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Keywords:

  • embryogenesis;
  • pregnancy;
  • hemostasis;
  • mouse;
  • fibrinogen;
  • platelets

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Yolk sac vessel injury model
  7. Histology
  8. Platelet count
  9. Results
  10. Normal development in embryos with combined platelet and fibrinogen deficiency
  11. Postnatal lethality of embryos with combined platelet – fibrinogen deficiency
  12. Residual vaso-occlusive function in embryos with individual platelet or fibrinogen deficiency, but not in embryos with combined platelet-fibrinogen deficiency
  13. The essential role of maternal fibrinogen in placental function is independent of platelets
  14. Embryonic, not maternal, NF-E2-deficiency reduces placental vascularization
  15. Discussion
  16. Acknowledgements
  17. References

Summary.  Studies of mice with genetic deficits in specific coagulation factors have shown that many, but not all, components of the hemostatic system serve an essential role in mouse embryogenesis and pregnancy. Although the developmental failures observed in these mice are typically associated with severe hemorrhage, it is uncertain whether the role of coagulation factors in embryogenesis and reproduction is specifically tied to their function in thrombus formation and prevention of blood loss (i.e. hemostasis). Here, we show (i) that a complete loss of fibrinogen- and platelet-dependent hemostatic capacity does not reproduce the developmental defects occurring in mice with either deficits in thrombin generation or unfettered thrombin consumption; (ii) that the essential role of fibrinogen in the maintenance of pregnancy does not involve interaction with platelets; and (iii) that the previously described in utero growth retardation of gene-targeted mice lacking NF-E2, a transcription factor critical for megakaryopoieis, is not caused by a loss of platelet-dependent hemostatic function. In addition, we demonstrate (iv) that fibrinogen can confer physiologically relevant hemostatic function in the absence of platelets, but that a complete loss of hemostatic capacity results if a combined absence of these components is genetically imposed. These findings support the notion that the function of coagulation factors for in utero development and pregnancy is mechanistically distinct from their ability to mediate the formation of hemostatic platelet-fibrin(ogen) aggregates.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Yolk sac vessel injury model
  7. Histology
  8. Platelet count
  9. Results
  10. Normal development in embryos with combined platelet and fibrinogen deficiency
  11. Postnatal lethality of embryos with combined platelet – fibrinogen deficiency
  12. Residual vaso-occlusive function in embryos with individual platelet or fibrinogen deficiency, but not in embryos with combined platelet-fibrinogen deficiency
  13. The essential role of maternal fibrinogen in placental function is independent of platelets
  14. Embryonic, not maternal, NF-E2-deficiency reduces placental vascularization
  15. Discussion
  16. Acknowledgements
  17. References

The current communication addresses unresolved questions about the role of hemostasis, in particular the role of fibrinogen and platelets, in mammalian development and reproduction. Studies of genetically altered mice with deficits in hemostatic system function have revealed that some, but not all coagulation factors serve an essential role in mammalian embryogenesis and pregnancy. The genetic abrogation of factors with an essential function in thrombin generation (i.e. tissue factor, factor (F)V and fX, and prothrombin itself) causes lethal developmental defects that lead to termination of pregnancy in midgestation [1–6]. A common observation in these hemostatic factor-deficient mice is severe hemorrhage, possibly associated with a defect in the integrity of blood vessels in the fetal yolk sac.

Remarkably, excessive coagulation system activation secondary to genetic elimination of endogenous inhibitors of thrombin formation or activity also causes intrauterine lethality in mid- to late-gestation. Mouse embryos lacking tissue factor pathway inhibitor (TFPI) [7], antithrombin III [8], or thrombomodulin (TM) [9] develop thrombo-hemorrhagic complications associated with augmented fibrin deposition within the vasculature of the embryo proper. Indeed, TFPI-null mice appear to suffer from defects of yolk sac blood vessel function that are surprisingly similar to that observed in mice lacking the targets of TFPI's inhibitory function, tissue factor [1–3] and fX [10]. One hypothesis that can explain this apparent paradox is that uncontrolled thrombin generation/activity leads to consumptive depletion of coagulation systems components, including thrombin, thus recapitulating the phenotype of knockout mice lacking these factors. Alternatively, inappropriate thrombin signaling may compromise embryonic development, independent of the platelet/fibrinogen axis.

A particularly perplexing finding is that the developmental consequences of genetically impeding thrombin generation appear to be dramatically different from the developmental consequences of eliminating the individual targets of thrombin that are the key building blocks of any thrombus, including fibrinogen [11,12], fXIII [13], platelet signal transduction molecules [14] and even platelets per se[15]. Notably, mice lacking fibrinogen [11,12] or platelets [14,15] successfully complete intrauterine development, and may even survive to adulthood, suggesting that the developmental function of thrombin may be distinct from its role in hemostasis. Arguably the best evidence for this hypothesis stems from the analysis of mice lacking the thrombin-activatable protease receptor, PAR. Approximately half of PAR-1–/– mice die in mid-gestation with a failure of vascular integrity that is similar to that observed in mice devoid of tissue factor [1–3] or prothrombin [5,6]. This failure appears to be distinct from platelet function, and reconstitution of PAR-1 expression in endothelial cells restores normal development of otherwise PAR-1-deficient mice [16].

Taken together, findings from the above studies have led to the inference that coagulation factors may contribute to embryogenesis in a way that is utterly distinct from their role in the formation of a hemostatic platelet–fibrin aggregate, and that hemostatic platelet-fibrin(ogen) interactions do not play any appreciable role in mammalian development. This conclusion is consistent with the observation that mice deficient in both PAR-4 and fibrinogen develop normally to term [17]. However, agonists other than thrombin, including epinephrine, ADP and collagen, may activate platelets in PAR-4-deficient mice. This may provide sufficient capacity for thrombus formation to allow for normal intrauterine development. Thus, an alternative explanation for the developmental success of mice lacking fibrinogen, platelets or PAR-4/fibrinogen is that the residual hemostatic function provided by just one of these components is adequate to support embryos to term, but the loss of both of these thrombin targets compromises development. Such a residual hemostatic capacity is clearly documented by intravital microscopy studies showing that platelet deposition and aggregation within arterioles in vivo remains remarkably robust in mice lacking fibrinogen [18]. Therefore, the role of thrombin in development independent of hemostatic plug formation has remained formally unresolved.

Similarly, the role of the hemostatic system in placental development and function also remains poorly defined. Placental defects have been established in embryos lacking TF [1–3] or TM [9], and – most importantly – the absence of fibrinogen [11,12] from the maternal circulation leads to fatal intrauterine bleeding from the placenta. In mice, this defect invariably causes death of the mother in midgestation; in afibrinogenemic human females, the mother may survive, but loss of pregnancy ensues [19,20]. It is completely unknown whether this essential role of fibrinogen in reproduction depends on fibrin(ogen)–platelet interactions and hemostatic function, or whether it is related to non-hemostatic function(s) of fibrinogen.

Here, we attempt to resolve these lingering questions about the individual and combined role of platelets and fibrinogen in embryonic and placental development. To do this we interbred fibrinogen-deficient mice (fibrinogen Aα-knockout mice) with gene-targeted mice deficient in either NF-E2 or Gαq. NF-E2 is a transcription factor critical for megakaryopoiesis, and consequently NF-E2-deficient mice have a severe quantitative platelet defect. Alternatively, deletion of the signal transducer, Gαq, results in normal numbers of circulating platelets which do not activate in response to any physiologically relevant agonist, including thrombin, ADP and collagen.

Mice

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Yolk sac vessel injury model
  7. Histology
  8. Platelet count
  9. Results
  10. Normal development in embryos with combined platelet and fibrinogen deficiency
  11. Postnatal lethality of embryos with combined platelet – fibrinogen deficiency
  12. Residual vaso-occlusive function in embryos with individual platelet or fibrinogen deficiency, but not in embryos with combined platelet-fibrinogen deficiency
  13. The essential role of maternal fibrinogen in placental function is independent of platelets
  14. Embryonic, not maternal, NF-E2-deficiency reduces placental vascularization
  15. Discussion
  16. Acknowledgements
  17. References

Mice lacking fibrinogen [11], the transcription factor NF-E2 [15,21,22], and the signal transducer Gαq [14] have been previously described. Mice with deficits in NF-E2, fibrinogen, or both were generated by interbreeding fibrinogen-deficient and NF-E2-deficient mice. These animals were backcrossed to a C57Bl/6 background at least seven generations, reducing the 129Sv contribution at loci other than fibrinogen and NF-E2 to less than 1%. Mice with single and combined deficits in Gαq and fibrinogen were generated in a similar fashion in a mixed C57BL/6/129 background, and therefore sibling controls were used for all experiments involving these particular mice. Genotypes were determined by PCR using genomic DNA obtained either from the embryo proper or tail/ear biopsies as previously described [11,14,15].

Yolk sac vessel injury model

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Yolk sac vessel injury model
  7. Histology
  8. Platelet count
  9. Results
  10. Normal development in embryos with combined platelet and fibrinogen deficiency
  11. Postnatal lethality of embryos with combined platelet – fibrinogen deficiency
  12. Residual vaso-occlusive function in embryos with individual platelet or fibrinogen deficiency, but not in embryos with combined platelet-fibrinogen deficiency
  13. The essential role of maternal fibrinogen in placental function is independent of platelets
  14. Embryonic, not maternal, NF-E2-deficiency reduces placental vascularization
  15. Discussion
  16. Acknowledgements
  17. References

Following a methodology adapted from Ni et al. [18], embryos with single and combined deficits in fibrinogen and NF-E2 were isolated shortly before birth (i.e. at day 18.0 after detection of a vaginal plug), placed in a Petri dish, and covered with PBS. Only embryos with entirely intact yolk sacs were used, and the analysis was confined to superficial yolk sac vessels with an average vessel diameter of 0.2 mm (range 0.15–0.25 mm). PBS was removed from the Petri dish to expose the blood vessels above the fluid level. A 7 mm diameter circle of Whatman 3 M paper was immersed for 10 s into a 1 m FeCl3 solution, and then placed on the yolk sac for exactly 1 min. The area was rinsed with PBS and the fluid level in the Petri dish replenished to cover the yolk sac. Thrombus formation in the injured yolk sac blood vessels was monitored using a stereomicroscope (Nikon SMZ-U) with an attached video camera for a period of 10 min after application of FeCl3. The viability of the embryo at this time was ascertained by the presence of pulsatile blood flow in the umbilical cord and embryonic heart. Blood flow in yolk sac vessels was qualitatively categorized as either normal, sluggish, or absent, based on visual monitoring of cell movement in the vessel lumen. Sluggish blood flow was defined as transitory blood vessel occlusion caused by intravascular thrombi. The presence of predominately white or predominately red-appearing thrombi (as judged by visual inspection) was recorded. Embryos with the following allele combinations were analyzed: Fib+/NF-E2+ [embryos expressing both fibrinogen (Fib+/+ or Fib+/–) and platelets (NF-E2+/+ or NF-E2+/–)], Fib+/NF-E2–[embryos expressing fibrinogen only (Fib+/+ or Fib+/–, NF-E2–/–)], Fib–/NF-E2+ [embryos with platelets only (NF-E2+/+ or NF-E2+/–, Fib–/–)], or Fib–/NF-E2–[embryos without fibrinogen and platelets (NF-E2–/–, Fib–/–)]. Blood in the yolk sac vasculature is exclusively of embryonic origin, and therefore thrombus formation was only correlated with the embryonic genotype. Statview Software (SAS Institute, Cary, NC, USA) was used for statistical analyses (unpaired t-test or chi-squared test as indicated).

Histology

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Yolk sac vessel injury model
  7. Histology
  8. Platelet count
  9. Results
  10. Normal development in embryos with combined platelet and fibrinogen deficiency
  11. Postnatal lethality of embryos with combined platelet – fibrinogen deficiency
  12. Residual vaso-occlusive function in embryos with individual platelet or fibrinogen deficiency, but not in embryos with combined platelet-fibrinogen deficiency
  13. The essential role of maternal fibrinogen in placental function is independent of platelets
  14. Embryonic, not maternal, NF-E2-deficiency reduces placental vascularization
  15. Discussion
  16. Acknowledgements
  17. References

Tissues were fixed in 10% neutral buffered formalin and processed for paraffin embedding and sectioning. Immunohistochemical detection of fibrin(ogen) with a polyclonal rabbit antihuman fibrinogen antibody (Dako Corp.) was performed as previously described [23]. Immunohistochemical detection of platelets was performed with polyclonal rat anti-mouse GPIb antibody (gift of B. Nieswandt, University of Würzburg, Germany) and secondary antibody/horseradish peroxidase conjugates (Vectastain ABC, Vector Laboratories). Vascular density in the placental labyrinthine layer was determined using hematoxylin/eosin stained sections. With each embryo, three images were captured from three different sections (total nine images per embryo) and the relative area covered by blood vessels was determined with Metamorph Image analysis software (Universal Imaging Corporation, West Chester, PA, USA).

Platelet count

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Yolk sac vessel injury model
  7. Histology
  8. Platelet count
  9. Results
  10. Normal development in embryos with combined platelet and fibrinogen deficiency
  11. Postnatal lethality of embryos with combined platelet – fibrinogen deficiency
  12. Residual vaso-occlusive function in embryos with individual platelet or fibrinogen deficiency, but not in embryos with combined platelet-fibrinogen deficiency
  13. The essential role of maternal fibrinogen in placental function is independent of platelets
  14. Embryonic, not maternal, NF-E2-deficiency reduces placental vascularization
  15. Discussion
  16. Acknowledgements
  17. References

Blood samples were collected from the inferior vena cava into 3.2% sodium citrate. Automated platelet counts were performed using a Heska VetABC Diff Haematology Analyzer (Heska, CO, USA) calibrated for mouse samples. For FACS analyzes erythrocytes were lysed in 100 µL of anticoagulated whole blood (PharM Lyse, PharMingen), platelets labeled with a FITC-conjugated rat anti-mouse αIIb antibody (PharMingen) and FITC-positive platelets were counted (FACScan, BD PharMingen, CA, USA).

Normal development in embryos with combined platelet and fibrinogen deficiency

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Yolk sac vessel injury model
  7. Histology
  8. Platelet count
  9. Results
  10. Normal development in embryos with combined platelet and fibrinogen deficiency
  11. Postnatal lethality of embryos with combined platelet – fibrinogen deficiency
  12. Residual vaso-occlusive function in embryos with individual platelet or fibrinogen deficiency, but not in embryos with combined platelet-fibrinogen deficiency
  13. The essential role of maternal fibrinogen in placental function is independent of platelets
  14. Embryonic, not maternal, NF-E2-deficiency reduces placental vascularization
  15. Discussion
  16. Acknowledgements
  17. References

Afibrinogenemic mice lacking the fibrinogen Aα-chain [11] were crossed with either Gαq knockout mice [14] or with NF-E2 knockout mice [15] to generate second generation offspring with a compound deficiency in fibrinogen and platelet function. Mice lacking the α-subunit of the heterotrimeric guanine-nucleotide-binding protein Gαq (Gαq–/– mice) have normal platelet numbers, but platelets are unresponsive to physiologically relevant agonists (including ADP, collagen and thrombin) [12]. Absence of the transcription factor NF-E2 (NF-E2–/– mice) causes a defect in megakaryocyte maturation that results in a complete absence of normal platelets. Residual platelet-like fragments generated from NF-E2-null megakaryocytes appear to be unresponsive to thrombin [21]. Thus, Gαq- and NF-E2-null mice represent two non-identical, yet complementary hemostatic mutants with a qualitative and quantitative platelet defect, respectively. Hybrid animals with combined deficits in platelet function and fibrinogen provided an opportunity to test whether the fibrinogen–platelet interaction leading to thrombus formation – the traditional primary endpoint of hemostasis – is required for embryonic development.

Mating of mice hemizygous for both Gαq and fibrinogen produced the expected number of Gαq–/–/Fib–/– embryos at 18.5 days post coitum (p.c.), assuming Mendelian inheritance (i.e, ∼1 in 16 or ∼6.25%) (Table 1). Likewise, crosses between NF-E2+/–/Fib+/– females and NF-E2+/–/Fib–/– males yielded double knockout embryos (NF-E2–/–/Fib–/–) with a normal Mendelian frequency of approximately one in eight (Table 2). Consistent with previous results [11,14,15], all embryos with single deficits in either fibrinogen or platelets also succeeded in developing to term.

Table 1.  Distribution of genotypes form Gαq+/− X Gαq+/−/Fib+/− crosses
AgeTotalGenotype Distribution
Gαq(+/+)(+/+)(+/+)(+/−)(+/−)(+/−)(−/−)(−/−)(−/−)
Fib(+/+)(−/−)(+/−)(+/+)(+/−)(−/−)(+/−)(+/+)(−/−)
%16.256.2512.512.52512.512.56.256.25
  1. 1. Percentages based on a Mendelian inheritance pattern.

  2. 2. Expected numbers based on Mendelian distribution.

  3. 3. P > 0.9 for expected vs. observed (Chi squared analysis).

  4. 4. P < 0.001 for expected vs. observed (Chi squared analysis) based on the assumption that 100% of Gαq+/−/Fib+/− are alive at 21 days.

  5. 5. Expected numbers were calculated based on the assumption that 100% of the Gαq+/−/Fib+/− mice survived to weaning.

  6. 6. E18.5 refers to embryos 18.5 days post conception and D21 refers to live pups 21 days after birth.

E18.5 Observed 215n (%)18 (8.4%)11 (5.1%) 22 (10.2%) 29 (13.5%) 54 (25%) 22 (10.2%) 28 (13%)16 (7.4%)153 (7.1%)
E18.5 Expected2 n1313 26 26 52 26 261313
D21 Observed 694n (%)72 (10.4%)274 (3.9%)136 (19.7%)125 (18.1%)250 (36.2%) 244 (3.5%) 424 (6.1%)184 (2.6%) 04 (0%)
D21 Expected51000n62.562.512512525012512562.562.5
Table 2.  Distribution of genotypes from NF-E2+/−/Fib+/− X NF-E2+/−/Fib−/−
AgeTotalGenotype Distribution
NF-E2(+)(−/−)(+)(−/−)
Fib(+/−)(+/−)(−/−)(−/−)
%137.512.537.512.5
  1. 1. Percentages based on a Mendelian inheritance pattern.

  2. 2. Expected number of embryos retrieved 18.5 days after conception, based on Mendelian inheritance of alleles.

E18.5 Observed215n (%)35 (38%)11 (11.9%)35 (38%)11 (11.9%)
E18.5 Expeted2 n34.511.534.511.5

Doubly deficient NF-E2–/–/Fib–/– and Gαq–/–/Fib–/– embryos isolated at day 18.5 p.c. were indistinguishable from wild-type animals at a gross morphological level (Fig. 1A,D). However, reproducing previous findings by others [15], NF-E2-null embryos were on average slightly smaller at term, as compared with wild-type embryos (Fig. 1G). In contrast, the size of Gαq-null embryos at term was indistinguishable from that of Gαq-expressing littermates. Histological analysis of thoracic and abdominal organs and the brain revealed no genotype-dependent abnormalities (Fig. 1B,C,E,F and data not shown). The yolk sac vasculature of NF-E2–/–/Fib–/– and Gαq–/–/Fib–/– embryos appeared macroscopically and microscopically intact and lacked signs of hemorrhage (Fig. 1D). Localized hemorrhagic lesions in the embryo proper were seen in less than 5% of all NF-E2–/–/Fib–/– and Gαq–/–/Fib–/– embryos, but these appeared to be due to investigator manipulation and occurred with a similar frequency in animals with isolated deficiency of fibrinogen, Gαq, or NF-E2, as well as in control animals.

image

Figure 1. Normal appearance of embryos with combined fibrinogen and platelet defects. Embryos lacking both NF-E2 and fibrinogen (D) appear grossly normal and lack signs of hemorrhage when compared with NF-E2 and fibrinogen expressing littermates (A). Note the normal appearing yolk sac vessels in both panel A and D (arrows). Both large vessel and microvascular development were unaffected by combined defects in NF-E2 and fibrinogen. Shown are representative photomicrographs of hematoxylin and eosin stained heart (B, E) and lung tissue (C, F). Note the normal appearing ventricles (arrows), atria (double arrow), and large thoracic blood vessels (pointed arrowheads) all with intact endothelium. Small blood vessels and lung capillaries were also intact (C, F arrowhead). (G) The average size of NF-E2–/– mice is reduced at term in comparison to NF-E2 expressing littermates. The horizontal bars represent 0.4 mm and 0.02 mm in C, G and E, F, respectively.

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These findings document that the combined loss of thrombin-mediated conversion of fibrinogen to fibrin and of hemostatic platelet function is entirely compatible with intrauterine development to term. The simultaneous loss of fibrinogen and platelet function neither reproduces the developmental defects occurring in mice with defective thrombin generation [1–5,10], nor resembles the developmental failure of mice with unfettered thrombin consumption [7–9].

Postnatal lethality of embryos with combined platelet – fibrinogen deficiency

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Yolk sac vessel injury model
  7. Histology
  8. Platelet count
  9. Results
  10. Normal development in embryos with combined platelet and fibrinogen deficiency
  11. Postnatal lethality of embryos with combined platelet – fibrinogen deficiency
  12. Residual vaso-occlusive function in embryos with individual platelet or fibrinogen deficiency, but not in embryos with combined platelet-fibrinogen deficiency
  13. The essential role of maternal fibrinogen in placental function is independent of platelets
  14. Embryonic, not maternal, NF-E2-deficiency reduces placental vascularization
  15. Discussion
  16. Acknowledgements
  17. References

Confirming previous observations by others [11,14,15], about half of mice with isolated deficiency of Gαq, NF-E2 or fibrinogen survived until weaning age (21 days; data not shown and Table 1). In contrast, mice with combined platelet-fibrinogen deficiency never survived beyond their first 48–72 h of life. Monitoring of litters after delivery lead to the identification of live-born Fib–/–/NF-E2–/– and Fib–/–/Gαq–/– pups that died within hours of birth with signs of severe hemorrhage (Fig. 2). The pattern of bleeding was similar to that previously described for mice with single deficits in platelets or fibrinogen, most commonly intra-abdominal hemorrhage. A significant proportion of these mice also had evidence of subcutaneous bleeding in the head and neck region. Occasionally soft tissue bleeding around joints, pericardial bleeding and liver hematomas were observed in newborn double knockout mice.

image

Figure 2. Appearance of newborn double knockout mice lacking functional platelets and fibrinogen. (A) Example of a pale appearing, but living NF-E2–/–/Fib–/– neonate with signs of hemorrhage (double arrow), which later died. (B) Dead embryos with single and combined deficits in NF-E2 and fibrinogen display pallor and hemorrhage with a typical distribution; subcutaneous over the head (pointed arrowheads), intra-abdominal (star), and peri-articular (arrow). (C) Gross appearance of live mice with single and combined deficits in Gαq and fibrinogen collected within hours of birth. Note the areas of intra-abdominal (*) and cranial subcutaneous hemorrhage (arrowhead), which is observed in mice with single and combined deficits in Gαq and fibrinogen. Also highlighted is a peri-articular hemorrhage (arrow).

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The inability of mice with a combined loss of fibrinogen and platelet function to control hemorrhagic events provides clear evidence for biologically significant residual hemostatic function in mice with isolated fibrinogen or platelet deficits. The residual hemostatic function associated with either clotting function or platelets can suffice to control bleeding resulting from vaginal delivery and/or minor trauma ex utero.

Residual vaso-occlusive function in embryos with individual platelet or fibrinogen deficiency, but not in embryos with combined platelet-fibrinogen deficiency

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Yolk sac vessel injury model
  7. Histology
  8. Platelet count
  9. Results
  10. Normal development in embryos with combined platelet and fibrinogen deficiency
  11. Postnatal lethality of embryos with combined platelet – fibrinogen deficiency
  12. Residual vaso-occlusive function in embryos with individual platelet or fibrinogen deficiency, but not in embryos with combined platelet-fibrinogen deficiency
  13. The essential role of maternal fibrinogen in placental function is independent of platelets
  14. Embryonic, not maternal, NF-E2-deficiency reduces placental vascularization
  15. Discussion
  16. Acknowledgements
  17. References

In light of the above findings, we examined whether the successful intrauterine development of Fib–/–/NF-E2–/– and of Fib–/–/Gαq–/– mice may be explained by any residual ability to form a thrombus, or to form some kind of a functional thrombus substitute. To this end, in vivo thrombus formation, and qualitative changes in blood flow in response to FeCl3-induced injury were examined in yolk sac blood vessels of day 18.5 p.c. embryos with single and combined deficits in NF-E2 and fibrinogen by intravital microscopy (see Methods yolk sac vessel injury).

Formation of mural thrombi with minimal reduction of blood flow was noted within the first minute after application of FeCl3 in control mice and in fibrinogen-deficient mice, but not in platelet-deficient NF-E2 mice (not shown). After 10 min, blood flow was overtly diminished in 73% of blood vessels in control embryos, in 80% of blood vessels in Fib–/– mice embryos, but only in 36% of blood vessels in NF-E2–/– mice (Fig. 3A,C). Within the entire 10 min observation period, flow completely ceased in 30% of vessels in control embryos, as compared with 20% of vessels in embryos lacking fibrinogen alone, and 26% of vessels in embryos lacking platelets alone. In striking contrast, blood flow consistently remained unaltered in FeCl3-treated yolk sac vessels of NF-E2–/–/Fib–/– embryos. These functional differences between mice of each genotype correlated with qualitative differences in overall thrombus composition. Control embryos exhibited mixed platelet fibrin thrombi, as evidenced by immuno-detection of platelets and fibrin with specific antibodies (Fig. 3B). As predicted, thrombi in mice with isolated fibrinogen- or platelet-deficiency consisted predominantly of platelets or fibrin, respectively. Composition was also reflected in the gross appearance of thrombi, i.e. predominately red (platelet-poor) in NF-E2-null mice, predominantly white (platelet-rich) in fibrinogen-null mice, and variable appearance in control mice. Platelet-deficient NF-E2-null embryos were still able to form occlusive fibrin clots (see above), but the absolute incidence of (occlusive and non-occlusive) thrombosis (67%) was significantly reduced as compared with fibrinogen-null mice and control mice, in which all vessels exhibited mural thrombi. Interestingly, the fibrin-rich occlusions formed in NF-E2-null mice were highly unstable; less than half of the fibrin aggregates (42%) remained localized. The majority of these platelet-free thrombi completely detached over the observation period. Most importantly, there was a complete absence of any kind of occlusive or non-occlusive thrombus in NF-E2–/–/Fib–/– mice, demonstrating that mice with combined platelet and fibrinogen deficiency have no residual ability to form occlusive intravascular deposits in vivo, consistent with a complete loss of hemostatic capacity.

image

Figure 3. Absence of in vivo blood clot/thrombus formation in NF-E2–/–/Fib–/– embryos following FeCl3 injury. (A) Captured video images of before (0 min) and 1 min, and 10 min after yolk sac vessel injury (FeCl3 exposure). Examples of predominately white (arrow) or red appearing (double arrow) thrombi are shown. Note the absence of intravascular thrombi in NF-E2–/–/Fib–/– embryos. (B) The presence of intravascular platelet (arrow) and/or fibrin(ogen) (double arrow) aggregates was confirmed by immunohistochemistry in tissue samples obtained at the 10-min time point. Notably, no platelet or fibrinogen aggregates are discernible in NF-E2–/–/Fib–/– mice. Horizontal bar represents 0.01 mm (C) Quantitation of the percentage of yolk sac vessels from control mice or mice with single and combined deficits in NF-E2 and fibrinogen which maintained normal blood after FeCl3 challenge. Each data point represents the mean of observations of at least 25 vessels from each genotype with standard deviations. All of the vessels examined in NF-E2–/–/Fib–/– embryos (◆) maintained normal blood flow after FeCl3 challenge. This was significantly greater (*P < 0.005) than the percentage of vessels occluded in either control (▪), NF-E2–/– (•) or Fib–/– (bsl00066) after parallel FeCl3 challenges.

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The essential role of maternal fibrinogen in placental function is independent of platelets

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Yolk sac vessel injury model
  7. Histology
  8. Platelet count
  9. Results
  10. Normal development in embryos with combined platelet and fibrinogen deficiency
  11. Postnatal lethality of embryos with combined platelet – fibrinogen deficiency
  12. Residual vaso-occlusive function in embryos with individual platelet or fibrinogen deficiency, but not in embryos with combined platelet-fibrinogen deficiency
  13. The essential role of maternal fibrinogen in placental function is independent of platelets
  14. Embryonic, not maternal, NF-E2-deficiency reduces placental vascularization
  15. Discussion
  16. Acknowledgements
  17. References

The presence of fibrinogen in maternal blood is absolutely required for the successful maintenance of pregnancy in mice [11,12], as well as in humans [19]. In mice, maternal afibrinogenemia causes fatal intrauterine bleeding around day 10 of gestation, due to hemorrhage from placental blood vessels. To investigate whether this essential function of fibrinogen in the placenta involves the interaction of fibrin(ogen) with platelets, we examined the effect of maternal platelet-deficiency on pregnancy outcome. NF-E2–/– females mated with NF-E2+/– or NF-E2–/– males successfully carried pregnancies to term, and in some cases gave rise to multiple litters. No significant difference in average litter-size was observed in NF-E2–/– females when compared with NF-E2+/– females, regardless of the genotype of the breeding partner (data not shown). The absence of platelets in NF-E2–/– females used for these experiments was confirmed after weaning by automated and manual platelet count and by FACS analyses using anti-mouse GPIIb antibodies (data not shown).

Consistent with their general reproductive success, pregnant NF-E2-null females did not exhibit vaginal bleeding or hemorrhage into the uterine cavity (Fig. 4A,B). However, analyzes of placental tissue sections obtained from embryos at day 14.5 p.c. and 18.0 p.c. showed engorged lacunar spaces within the labyrinthine layer of the placenta filled exclusively with maternal blood (as indicated by the absence of nucleated red cells at day 14.5 p.c. (Fig. 4C–I). These abnormalities occurred in approximately half of the placentae in NF-E2-null females, and presence of these lesions did not correlate with embryonic genotype. The above histological analysis did not provide further insights into whether these blood-filled spaces are the consequence of vascular leakage, or reflect an altered architecture of the maternal blood vessels.

image

Figure 4. Placental hemorrhage in the absence of maternal platelets. (A and B) No hemorrhage into the intrauterine cavity is noticed in the presence (NF-E2+/–) or absence (NF-E2–/–) of maternal platelets at day 18.5 p.c.; (C–I) Hemorrhage into the labyrinthine layer of the placenta occurred in the absence of maternal platelets (E, F, H, I) regardless of the presence or absence of embryonic NF-E2. Survival of embryos or mothers was not compromised despite the prominent placental hemorrhage observed in many individuals (C). Genotype shown in (A) and (B) indicates the maternal genotype, genotypes of placenta whole mount pictures and histology in D and G, E and H, or F and I are indicated above the pictures. (G–I) H & E stains; size bar: 0.5 mm.

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The outcome of this experiment shows (i) that the essential role of fibrin(ogen) in the placenta does not strictly depend on its interaction with platelets; (ii) that platelets are required for maintaining the architecture, and possibly the integrity, of the maternal component of the placental vascular bed; and (iii) that the complete loss of hemostatic platelet function is nevertheless compatible with successful pregnancy outcome.

Embryonic, not maternal, NF-E2-deficiency reduces placental vascularization

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Yolk sac vessel injury model
  7. Histology
  8. Platelet count
  9. Results
  10. Normal development in embryos with combined platelet and fibrinogen deficiency
  11. Postnatal lethality of embryos with combined platelet – fibrinogen deficiency
  12. Residual vaso-occlusive function in embryos with individual platelet or fibrinogen deficiency, but not in embryos with combined platelet-fibrinogen deficiency
  13. The essential role of maternal fibrinogen in placental function is independent of platelets
  14. Embryonic, not maternal, NF-E2-deficiency reduces placental vascularization
  15. Discussion
  16. Acknowledgements
  17. References

As described by others [15] and confirmed in the current analysis (see above), term NF-E2-null embryos were smaller than NF-E2-expressing littermates, indicating an intrauterine retardation of growth. Histological survey of placental tissue sections suggested an altered cyto-architecture within the placental labyrinthine layer in NF-E2-null mice. The labyrinth seemed to contain smaller blood filled spaces (Fig. 5A,B); this apparent reduction of perfused cross-sectional area was confirmed by computer-assisted, quantitative image analyses (Fig. 5C). On the other hand, the number of blood vessels per cross-sectional area was identical in mutant and wild-type mice (Fig. 5D). These architectural abnormalities of the vascular bed in the placental labyrinthine layer were apparent both in the day 14.5 and in the term 18.5 p.c. placenta. Unlike the placental hemorrhage described above, this particular phenotype was independent of the maternal genotype, and correlated strictly with embryonic NF-E2 deficiency. The above abnormalities were observed even in the absence of placental hemorrhage (maternal genotype NF-E2+/–), excluding the possibility that this phenotype is the consequence of tissue compression secondary to placental hemorrhage. A corresponding analysis of placentas from age-matched Gαq–/– embryos did not demonstrate any significant differences as compared with controls (Fig. 5E–G). These data suggest that embryonic, but not maternal NF-E2 deficiency causes alterations in the cyto-architecture of the placental labyrinth associated with reduced perfusion, which may then lead to diminished growth of the embryo. Neither intrauterine growth restriction, nor alterations in labyrinth structure were present in Gαq-null mice with a loss of hemostatic platelet function.

image

Figure 5. Abnormal vascularization of the labyrinthine layer of NF-E2-null embryos. (A–C) representative sections of the placental labyrinth in NF-E2-expressing (A) and NF-E2-deficient (B) embryos (E 18.5). Quantitative morphometry showed a reduction of perfused cross-sectional area by ∼ 20% at E18.5 (C), however, the number of blood vessels per cross sectional area was identical to that of wild-type mice (D). No reduction of vascular density was observed in the placenta of Gαq-deficient mice relative to controls (E–G). Size bar: 0.02 mm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Yolk sac vessel injury model
  7. Histology
  8. Platelet count
  9. Results
  10. Normal development in embryos with combined platelet and fibrinogen deficiency
  11. Postnatal lethality of embryos with combined platelet – fibrinogen deficiency
  12. Residual vaso-occlusive function in embryos with individual platelet or fibrinogen deficiency, but not in embryos with combined platelet-fibrinogen deficiency
  13. The essential role of maternal fibrinogen in placental function is independent of platelets
  14. Embryonic, not maternal, NF-E2-deficiency reduces placental vascularization
  15. Discussion
  16. Acknowledgements
  17. References

The current work addresses unresolved questions about the role of hemostasis, i.e. the role of fibrinogen and platelets, in mammalian development and reproduction. In particular, we sought to resolve (i) whether a complete loss of hemostatic function provided by platelets and fibrinogen reproduces the lethal mid-gestation failures observed in mice with a defect in either thrombin generation or inhibition, and (ii) whether platelet–fibrinogen interactions are required for the successful maintenance of pregnancy.

Here, we show that the combined loss of thrombin-mediated conversion of fibrinogen to fibrin and of hemostatic platelet function is entirely compatible with intrauterine development to term. This observation dovetails the recent finding that the combined loss of the primary platelet thrombin receptor, PAR-4, and fibrinogen does not impair embryogenesis [17], but in addition rules out the possibility that platelet activation by agonists other than thrombin could account for the developmental success of animals lacking both PAR-4 and fibrinogen. The yet to be fully defined mechanism underlying the failure of blood vessel development in the yolk sac of mice lacking prothrombin or mediators of thrombin generation must therefore indeed be distinct from the role of thrombin in thrombus formation. The normal development of mice lacking both of the key targets of thrombin in the context of hemostasis points toward PAR-mediated thrombin signaling in endothelial cells as the primary thrombin target important in vascular development [16,24–26].

Our data also provide conclusive evidence that the developmental defects in mice with deficits in natural anticoagulant pathways [7–9] are not caused by the loss of fibrinogen- and platelet-dependent hemostatic capacity that occurs secondary to the consumption of coagulation factors. Rather, the normal intrauterine development of mice lacking both fibrinogen [11] and platelet function [14,15,22] strongly indicates that PAR-mediated signaling may also be at the root of the embryonic failure observed in mice with deficits in anticoagulant factors. Maintenance of vascular integrity might require constitutive PAR-engagement, whereby cessation of signaling could provoke vascular instability. Cessation of signaling could result from the progressive, consumptive depletion of ligands. Conversely, excessive ligand production, without complete consumption, might desensitize the same receptor pathways, producing a similar suppression of PAR-dependent signaling. Furthermore, coagulation components such as FXa or bioactive components released from platelets could exert a direct detrimental effect on vascular integrity, for example by inducing uncompensated changes in vascular permeability and endothelial viability. These potential pathways, i.e. deranged PAR-signaling and ‘coagulotoxicity’, are not mutually exclusive, but may compromise vascular function in a cooperative manner. Lack of PAR-mediated stability signals may sensitize the blood vessel to the coagulation insult, or the latter might interfere with PAR-dependent repair mechanisms [27]. While the precise pathological mechanism is still unresolved, our results make it clear that a complete loss of hemostatic function is not sufficient to reproduce the developmental defects occurring in mice with defective thrombin generation, or that in mice with unfettered thrombin consumption.

Despite the normal intrauterine development of mice with combined platelet- and fibrinogen deficiency (Fib–/–/NF-E2–/–), and of mice with combined fibrinogen-deficiency and a loss of hemostatic platelet function (Fib–/–/Gαq–/– mice) this ‘success’ is rather short-lived, as these mice do not survive beyond the first few days of life after birth. The uniform postnatal lethality of mice lacking both fibrinogen and platelets contrasts the potential for long-term survival of afibrinogenemic mice [11], and that of animals lacking platelets (NF-E2–/– mice) [15], or having platelets with severely compromised hemostatic function (Gαq–/– mice) [14]. In theory, one could argue that the sudden development of hemorrhage may be caused by the sudden dissociation of fetal and maternal circulation occurring at birth, and the concomitant loss of some residual fibrinogen function provided via transplacental transfer of maternal fibrinogen. However, this explanation is extremely unlikely, since such a transfer would at best yield embryonic fibrinogen concentrations five orders of magnitude below normal levels [11]. Even if one presumes that fibrinogen is transferred just below this detection limit, such concentrations would range orders of magnitude below the Km for the fibrinogen–thrombin interaction [28] and therefore would not be of biological significance. Furthermore, residual platelet function has essentially been excluded in the case of NF-E2 deficient mice with a quantitative platelet deficit [21], and the absence of ‘residual clotting function’ was persuasively established from the lack of any local matrix deposition in the FeCl3-yolk sac injury model. Thus, the most straightforward explanation for the rapid onset of fatal bleeding complications in the neonatal period is that vaginal delivery and/or the continued ex utero exposure to minor trauma to joints and other critical sites provide novel challenges that the animals are unable to cope with. Therefore, the above findings provide a direct experimental demonstration that platelets and fibrinogen alone can provide physiologically relevant hemostatic function, but that a concurrent deficit in both components results in a complete loss of hemostatic capacity. This notion is supported by the current analysis of the hemostatic response to chemical injury of yolk sac blood vessels.

The observed formation of unstable platelet thrombi in the absence of fibrinogen is congruent with previous results obtained in blood vessels of adult fibrinogen-deficient mice [18], and extends these finding to the embryonic (yolk sac) vasculature. Conversely, the ability of NF-E2-deficient mice to form occlusive blood clots is a novel finding. The majority of these clots in yolk sac blood vessels were unstable and rapidly detached, and these findings likely cannot be extrapolated to vessels with a different geometry or flow conditions. Nevertheless, fibrinogen-mediated clot formation must confer physiologically relevant hemostatic function, since only the combined loss of fibrinogen and platelet function causes hemorrhage of a severity that is incompatible with postnatal survival. These data further demonstrate that other blood components, like fibronectin and leukocytes, cannot provide effective hemostatic function in the absence of platelets and fibrinogen.

Our analysis further provides novel insights into the pathogenesis of pregnancy loss caused by afibrinogenemia [11,19]. We demonstrate that platelet-deficient NF-E2/females, unlike afibrinogenemic mice, are able to successfully carry pregnancy to term, and deliver even multiple litters without succumbing to fatal postpartum bleeding. This implies that the essential role of fibrin(ogen) in the placenta does not depend entirely on its interaction with platelets, and that platelet-dependent hemostasis is not strictly required for successful reproduction. These observations in gene-targeted mice are congruent with the clinical experience: Afibrinogenemia in pregnant women results in hemorrhage and fetal loss during early gestation [19,20], while pregnant women with severe platelets defects, such as Glanzman thrombasthenia [29], Bernhard–Soulier syndrome [30], or a congenital absence of platelets [31] generally experience uneventful pregnancies until term. In light of our above demonstration of platelet-independent hemostatic function of fibrinogen, it is difficult to distinguish whether fibrinogen's function in placentation reflects fibrinogen's hemostatic function, interactions of fibrinogen not related to hemostasis, or both. On a histological level, hemorrhagic lesions in the placenta of fibrinogen-deficient mothers [11] appear very similar to the lesions seen in NF-E2-null mothers; yet, virtually all fibrinogen-null females bleed to death, whereas vaginal bleeding or draining of blood into the uterine cavity are not observed in NF-E2-null mothers. This implies that the loss of hemostatic platelet function does not breach the overall continuity of the vascular bed in the placenta, while fibrinogen-deficiency causes a complete integrity breakdown and fatal hemorrhage. Moreover, this function of fibrinogen must be dependent on its conversion to fibrin, since inhibition of coagulation with heparin or warfarin precisely reproduces the consequences of fibrinogen-deficiency [32]. Fibrinogen may also have a non-haemostatic, platelet-independent function by anchoring the placenta to the maternal decidua. In the absence of fibrinogen, but not in the absence of platelets, the fetal aspect of the placenta detaches from the decidua, thereby separating the feto-placental unit from the uterine wall [11,12]. This particular aspect of fibrinogen function in the placenta may depend on its interaction with integrins, or other fibrinogen receptors such as VE-cadherin and ICAM-1 [33–36], which are expressed by fetal trophoblast cells and by maternal endometrium [37–39].

Finally, we show that the intrauterine growth restriction of platelet-deficient NF-E2-knockout mice is caused by a defect in the architecture of the placental labyrinth that results in reduced placental perfusion. Importantly, this defect is not reproduced in Gαq-null mice, and is therefore unlikely due to the loss of hemostatic platelet function. This notion is further supported by the lack of placental abnormalities in a variety of other established mouse mutants with profound platelet defects, including the loss of Par-3 and Par-4 mediated platelet activation [25,26]. In contrast to the placental hemorrhage phenotype observed in fibrinogen- and NF-E2-deficient mothers, the defects in the labyrinthine layer vasculature in the placenta of NF-E2 deficient embryos strictly correlate with the fetal genotype, and occur independently of placental hemorrhage. This suggests that the defects in the placenta of NF-E2-deficient embryos reflect a defect intrinsic to the embryo-derived components of the placenta, i.e. placental trophoblast cells or endothelium. For example, NF-E2 might have some unknown role in trophoblast cells where this transcription factor is indeed expressed (author's unpublished observations). While the mechanism by which NF-E2 contributes to placental development remains to be investigated, our findings show that the intrauterine growth restriction of NF-E2-null mice is likely caused by reduced placental perfusion, and thus impaired feto-maternal exchange of nutrients.

In summary, our work has led to several novel insights into the function of platelets and fibrinogen in embryogenesis and reproduction. First, the above data formally demonstrate that the developmental defects in mice lacking critical hemostatic factors cannot be attributed to the loss of hemostatic capacity. Second, we show that fibrinogen can provide platelet-independent, biologically meaningful hemostatic function, which enables mice with quantitative or qualitative platelet defects to survive well into adulthood. Third, our findings suggest that the essential role of fibrinogen in preventing fatal mid-gestation hemorrhage from the placenta is mechanistically distinct from its function in mediating the formation of fibrin(ogen)-platelet aggregates. Finally, we provide evidence that the earlier described intrauterine growth restriction of NF-E2-deficient embryos cannot be attributed to the loss of hemostatic platelet function, but rather reflects a previously unknown role of this transcription factor in placental development.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Mice
  6. Yolk sac vessel injury model
  7. Histology
  8. Platelet count
  9. Results
  10. Normal development in embryos with combined platelet and fibrinogen deficiency
  11. Postnatal lethality of embryos with combined platelet – fibrinogen deficiency
  12. Residual vaso-occlusive function in embryos with individual platelet or fibrinogen deficiency, but not in embryos with combined platelet-fibrinogen deficiency
  13. The essential role of maternal fibrinogen in placental function is independent of platelets
  14. Embryonic, not maternal, NF-E2-deficiency reduces placental vascularization
  15. Discussion
  16. Acknowledgements
  17. References
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