An Experimental Model of Platelet Activation at the Feto-Maternal Interface

Background and Objectives: A developing fetus relies on the placenta for nutrition and other essential functions. To perform these functions human and rodent placenta forms unusual vascular spaces filled with maternal blood and directly accessible to specialized placental cells, called trophoblast cells. Such vascular spaces are not found anywhere else in the body. Trophoblast cells are of zygotic origin and they express a repertoire of molecules with anticoagulant and anti-platelet activities (PMID 16380449). In addition, they constitutively express Tissue Factor. Given these peculiarities, it is unclear how blood flow is maintained in these vascular spaces. The mechanisms by which activated coagulation causes placental pathology also remain undefined. We have previously developed and characterized a murine model of placental pathology precipitated by combining maternal and fetal prothrombotic mutations at the feto-maternal interface (PMID 17438064). We have shown that placental failure in this model can be prevented by depletion of maternal platelets or genetic ablation of thrombin receptor, Par4. Treatment of the mother with heparin significantly improves pregnancy outcome, but equivalent anticoagulation with fondaparinux, hirudin or direct Xa inhibitors is ineffective (PMID 23325830). We combine genetic tools with platelet transfusion experiments to present new data showing a critical role of Par4 and factor V carried by maternal platelets in placental failure and fetal death. We further show that the mechanism of placental failure, although mediated by maternal platelets, does not require integrin αIIb-mediated aggregation.

Methods and Results: Factor V Leiden and thrombomodulin Pro mice have been previously described (PMID 11110695, 9576763). Both strains are viable in homozygous state for the mutated allele on C57Bl6 genetic background. We combined factor V Leiden mutation in the mother (homozygous FV^Q/Q) with a fetal prothrombotic Glu387Pro mutation in thrombomodulin (homozygous Thbd^Pro/Pro) by crossing FV^Q/QThbd^Pro/+ females with Thbd^Pro/Pro males. Almost all Thbd^Pro/Pro embryos conceived from these pregnancies die in utero due to placental failure (Table 1, row 1). The near complete loss of Thbd^Pro/Pro embryos is maternal genotype specific and is not observed in FV^+/+Thbd^Pro/+ mothers. While >95% antibody-mediated platelet depletion in the mother rescues Thbd^Pro/Pro embryos (Table 1, row 2), we show that partial platelet depletion (~75%) is ineffective (Table 1, row 3). Genetic absence of Par4 in the mother completed rescues pregnancies (Table 1, row 4). We show that transfusion of Par4^+/+FV^Q/Q platelets to 25% of host platelets re-precipitates placental failure and death of Thbd^Pro/Pro embryos (table 1, row 5). In contrast, transfusion of wild type (Par4^+/+FV^+/+) platelets did not precipitate placental failure and death of Thbd^Pro/Pro embryos (Table 1, row 6). Genetic absence of integrin αIIb in the mother did not protect Thbd^Pro/Proembryos from intrauterine death.

Conclusions: We explore pathogenic mechanisms in a murine model of platelet-mediated placental development failure. Our results demonstrate that placental failure in this model is caused by Par4-mediated activation of maternal platelets. The mechanism, however, does not involve αIIb-mediated platelet aggregation. Our results suggest that platelet factor V makes an important contribution to the development of placental pathology. These observations demonstrate the existence of vascular bed specific pathogenic mechanisms. Involvement of platelet secretion and/or recruitment of immune cells is being explored.