and STA PTT reagent (Diagnostica Stago,
Parsippany, NJ).
Statistical Analysis: P<0.05 was used to establish significance for all experiments. Chi-squaredGOF (goodness of fit) test was used to determine deviation from expected Mendelian proportions.
Page 5 of 24
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Exact binomial 95% Confidence Intervals (CI) were computed where appropriate. Chi-squared
test of independence was used for comparing two treatments or treated versus untreated groups.
The exact binomial for goodness of fit or the Fisher’s exact test of independence were used in
experiments where the expected number in any cell was lower than 5. Student’s t-test (two-tailed
with unequal variance) was used for comparing embryonic and placental sizes.
RESULTS
LMWH rescues pregnancies in a murine model of thrombophilia-associated fetal loss:
ThbdPro/Pro embryos conceived from crosses between ThbdPro/Pro males and FVQQThbdPro/+ females
are growth retarded by 9.5 days post coitum (dpc)20 and all or most are dead and resorbed by 12.5
dpc (Table 1, row 1; P=0.000003, chi-squared-GOF; 95% CI of 0 to 15.4%). We treated pregnant
FVQQThbdPro/+ females with LMWH, and examined its ability to rescue ThbdPro/Pro embryos from
intrauterine death. Treatment with LMWH resulted in marked anticoagulation in maternal
circulation (measured at 0.3 to 0.5 IU anti factor Xa activity per ml plasma) and significantly
improved the yield of live ThbdPro/Pro fetuses examined at 16.5 dpc (Table 1, row 2; P=0.0006
compared to untreated in row 1, chi-squared test of independence; 95% CI of 23.9% to 57.9%
with treatment). The ThbdPro/Pro embryos obtained after LMWH treatment appeared
morphologically normal (Figure 1 A, B). Treatment significantly reduced the abortion rate from
69.9% to 34% (P=0.00006, chi-squared test of independence; untreated 22 live, 51 aborted versus
treated 35 live, 18 aborted), but did not bring it down to background levels (<5%) observed in
controls pregnancies. Thus, LMWH treatment significantly improved survival of ThbdPro/Pro
embryos and reduced the overall abortion rate in pregnancies of FVQ/Q mothers.
Rescue by LMWH is not replicated by other anticoagulants that inhibit thrombin activity
or generation: Since heparins also possess biological effects unrelated to anticoagulation17, we
examined whether the ability of LMWH to rescue pregnancies is mediated through
anticoagulation. LMWH inhibits FXa and thrombin, in an antithrombin-dependent manner. We
treated pregnant FVQQThbdPro/+ females, mated to ThbdPro/Pro males, with a direct thrombin
inhibitor, lepirudin. Treatment effectively anticoagulated pregnant females (measured by 2 to 3
fold prolongation in PTT) but did not result in live ThbdPro/Pro embryos (Table 1, row 3; 95% CI
of 0 to 16.1%). The abortion rate in lepirudin treated pregnancies was somewhat reduced (56.2%)
but was not significantly different from untreated pregnancies (69.9%) (P=0.13, chi-squared test
of independence; untreated 22 live, 51 aborted versus treated 21 live, 27 aborted). Lepirudin may
cross the placenta. No obvious bleeding was observed in the embryos with lepirudin treatment.
Page 6 of 24
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We next used fondaparinux, a synthetic pentasaccharide composed of minimal antithrombin
binding subunit of heparin that retains the ability to inhibit FXa activity. Treatment of
pregnancies with fondaparinux resulted in anticoagulation comparable to LMWH (measured at
0.5 to 0.7 IU anti factor Xa activity per ml plasma), but did not result in comparable rescue of
ThbdPro/Pro embryos (Table 1, row 4; P=0.002 compared to LMWH treated in row 2, chi-squared
test of independence). Only one of 26 live embryos from fondaparinux treated pregnancies was
determined to be ThbdPro/Pro. Thus, despite equivalent anticoagulation, the ability of LMWH to
ameliorate fetal loss far exceeded that of fondaparinux.
FXa as part of the prothrombinase complex is 300,000 fold more efficient in thrombin
generation than FXa alone24. However, once incorporated into the prothrombinase complex, FXa
is protected from antithrombin-LMWH and antithrombin-fondaparinux mediated inactivation25.
To overcome this limitation we treated pregnant females with C921-78, a synthetic peptide and
direct Xa inhibitor that efficiently inhibits Xa in solution phase and in membrane bound
prothrombinase complexes26. Treatment with C921-78 resulted in high steady state levels of
circulating Xa inhibitory peptide (2.65 ± 0.38 μM/L plasma) and more than 1 IU/ml of plasma Xa
inhibitory activity. C921-78 treated pregnancies produced 4 ThbdPro/Pro embryos out of a total of
40 live (Table 1, row 5; P=0.0000004; chi-squared-GOF; 95% CI of 2.8% to 23.7%). Comparison
with pooled historical data from our lab (0 ThbdPro/Pro/55 live embryos, 95% CI of 0% to 6.5%)
suggests a statistically significant low level rescue with C921-78 (P=0.029; Fisher’s exact test).
However, despite more efficient anticoagulation, C921-78 treatment resulted in significantly
fewer live ThbdPro/Pro embryos as compared to treatment with LMWH (P= 0.002, Table 1 row 5
compared to row 2, chi-squared test of independence). Taken together, these data show that
placental pathology in the murine model of thrombophilia-associated fetal loss persists even after
treatments that significantly reduce thrombin generation or activity. In contrast, LMWH affords
rescue in this model.
Anticoagulation protects surviving littermates and reduces overall abortion rate: We noticed
an abortion rate of ~70% in crosses between ThbdPro/Pro males and FVQ/QThbdPro/+ females (Table
1, row 1), higher than accountable from loss of ThbdPro/Pro embryos alone. Although treatment
with Fondaparinux or C921-78 did not improve specific survival of ThbdPro/Pro embryos, the
overall abortion rates were significantly reduced (44% with Fondaparinux; P=0.004, chi-squared
test of independence; untreated 22 live, 51 aborted versus treated 26 live, 20 aborted) (47% with
C921-78; P=0.004, chi-squared test of independence; untreated 22 live, 51 aborted versus treated
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40 live, 35 aborted). These data suggested that in addition to ThbdPro/Pro embryos some of the
ThbdPro/+ embryos are also aborted in pregnancies of FVQ/Q mothers, and that the death of
ThbdPro/+ embryos is ameliorated with anticoagulation treatment. To compare the survival of
ThbdPro/+ with littermate Thbd+/+ embryos, ThbdPro/+ males were mated to FVQ/QThbdPro/+ females
and pregnancies were analyzed at 15.5 dpc. No live ThbdPro/Pro embryos were observed in this
cross (Table 2, row 1). Although fewer than expected live ThbdPro/+ embryos were observed, this
reduction did not reach statistical significance (Table 2, row 1; P=0.18, chi-squared-GOF, 20
Thbd+/+ and 27 ThbdPro/+). Measurement of the embryonic and placental sizes in these
pregnancies, however, revealed a significant growth retardation of ThbdPro/+ embryos and
placentae, as compared to littermate Thbd+/+ controls (Figure 2 and supplemental Figure 1).
Growth retardation was not observed in pregnancies analyzed at 12.5 dpc (ThbdPro/+ males mated
to FVQ/QThbdPro/+ females; 9 Thbd+/+, 14 ThbdPro/+, 0 ThbdPro/Pro, 15 aborted not genotyped, 4
pregnancies analyzed; measurement data not shown) or in the reverse genetic crosses where
FVQ/QThbdPro/+ males were mated to ThbdPro/+ females (supplemental Figure 2). All three
genotypes, Thbd+/+, ThbdPro/+ and ThbdPro/Pro embryos were obtained in normal Mendelian
proportions from the reverse cross (Table 2, row 2; P=0.42, chi-squared-GOF). These
observations suggested that the growth retardation of ThbdPro/+ embryos, like the fetal demise of
ThbdPro/Pro embryos, only occurs in pregnancies of FVQ/Q mothers and is, therefore, specific to the
mother-fetus pair. However, since placentae of dead or aborted embryos can activate coagulation
in the mother27, we examined the possibility that dead littermates play a role in growth retardation
of ThbdPro/+ placentae in these pregnancies. To address this question we set up a different genetic
cross. FVQ/QThbdPro/+ females were mated to wild type males and growth retardation and survival
of ThbdPro/+ embryos was assessed in comparison to their Thbd+/+ littermates. No ThbdPro/Pro
embryos are expected from this cross. Analysis of these pregnancies revealed normal numbers
and sizes of ThbdPro/+ embryos and placentae as compared to littermate Thbd+/+controls. (Thbd+/+
males mated to FVQ/QThbdPro/+ females; 35 Thbd+/+, 41 ThbdPro/+, 12 aborted not genotyped;
P=0.49, chi-squared-GOF; Supplemental Figure 3). These data demonstrate that ThbdPro/+
embryos are smaller in size and tend to have reduced survival in pregnancies shared with dead
ThbdPro/Pro embryos, but not in comparable pregnancies that do not include ThbdPro/Pro
conceptuses. ThbdPro/+ conceptuses with reduced anticoagulation ability are more susceptible than
Thbd+/+ conceptuses to the adverse effects of “toxic factors” produced by their dead littermates. In
multifetal human pregnancies, retention of a dead fetus is associated with increased platelet
activation, thrombin generation, and a higher risk of disseminated intravascular coagulation in the
mother, and morbidity of the surviving fetus27,28. Our data shows that treatments with
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Fondaparinux or C921-78, although not very effective in protecting ThbdPro/Pro fetuses from
intrauterine death, do protect ThbdPro/+ fetuses from morbidity associated with sharing the womb
with one or more dead ThbdPro/Pro fetuses.
Attenuation of thrombin signaling in the mother partially rescues fetal loss: The inability of
anticoagulation therapy to protect ThbdPro/Pro embryos was unexpected given the role of platelets
and Par4 in their demise. It prompted us to re-evaluate the involvement of thrombin in the loss of
ThbdPro/Pro embryos. Although generally recognized as a thrombin receptor, Par4 is also activated
by other proteases such as neutrophilic granule protease cathepsin G, tissue Kallikrein and trypsin
29,30
. Thrombin triggers activation of murine platelets via two receptors, Par3 and Par4. In contrast
to Par4, thrombin is the only known agonist for Par3. To more rigorously evaluate the role of
thrombin in placental pathology, we conducted genetic experiments to determine whether Par3
deficiency in the mother ameliorates placental phenotype and fetal death. ThbdPro/Pro males were
mated to Par3-/-FVQ/QThbdPro/+ females and pregnancies were analyzed at 15.5 dpc. Several live
ThbdPro/Pro embryos were obtained from this cross and rescue was found to be highly significant
(Table 1, row 6; P=0.0001 compared to untreated in row 1, chi-squared test of independence).
Consistent with this observation, the genetic absence of Par3 in the mother significantly reduced
the abortion rate (33.3% compared to 69.9% in untreated pregnancies; P=0.0000007, chi-squared
test of independence; untreated 22 live, 51 aborted versus treated 82 live, 41 aborted). Rescue of
ThbdPro/Pro embryos observed in the absence of maternal Par3 was comparable to LMWH
treatment (P=0.55, chi-squared test of independence, Table 1 row 6 compared to row 2), but
partial as compared to expected Mendelian proportions (Table 1, row 6; P=0.004, chi-squaredGOF; 95% CI of 24% to 45%). Thus, even a partial loss of thrombin signaling allows placental
development to proceed and results in significant rescue. These data provide strong evidence that
thrombin-mediated activation of maternal platelets causes placental failure and fetal death in this
model.
We investigated whether the ThbdPro/Pro embryos that survive in utero death in
pregnancies carried by FVQ/Q mothers exhibit normal placental and embryonic growth. For these
experiments Par3-/-FVQ/QThbdPro/+ females were mated to ThbdPro/+ males to generate Thbd+/+
littermate controls. Only 25% of all conceptuses in this cross are expected to be ThbdPro/Pro. Once
again, live ThbdPro/Pro embryos were observed in late pregnancies (Table 2, row 3; P=0.236, chisquared-GOF). The abortion rate was reduced as compared to untreated (31.2% in Table 2 row 3
compared to 47.2% in row 1; 53 live and 24 aborted versus untreated 47 live and 42 aborted;
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P=0.035, chi-squared test of independence) but remained higher than background (31.2% in
Table 2 row 3 compared to 4.3% in row 2; 53 live and 24 aborted versus 88 live and 4 aborted;
P=0.000003, chi-squared test of independence). Although ThbdPro/Pro embryos survived in
statistically significant numbers, these were growth restricted as compared to the Thbd+/+
littermates and tended to have smaller placentae (Figure 1 C-D; Figure 3 A-C). Histological
evaluation of ThbdPro/Pro placentae did not reveal any evidence of increased thrombosis (Figure 1
E-F). ThbdPro/Pro neonates born from pregnancies of FVQ/QThbdPro/+ mothers lacking Par3
appeared normal, but exhibited lower birth weights as compared to their littermates (Table 2, row
4; Figure 3D). These data suggest that although several ThbdPro/Pro embryos survive in
pregnancies of FVQ/Q mothers that lack Par3, their placental function is not fully restored,
resulting in fetal growth retardation. For comparison, we also weighed neonates from pregnancies
of Par4-/-FVQ/QThbdPro/+ females mated to ThbdPro/+ males (Table 2, row 5). These mothers
completely lack platelet responsiveness to thrombin. ThbdPro/Pro neonates did not exhibit smaller
birth weights (supplemental Figure 4). Our observations underscore the sensitivity of placental
growth and development to maternal platelet activation. The mechanisms by which activated
platelets alter placental growth are currently unknown.
Attenuation of platelet aggregation does not rescue fetal loss: Thrombotic clogging of
placental vessels and local hypoxia, albeit transient, may explain the failure of ThbdPro/Pro
placentae to complete morphogenesis in FVQ/Q mothers. In our previous work, staining for fibrin
clots did not reveal any evidence of increased thrombosis in ThbdPro/Pro placentae in pregnancies
of FVQ/QThbdPro/+ females20. Par cleavage on platelets potently activates αIIbβ3 integrin by insideout signaling leading to platelet aggregation. We examined if platelet aggregation plays a role in
fetal loss. The L746A mutation in the β3 subunit (homozygous, β3LA/LA) impairs agonist-induced
inside-out activation of αIIbβ322. Homozygous animals show attenuated platelet aggregation in
vitro and in vessel injury models22,31. We used the β3(L746A) mutation as a genetic tool to
investigate the role of maternal platelet aggregation in placental failure and fetal loss. ThbdPro/Pro
males were mated to β3LA/LAFVQ/QThbdPro/+ females and pregnancies were analyzed at 15.5 dpc.
Of the 101 embryos analyzed in a total of 12 pregnancies, only 2 live ThbdPro/Pro embryos were
obtained (Table 1, row 7; P=0.000003, chi-squared-GOF). The survival of ThbdPro/Pro embryos
was significantly lower than observed with the absence of maternal Par3 (P=0.005, chi-squared
test of independence, Table 1 row 7 compared to row 6) or with LMWH treatment (P=0.002, chisquared test of independence, Table 1 row 7 compared to row 2). The placentae of the two
surviving ThbdPro/Pro embryos did not show any evidence of overt thrombosis (data not shown). In
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a different cross, β3LA/LAFVQ/QThbdPro/+ females were mated to ThbdPro/+ males and analyzed at
15.5 dpc. No ThbdPro/Pro embryos were obtained from this cross (Table 2, row 6; P=0.000003, chisquared-GOF). Analysis of pregnancies at 12.5 dpc also did not reveal any live ThbdPro/Pro
embryos (Table 1, row 8; P=0.004, exact binomial test for goodness of fit; 95% CI of 0.4% to
12.1% from pooled data from all three crosses). Thus, attenuation of maternal platelet aggregation
due to β3(L746A) mutation produced little improvement in the yield of live ThbdPro/Pro embryos.
DISCUSSION
Antithrombotic prophylaxis of pregnant women with inherited thrombophilia and RPL is
an intensely debated issue in obstetric medicine8-11. LMWH is suspected to be beneficial in a
subgroup of high-risk pregnancies, but the multi-factorial nature of RPL and the inherent
heterogeneity in study populations precludes easy identification of this subgroup. It has also been
questioned if thrombophilia mutations disrupt placental function via thrombotic processes.
Evaluation of placental pathologies has not revealed a clear correlation between maternal
inherited thrombophilia and increased thrombotic lesions in the placenta12-14. Meanwhile, animal
models have identified a critical role of coagulation components in the development and function
of extraembryonic tissues. Complete absence of Thbd or the Endothelial Protein C Receptor
(EPCR) results in embryonic lethality in mice, secondary to placental defects. Treatment with
heparin extends survival of a small fraction of EPCR-/- embryos32, and does not improve survival
of Thbd-/- embryos33. These observations are often quoted as evidence against the role of
thrombotic processes in thrombophilia-associated placental disorder10. Since these are gene
knock-out models, the inability of anticoagulation to rescue these pregnancies may reflect other
essential functions of the missing gene products. In contrast to the complete absence of Thbd and
EPCR, reduced expression or compromised function of these components allows placental and
embryonic development to proceed and results in viable adult animals34,35. Placental failure and
embryonic death is once again observed when reduced thrombomodulin function or reduced
EPCR expression in the fetus is combined with the prothrombotic Factor V Leiden mutation in
the mother20. These studies provided us with an animal model of high-risk pregnancy where a
combination of maternal and fetal inherited thrombophilia causes placenta developmental failure.
We asked if placental failure and fetal loss can be counteracted with antithrombotic therapy.
We demonstrate, first, that LMWH unequivocally protects pregnancies in the murine
model, lending support to its reported efficacy in improving pregnancy outcome in thrombophilic
women5,6. Protection was observed both in terms of rescue of ThbdPro/Pro embryos from
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intrauterine death in pregnancies of FVQ/Q mothers and a significantly improved overall abortion
rate. Nonetheless, our data shows that the observed therapeutic effect of LMWH is not replicated
by anticoagulation alone. Factor Xa and thrombin inhibition are the two most well known
anticoagulant functions of LMWH. We used Fondaparinux to address whether the Xa-inhibitory
activity of LMWH is sufficient to rescue pregnancies. Treatment with Fondaparinux resulted in
anticoagulation comparable to LMWH, but did not protect ThbdPro/Pro embryos from in utero
death. Inhibition of thrombin activity using the direct thrombin inhibitor, lepirudin, was similarly
effective in anticoagulation but did not ameliorate pregnancy loss. Using the direct Xa inhibitor,
C921-78, resulted in anticoagulation much higher than achieved with LMWH but led to a
significantly lower rescue of ThbdPro/Pro embryos. These observations are striking in the extent of
rescue observed with LMWH, all or most of which could not be reproduced with thrombin or
FXa inhibition alone. The molecular basis of LMWH's ability to protect pregnancies remains to
be determined and is a subject of our ongoing research. LMWH rescues pregnancies in a murine
model of antiphospholipid antibody-induced fetal loss by suppressing complement activity36. In
preliminary studies, we treated pregnant factor V Leiden females with complement-inhibitory
anti-C5 monoclonal antibodies. This treatment has been previously shown to prevent
antiphospholipid antibody-induced fetal loss in mice37. Anti-C5 mAb treatment was ineffective
in preventing fetal loss of ThbdPro/Pro embryos (Sood et al, unpublished observations), indicating
that the pathological mechanism of fetal loss in our model of inherited thrombophilia is distinct
from the complement-driven mechanism operating in the murine model of antiphospholipid
antibody-induced fetal loss, where C5 activation is a critical effecter. Other candidate
mechanisms include inhibition of p-selectin-mediated platelet tethering to trophoblast cells or to
immune cells38,39, and possible direct actions of LMWH on trophoblast cells17,40,41.
The failure to suppress placental pathology with lepirudin, fondaparinux or C921-78,
despite demonstrated efficacy of anticoagulation, was puzzling. Since Par4 can also be activated
by agonists other than thrombin29,30, we asked whether genetic absence of Par3, a second
thrombin receptor on mouse platelets, ameliorates fetal loss. Par3 and Par4 on mouse platelets are
analogous to PAR1 and PAR4 on human platelets in their threshold for activation by thrombin.
Par3 promotes cleavage and activation of Par4 at low concentrations, such that the EC50 of
platelet activation by thrombin increases from 0.10 ± 0.08 nM in WT platelets to 1.5 ± 0.7 nM in
Par3-/- platelets21. We demonstrate, second, that the genetic absence of Par3 in the mother allows
placental development to proceed and significantly improves the survival of ThbdPro/Pro fetuses.
The observation that ThbdPro/Pro embryos in pregnancies of FVQ/Q mothers survive in each case –
Page 12 of 24
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in the absence of platelets or in the absence of Par4 or Par3 in the mother - provides a strong
argument that placental developmental failure in this model is driven by activation of maternal
platelets. Thrombin activates both receptors and is the most likely mediator of platelet activation
in this scenario. Our observations demonstrate that even a slight elevation in EC50 of maternal
platelet activation by thrombin affords significant rescue. Thus, placental development is highly
vulnerable to slight increases in thrombin generation and platelet activation. ThbdPro/Pro embryos
that escape arrest of placental morphogenesis in the absence of maternal Par3 are not completely
normal. These are growth restricted at late gestation and at birth. A similar reduction in birth
weight was not detected in ThbdPro/Pro neonates that survive in the absence of maternal Par4.
Although our data does not formally prove that the Par3- and Par4-dependent effects are mediated
via activation of these receptors on platelets, it associates residual reactivity of maternal platelets
to thrombin with suboptimal placental function in a prothrombotic setting. These data, once
again, demonstrate that thrombophilia-associated placental dysfunction correlates with
developmental or structural abnormalities, rather than thrombotic clogging.
If thrombin is the critical mediator of placental dysfunction, why is anticoagulation not
effective? This likely reflects the low EC50 of thrombin concentration required for platelet
activation. While hirudin, Fondaparinux and direct thrombin inhibitors are increasingly efficient
in delaying the onset of the propagation phase and reducing the total amount of thrombin, a low
level of thrombin generation and platelet activation is likely to continue in their presence.
Notably, C921-78 effectively inhibits fibrin-rich venous clots in a rabbit deep vein thrombosis
model, but severely attenuates and does not completely inhibit platelet-rich arterial clots in an
arteriovenous shunt model, even at the highest doses examined23. Similarly, C921-78 alone
significantly delays thrombus formation upon FeCl3-induced injury of mouse mesentric arteries,
but a combination of C921-78 and reduced ADP receptor expression is required to completely
abolish platelet aggregation and thrombus formation42. Observations such as these have suggested
a more crucial role of platelets in arterial thrombosis. The utero-placental circulation does not
conform to traditional definitions of arterial or venous blood flow, and the relative contribution of
platelets in causing placental pathology is unclear. Our data suggests that a low level of thrombin
generation and platelet activation, rather than accumulation of thrombin, causes the primary
pathology in our model. It also excludes the possibility that the contribution of activated platelets
in this pathology primarily resides in their ability to enhance thrombin generation, also referred to
as their procoagulant function.
Page 13 of 24
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We investigated the contribution of platelet aggregation to disease pathology. The αIIbβ3
complex plays a central role in firm adhesion of platelets to the vessel wall and in platelet
aggregation. We used β3(L746A) mutant mice that maintain αIIbβ3-mediated platelet adhesion,
but are attenuated in their ability to activate the integrin complex by inside-out signaling in
response to thrombin and other agonists. These mice show impaired platelet aggregation in vitro
and diminished thrombus growth in laser injury models22,31. We demonstrate, third, that FVq/q
mothers homozygous for the L746A mutation continue to abort ThbdPro/Pro embryos, despite
attenuated platelet aggregation. Taken together, our genetic and anticoagulation studies suggest
that PAR-mediated platelet activation at the feto-maternal interface precipitates placental
developmental failure, without the need for thrombotic clogging of utero-placental vessels.
Placental failure appears to involve an activation-dependent, but aggregation-independent,
function of platelets. Platelet activation triggers release of stored components from secretory
vesicles - α-granules, dense granules and lysosomes – that range from small molecules and ions
to enzymes and growth factors. The repertoire of the cargo stored in platelets includes potently
antiangiogenic platelet factor-4, endostatin, and thrombospondin-143-45. The ability of activated
platelets to degranulate and release factors that may affect placental development and the ability
of platelets to recruit immune cells are candidate mechanisms of placental failure.
Our observations may have relevance for human multifetal pregnancies affected by the
death of one or more fetuses. Intrauterine fetal death of one fetus in a multiple gestation is a rare
condition that complicates approximately 0.5% to 6.8% of twin pregnancies and 4.3% to 17% of
triplet pregnancies. With greater reliance on assisted reproductive techniques, twins and higher
order multiples are becoming increasingly widespread. In these pregnancies, retention of a dead
fetus is associated with increased coagulation activation in maternal circulation and morbidity for
the surviving fetuses27,28. Our data shows that ThbdPro/+ embryos and placentae with reduced
anticoagulation ability are more susceptible to hostile intrauterine environment associated with
fetal death in a multifetal pregnancy than their Thbd+/+ littermates. Antithrombotic therapy with
fondaparinux and direct Xa inhibitors is beneficial in this scenario.
In summary, we have presented data evaluating the role of thrombin and thrombotic
processes in placental developmental failure observed in a murine high-risk pregnancy model,
generated by combining maternal and fetal prothrombotic mutations in genes expressed at the
feto-maternal interface. Given the multi-factorial nature of thrombophilia-associated RPL and the
inherent deficiencies of animal models of human disease the exact implication of our
Page 14 of 24
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observations for human pregnancies is unclear. Our experiments should, therefore, only be
interpreted as proof-of-concept studies. Our data supports the clinical observation that LMWH
might, indeed, be beneficial for a subset of high-risk pregnancies with maternal thrombophilia
and a history of RPL. It further supports the stimulating possibility that LMWH may have
beneficial effects beyond anticoagulation and prompts an investigation into the mechanism by
which LMWH improves placental health. Two pilot studies have reported a beneficial effect of
LMWH in pregnancies of women with a history of placenta-mediated complications without
thrombophilia46,47, but no benefit was observed in three other studies48-50. We report that blocking
early steps of PAR-mediated platelet activation is beneficial in preventing fetal loss in mice.
Pharmacologically blocking thrombin receptor activation on human platelets may be potentially
beneficial for a subset of women with thrombophilia and RPL. Our data, however, cautions that
certain treatments may improve intrauterine survival but continue to be associated with
suboptimal growth and development.
Acknowledgements:
We thank Sara Szabo for examination of placental slides and expert pathology opinion;
the CRI Histology, Imaging and Pediatric Biobank & Analytical tissue cores for expert services;
Ann Diamond, Andrew Webb, Micah Tesdall and Eric Jukkala for administrative support; Mark
Ginsberg from University of California, San Diego, for β3 (L746A) mutant mice; Hartmut Weiler
from the Blood Center of Wisconsin for critical reading of the manuscript and for suggestions.
This work was supported by the Basil O’ Connor Starter Scholar Award #5-FY09-121
(March of Dimes Foundation), National Scientist Development Grant # 0930200N (American
Heart Association) and CRI Histology and Imaging Core grant to R.S.
Author Contributions:
J.A., M.W., M.B. and V.A. performed experiments; R.G.H. helped with statistical analysis; B.P.,
U.S. and P.N. provided critical reagents and reviewed the manuscript; R.S. designed and
performed experiments, analyzed data and wrote the manuscript.
The authors have no conflicting financial interests to declare. U. Sinha is an employee of
Portola Pharmaceutical Inc.
Page 15 of 24
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REFERENCES:
1.
Ngoc NT, Merialdi M, Abdel-Aleem H, et al. Causes of stillbirths and early neonatal
deaths: data from 7993 pregnancies in six developing countries. Bull World Health
Organ. 2006;84(9):699-705.
2.
Barker DJ, Osmond C, Kajantie E, Eriksson JG. Growth and chronic disease: findings in
the Helsinki Birth Cohort. Ann Hum Biol. 2009;36(5):445-458.
3.
Benedetto C, Marozio L, Tavella AM, Salton L, Grivon S, Di Giampaolo F. Coagulation
disorders in pregnancy: acquired and inherited thrombophilias. Ann N Y Acad Sci.
2010;1205:106-117.
4.
Robertson L, Wu O, Langhorne P, et al. Thrombophilia in pregnancy: a systematic
review. Br J Haematol. 2006;132(2):171-196.
5.
Brenner B, Hoffman R, Carp H, Dulitsky M, Younis J. Efficacy and safety of two doses
of enoxaparin in women with thrombophilia and recurrent pregnancy loss: the LIVEENOX study. J Thromb Haemost. 2005;3(2):227-229.
6.
Gris JC, Mercier E, Quere I, et al. Low-molecular-weight heparin versus low-dose aspirin
in women with one fetal loss and a constitutional thrombophilic disorder. Blood.
2004;103(10):3695-3699.
7.
de Vries JI, van Pampus MG, Hague WM, Bezemer PD, Joosten JH. Low-molecularweight heparin added to aspirin in the prevention of recurrent early-onset pre-eclampsia
in women with inheritable thrombophilia: the FRUIT-RCT. J Thromb Haemost.
2012;10(1):64-72.
8.
Gris JC. LMWH have no place in recurrent pregnancy loss: debate-against the motion.
Thromb Res. 2011;127 Suppl 3S110-112.
9.
Lindqvist PG, Merlo J. Low molecular weight heparin for repeated pregnancy loss: is it
based on solid evidence? J Thromb Haemost. 2005;3(2):221-223.
10.
Middeldorp S. Low-molecular-weight heparins have no place in recurrent miscarriage:
debate--for the motion. Thromb Res. 2011;127 Suppl 3S105-109.
11.
Walker ID, Kujovich JL, Greer IA, et al. The use of LMWH in pregnancies at risk: new
evidence or perception? J Thromb Haemost. 2005;3(4):778-793.
12.
Sikkema JM, Franx A, Bruinse HW, van der Wijk NG, de Valk HW, Nikkels PG.
Placental pathology in early onset pre-eclampsia and intra-uterine growth restriction in
women with and without thrombophilia. Placenta. 2002;23(4):337-342.
Page 16 of 24
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
13.
Morssink LP, Santema JG, Willemse F. Thrombophilia is not associated with an increase
in placental abnormalities in women with intra-uterine fetal death. Acta Obstet Gynecol
Scand. 2004;83(4):348-350.
14.
Rogers BB, Momirova V, Dizon-Townson D, et al. Avascular villi, increased syncytial
knots, and hypervascular villi are associated with pregnancies complicated by factor V
Leiden mutation. Pediatr Dev Pathol. 2010;13(5):341-347.
15.
Rodger MA, Paidas M. Do thrombophilias cause placenta-mediated pregnancy
complications? Semin Thromb Hemost. 2007;33(6):597-603.
16.
Gris JC. Thrombophilia and pregnancy loss: cause or association. Thromb Res. 2009;123
Suppl 2S105-110.
17.
Kingdom JC, Drewlo S. Is heparin a placental anticoagulant in high-risk pregnancies?
Blood. 2011;118(18):4780-4788.
18.
Georgiades P, Ferguson-Smith AC, Burton GJ. Comparative developmental anatomy of
the murine and human definitive placentae. Placenta. 2002;23(1):3-19.
19.
Sood R, Kalloway S, Mast AE, Hillard CJ, Weiler H. Fetomaternal cross talk in the
placental vascular bed: control of coagulation by trophoblast cells. Blood.
2006;107(8):3173-3180.
20.
Sood R, Zogg M, Westrick RJ, et al. Fetal gene defects precipitate platelet-mediated
pregnancy failure in factor V Leiden mothers. J Exp Med. 2007;204(5):1049-1056.
21.
Cornelissen I, Palmer D, David T, et al. Roles and interactions among protease-activated
receptors and P2ry12 in hemostasis and thrombosis. Proc Natl Acad Sci U S A.
2010;107(43):18605-18610.
22.
Petrich BG, Fogelstrand P, Partridge AW, et al. The antithrombotic potential of selective
blockade of talin-dependent integrin alpha IIb beta 3 (platelet GPIIb-IIIa) activation. J
Clin Invest. 2007;117(8):2250-2259.
23.
Sinha U, Ku P, Malinowski J, et al. Antithrombotic and hemostatic capacity of factor Xa
versus thrombin inhibitors in models of venous and arteriovenous thrombosis. Eur J
Pharmacol. 2000;395(1):51-59.
24.
Mann KG, Butenas S, Brummel K. The dynamics of thrombin formation. Arterioscler
Thromb Vasc Biol. 2003;23(1):17-25.
25.
Brufatto N, Ward A, Nesheim ME. Factor Xa is highly protected from antithrombinfondaparinux and antithrombin-enoxaparin when incorporated into the prothrombinase
complex. J Thromb Haemost. 2003;1(6):1258-1263.
Page 17 of 24
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
26.
Betz A, Wong PW, Sinha U. Inhibition of factor Xa by a peptidyl-alpha-ketothiazole
involves two steps. Evidence for a stabilizing conformational change. Biochemistry.
1999;38(44):14582-14591.
27.
Erez O, Gotsch F, Mazaki-Tovi S, et al. Evidence of maternal platelet activation,
excessive thrombin generation, and high amniotic fluid tissue factor immunoreactivity
and functional activity in patients with fetal death. J Matern Fetal Neonatal Med.
2009;22(8):672-687.
28.
Romero R, Copel JA, Hobbins JC. Intrauterine fetal demise and hemostatic failure: the
fetal death syndrome. Clin Obstet Gynecol. 1985;28(1):24-31.
29.
Sambrano GR, Huang W, Faruqi T, Mahrus S, Craik C, Coughlin SR. Cathepsin G
activates
protease-activated
receptor-4
in
human
platelets.
J
Biol
Chem.
2000;275(10):6819-6823.
30.
Oikonomopoulou K, Hansen KK, Saifeddine M, et al. Proteinase-activated receptors,
targets for kallikrein signaling. J Biol Chem. 2006;281(43):32095-32112.
31.
Nishimura S, Manabe I, Nagasaki M, et al. In vivo imaging visualizes discoid platelet
aggregations without endothelium disruption and implicates contribution of inflammatory
cytokine and integrin signaling. Blood. 2012;119(8):e45-56.
32.
Li W, Zheng X, Gu JM, et al. Extraembryonic expression of EPCR is essential for
embryonic viability. Blood. 2005;106(8):2716-2722.
33.
Isermann B, Sood R, Pawlinski R, et al. The thrombomodulin-protein C system is
essential for the maintenance of pregnancy. Nat Med. 2003;9(3):331-337.
34.
Weiler-Guettler H, Christie PD, Beeler DL, et al. A targeted point mutation in
thrombomodulin generates viable mice with a prethrombotic state. J Clin Invest.
1998;101(9):1983-1991.
35.
Castellino FJ, Liang Z, Volkir SP, et al. Mice with a severe deficiency of the endothelial
protein C receptor gene develop, survive, and reproduce normally, and do not present
with enhanced arterial thrombosis after challenge. Thromb Haemost. 2002;88(3):462472.
36.
Girardi G, Redecha P, Salmon JE. Heparin prevents antiphospholipid antibody-induced
fetal loss by inhibiting complement activation. Nat Med. 2004;10(11):1222-1226.
37.
Girardi G, Berman J, Redecha P, et al. Complement C5a receptors and neutrophils
mediate fetal injury in the antiphospholipid syndrome. J Clin Invest. 2003;112(11):16441654.
Page 18 of 24
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
38.
Koenig A, Norgard-Sumnicht K, Linhardt R, Varki A. Differential interactions of heparin
and heparan sulfate glycosaminoglycans with the selectins. Implications for the use of
unfractionated and low molecular weight heparins as therapeutic agents. J Clin Invest.
1998;101(4):877-889.
39.
Ludwig RJ, Alban S, Bistrian R, et al. The ability of different forms of heparins to
suppress P-selectin function in vitro correlates to their inhibitory capacity on bloodborne
metastasis in vivo. Thromb Haemost. 2006;95(3):535-540.
40.
Drewlo S, Levytska K, Sobel M, Baczyk D, Lye SJ, Kingdom JC. Heparin promotes
soluble VEGF receptor expression in human placental villi to impair endothelial VEGF
signaling. J Thromb Haemost. 2011;9(12):2486-2497.
41.
Sobel ML, Kingdom J, Drewlo S. Angiogenic response of placental villi to heparin.
Obstet Gynecol. 2011;117(6):1375-1383.
42.
Andre P, LaRocca T, Delaney SM, et al. Anticoagulants (thrombin inhibitors) and aspirin
synergize
with
P2Y12
receptor
antagonism
in
thrombosis.
Circulation.
2003;108(21):2697-2703.
43.
Bikfalvi A. Platelet factor 4: an inhibitor of angiogenesis. Semin Thromb Hemost.
2004;30(3):379-385.
44.
O'Reilly MS, Boehm T, Shing Y, et al. Endostatin: an endogenous inhibitor of
angiogenesis and tumor growth. Cell. 1997;88(2):277-285.
45.
Zaslavsky A, Baek KH, Lynch RC, et al. Platelet-derived thrombospondin-1 is a critical
negative regulator and potential biomarker of angiogenesis. Blood. 2010;115(22):46054613.
46.
Rey E, Garneau P, David M, et al. Dalteparin for the prevention of recurrence of
placental-mediated complications of pregnancy in women without thrombophilia: a pilot
randomized controlled trial. J Thromb Haemost. 2009;7(1):58-64.
47.
Gris JC, Chauleur C, Molinari N, et al. Addition of enoxaparin to aspirin for the
secondary prevention of placental vascular complications in women with severe preeclampsia. The pilot randomised controlled NOH-PE trial. Thromb Haemost.
2011;106(6):1053-1061.
48.
Martinelli I, Ruggenenti P, Cetin I, et al. Heparin in pregnant women with previous
placenta-mediated pregnancy complications: a prospective, randomized, multicenter,
controlled clinical trial. Blood. 2012;119(14):3269-3275.
Page 19 of 24
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
49.
Clark P, Walker ID, Langhorne P, et al. SPIN (Scottish Pregnancy Intervention) study: a
multicenter, randomized controlled trial of low-molecular-weight heparin and low-dose
aspirin in women with recurrent miscarriage. Blood. 2010;115(21):4162-4167.
50.
Kaandorp SP, Goddijn M, van der Post JA, et al. Aspirin plus heparin or aspirin alone in
women with recurrent miscarriage. N Engl J Med. 2010;362(17):1586-1596.
Page 20 of 24
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TABLE LEGENDS:
Table 1: Pregnancy outcome of FVQ/QThbdPro/+ females mated to ThbdPro/Pro males:
ThbdPro/Pro embryos die in midgestation in pregnancies carried by FVQ/QThbdPro/+ females (row 1),
but survive if the mother is treated with LMWH (row 2) or is deficient in Par3 (row 6).
Anticoagulation with Hirudin (lepirudin) (row 3), Fondaparinux (row 4) or C921-78 (row 5), or
genetic attenuation of maternal platelet aggregation (rows 7 & 8) does not result in comparable
rescue. Asterix (*) denotes significant difference from expected proportions (P-value < 0.05)
based on chi-squared-GOF. Equal proportions (50% each) of ThbdPro/+ and ThbdPro/Pro embryos
are expected from this cross.
Table 2: Pregnancy outcome of FVQ/QThbdPro/+ females mated to ThbdPro/+ males: ThbdPro/Pro
embryos die in midgestation in pregnancies carried by FVQ/QThbdPro/+ mothers (row 1), but
survive in the reverse genetic cross (row 2) in pregnancies carried by FV+/+ mothers. Attenuation
of thrombin signaling by genetic absence of Par3 in the mother allows survival of several
ThbdPro/Pro embryos (row 3) and neonates (row 4) in these pregnancies, as does the genetic
absence of maternal Par4 (row 5). Attenuation of maternal platelet aggregation (row 6) does not
result in comparable rescue. Asterix (*) denotes significant difference from expected proportions
(P-value < 0.05) based on chi-squared-GOF. Based on Mendelian inheritance, Thbd+/+, ThbdPro/+
and ThbdPro/Pro embryos are expected to be born at 25%, 50% and 25% proportions, respectively,
from this cross.
Page 21 of 24
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Table 1:
FVQ/+
ThbdPro/+
FVQ/+
ThbdPro/Pro
22
(100%)
21
(60%)
21
(100%)
25
(96.2%)
36
(90%)
0*
1
(3.8%)
4*
(10%)
Number
(%age)
of
aborted
embryos
51
(69.9%)
18
(34%)
27
(56.2%)
20
(43.5%)
35
(46.7%)
ThbdPro/Pro
54
(65.9%)
28*
(34.1%)
41
(33.3%)
123 (13)
ThbdPro/Pro
27
(93.1%)
2*
(6.9%)
72
(71.3%)
101 (12)
ThbdPro/Pro
9
(100%)
0*
9
(50%)
18 (2)
Parental genotype
Experimental
manipulation
Stage of
Analysis
None
12.5 dpc
LMWH
16.5 dpc
Hirudin
16.5 dpc
Fondaparinux
16.5 dpc
Xa inhibitor
C921-78
15.5 dpc
Mother
Par3-/-
15.5 dpc
Mother
β3LA/LA
15.5 dpc
Mother
β3LA/LA
12.5 dpc
Female
FVQ/Q
ThbdPro/+
FVQ/Q
ThbdPro/+
FVQ/Q
ThbdPro/+
FVQ/Q
ThbdPro/+
FVQ/Q
ThbdPro/+
Par3-/FVQ/Q
ThbdPro/+
β3LA/LA
FVQ/Q
ThbdPro/+
β3LA/LA
FVQ/Q
ThbdPro/+
Male
ThbdPro/Pro
ThbdPro/Pro
ThbdPro/Pro
ThbdPro/Pro
ThbdPro/Pro
Genotype of live
embryos
Page 22 of 24
14
(40%)
0*
*
Number of
embryos
(pregnancies)
analyzed
73 (8)
53 (7)
48 (6)
46 (6)
75 (8)
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Table 2:
Stage of
Analysis
15.5 dpc
15.5 dpc
FVQ/+
Thbd+/+
20
(42.6%)
26
(29.5%)
FVQ/+
ThbdPro/+
27
(57.4%)
38
(43.2%)
FVQ/+
ThbdPro/Pro
24
(27.3%)
Number
(%age) of
aborted
embryos
42
(47.2%)
4
(4.3%)
ThbdPro/+
14
(26.4%)
31
(58.5%)
8
(15.1%)
24
(31.2%)
77 (8)
ThbdPro/+
16
(22.9%)
38
(54.3%)
16
(22.9%)
-
70 (9)
ThbdPro/+
7
(20%)
22
(62.9%)
6
(17.1%)
-
35 (7)
ThbdPro/+
19
(25%)
32
(42.1%)
0*
25
(32.9%)
76 (9)
Parental genotype
Female
Male
FVQ/Q
ThbdPro/+
ThbdPro/+
ThbdPro/+
Q/Q
FV
ThbdPro/+
Genotype of live embryos
0*
Number of
embryos
(pregnancies)
analyzed
89 (9)
92 (10)
-/-
15.5 dpc
Neonates
Neonates
15.5 dpc
Par3
FVQ/Q
ThbdPro/+
Par3-/FVQ/Q
ThbdPro/+
Par4-/FVQ/Q
ThbdPro/+
β3LA/LA
FVQ/Q
ThbdPro/+
Page 23 of 24
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FIGURE LEGENDS:
Figure 1: ThbdPro/Pro embryos survive pregnancies of FVQ/QThbdPro/+ females if the mother
is treated with LMWH or is deficient in Par3: ThbdPro/Pro (B, D) and littermate ThbdPro/+ (A)
and Thbd+/+ (C) embryos from representative pregnancies treated with LMWH (A, B) or with
maternal Par3 deficiency (C, D) are shown. These correspond to 16.5 dpc for LMWH treatment
and 15.5 dpc for maternal Par3 deficiency. ThbdPro/Pro embryos are growth restricted in
pregnancies of Par3-/- FVQ/QThbdPro/+ females (measurements shown in Figure 3A-B). Their
placentae tend to be smaller (measurements in Figure 3C) but do not present evidence of overt
thrombosis. Hematoxylin-Eosin stained histological sections of Thbd+/+ (E) and ThbdPro/Pro
placentae (F) corresponding to (C) and (D), respectively, are shown. The plane of sectioning is
through the center of the placenta and perpendicular to its flat surface. Regions of the placenta are
marked. Db, decidua basalis; jz, junctional zone corresponding to basal plate in human placenta;
lb, labyrinth or fetal placenta containing trophoblast covered fetal vessels bathed in maternal
blood. Scale bars represent 5 mm each in A-D and 1mm in E-F.
Figure 2: ThbdPro/+ embryos and placentae are smaller than littermate Thbd+/+ controls in
pregnancies of FVQ/QThbdPro/+ females mated to ThbdPro/+ males: The size distribution of
ThbdPro/+ and littermate Thbd+/+ embryos and placentae measured at 15.5 dpc is shown.
Lengthwise cross-sectional area (panel A) and length of the embryo (panel B) and placental
cross-sectional area (panel C) were measured from digital photographs taken at a known
magnification and resolution. P values were computed in comparison to Thbd+/+ controls. No live
ThbdPro/Pro embryos were obtained from these pregnancies; these die in utero before 12.5 dpc.
Figure 3: Par3 deficiency in the mother allows survival of ThbdPro/Pro embryos, but these are
growth retarded in utero and smaller at birth: Pregnancies of Par3-/-FVQ/QThbdPro/+ females
mated to ThbdPro/+ males were analyzed at 15.5 dpc. Lengthwise cross-sectional area (panel A)
and length of the embryo (panel B) and placental cross-sectional area (panel C) were measured
from digital photographs taken at a known magnification and resolution. Neonatal weight was
taken within 18 hours of birth (panel D). P values were computed in comparison to Thbd+/+
controls.
Page 24 of 24
A
E
B
C
db
jz
lb
Fetal Side
F
db
jz
lb
Fetal Side
Figure 1
D
A
*
Area (sq cm)
P=0.008
B
*
Length (cm)
P=0.01
*
P=0.046
Area (sq cm)
C
Thbd+/+ ThbdPro/+
FVQ/QThbdPro/+ mother
Figure 2
B
P=0.23
P=0.51
*
P=0.04
Length (cm)
Area (sq cm)
A
D
P=0.57
P=0.09
ThbdPro/+
Thbd+/+
ThbdPro/Pro
Neonatal Weight (g)
Area (sq cm)
C
*
P=0.003
P=0.95
*
P=0.008
ThbdPro/+
Thbd+/+
Par3-/- Mother
ThbdPro/Pro
Par3-/- Mother
Figure 3
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Prepublished online January 16, 2013;
doi:10.1182/blood-2012-08-448209
Heparin rescues factor V Leiden-associated placental failure independent
of anticoagulation in a murine high-risk pregnancy model
Jianzhong An, Magarya Waitara, Michelle Bordas, Vidhyalakshmi Arumugam, Raymond G. Hoffmann,
Brian G. Petrich, Uma Sinha, Paula North and Rashmi Sood
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