Our findings reveal several new aspects of the association between maternal thrombophilia and fetal loss. First, it lends direct experimental support to the notion that the FvL carrier status of the mother increases the risk of pregnancy failure, thereby establishing a cause–effect relationship for the epidemiologic association. Second, it demonstrates that hemostatic regulators on fetal trophoblast cells are dominant risk modifiers of fetal loss in FvL carriers. Thbd and Procr, allelic variants of which are suspected risk modifiers in human thrombophilia (21
), are experimentally validated in the current mouse model as modulators of pregnancy success in FvL mothers. The experimental demonstration of a synergistic adverse effect of maternal and fetal prothrombotic mutations in causing pregnancy failure has direct implications for the risk stratification of patient populations. It also validates the approach taken in the “NOHA first” study (26
), which systematically includes analysis of the paternal (in lieu of the fetal) genome to more accurately interpret the epidemiological association between maternal thrombophilia and fetal loss. Third, it establishes a novel and unique animal model of fetal loss associated with maternal inherited thrombophilia. Using this animal model, we have identified the Par4 receptor on maternal platelets as a key component of the pathogenic mechanism underlying fetal loss in FvL mice and have determined that, at least in mice, fetal loss occurs in the absence of overt thrombosis. The relevance of our observations for human FvL carriers remains to be determined.
Based on placental pathology, the pathogenesis of FvL-associated fetal loss in mice appears distinct from the complement-driven mechanism operating in fetal loss induced by antiphospholipid antibodies (27
). Consistent with this notion, platelet depletion did not ameliorate fetal loss in the antiphospholipid model, and anti-C5 antibody treatment was insufficient to rescue fetal loss in the FvL model (unpublished data).
In striking contrast to fetal loss caused by reduced function or expression of Thbd or Procr on trophoblast cells in FvL mothers, the combination of the same risk factors does not cause acute thrombosis in the systemic vasculature. The reverse is true for Tfpi, where lethal thrombosis ensues when heterozygous Tfpi deficiency is combined with FvL homozygosity in the same animal (18
) but is inconsequential with respect to pregnancy outcome. The absence of a measurable consequence of embryonic Tfpi heterozygosity on pregnancy outcome in FvL mothers may reflect a substantial contribution of maternally derived plasma Tfpi in local hemostasis at the fetomaternal interface. Collectively, these observations demonstrate a marked discrepancy of consequences (i.e., thrombosis vs. pregnancy loss) triggered by the interaction of thrombosis risk factors in different vascular beds and suggest that the effect of a given risk modifier on venous or arterial thrombosis is not necessarily predictive of pregnancy outcome.
The placental abnormalities of embryos with reduced Thbd activity carried by FvL mothers and those associated with Thbd-null embryos (15
) share characteristic features suggestive of a common underlying pathogenic mechanism. Both occur in the absence of overt thrombosis. Embryos with reduced Thbd activity in FvL mothers can be rescued by platelet depletion at E7.5 but not at E9.5, indicating that the pathogenic insult has already occurred by E9.5. Growth retardation of Thbd-null embryos is observed as early as E8.5, suggesting a similar time of pathogenic insult. In both cases, placental morphogenesis is disrupted at the time of labyrinth layer formation. The combined results from the analysis of fetal loss in these two models suggest that the expression of tissue factor on placental trophoblast cells provides a constitutive procoagulant stimulus in the placental vascular bed (15
). The protein C anticoagulant pathway sustained by expression of Thbd and Procr on trophoblast cells appears to be necessary to suppress this stimulus. Localized disruption of this pathway at the fetomaternal interface—either caused by the complete lack of Thbd or Procr from trophoblast cells or a combination of fetal and maternal risk factors—permits amplification of the coagulation reaction and formation of activated clotting factors, including thrombin. We find that platelets and Par4 are critical mediators of placental failure. The impairment of placental growth and morphogenesis is therefore caused by a process similar to the initial tissue factor– and thrombin-dependent stages of arterial thrombosis, likely involving platelet activation, but does not require thrombotic occlusion. Possible explanations for the lack of occlusive thrombi in the setting of an active coagulation cascade include inhibition of ADP-driven platelet aggregation caused by abundant expression of ecto-ATPDase/CD39 on the trophoblast cell surface (19
The nature of the mechanism by which activation of maternal platelets causes pregnancy failure is unknown, but the possibility that platelet-released factors negatively influence placental development or function is an obvious candidate. Peptide mediators with (anti)angiogenic activity have been suggested to alter trophoblast function and contribute to the etiology of pregnancy disorders such as preeclampsia (28
). Of note, platelets have been shown to adhere to endovascular trophoblasts within the lumen of spiral arteries in normal pregnancy (30
), indicating that the recruitment of platelets to the fetomaternal interface is a physiological process that links platelet function to the regulation of placental development.
It is noteworthy that fetal loss in FvL animals occurs in the absence of overt thrombosis in the mother's systemic vasculature and is caused by a highly localized defect precipitated by the fetal genotype, which affects individual placental beds. Local dysregulation of the protein C anticoagulant pathway at the fetomaternal interface alone (Thbd−/−
embryos) is also sufficient to cause fetal loss in a mother with near-normal hemostatic function (Thbd+/−
). Complete disruption of the protein C pathway (≤3% of protein C), on the other hand, is accompanied by active thrombotic disease, consumptive coagulopathy, and a severe proinflammatory phenotype (31
). These animals suffer fetal genotype-independent early pregnancy loss (E6.5) with a markedly different disease etiology caused by the pronounced secondary effects of acute protein C deficiency in the mother. Increasing maternal protein C to 18% of wild-type levels overcomes systemic thrombosis and inflammation and restores normal pregnancies (31
In summary, we have presented animal models of placental malfunction and fetal loss secondary to inherited defects in the protein C anticoagulant pathway, including a clinically relevant gene defect (FvL) in the mother. The loss of ThbdProPro embryos in FvL mothers correlates with the early fetal loss observed in women with the Leiden mutation. We also observed a partial loss of embryos with a heterozygous defect in Thbd function (ThbdPro+) later in pregnancy (around E12.5, correlating with Leiden-associated late fetal loss in women) that we did not characterize further in this study. The early fetal loss in FvL mice is preceded by disrupted placental growth and morphogenesis during developmental events that lead to the formation of a functional labyrinth and is mediated through a platelet and Par4 receptor–driven mechanism but does not involve formation of occlusive thrombi in the placenta. The described animal model of thrombophilia-associated fetal loss provides an opportunity to test the efficacy of antithrombotic treatments and, possibly, of antiplatelet treatments targeting the recruitment and activation of platelets in the placental vascular bed.