Heparin rescues factor V Leiden-associated placental failure independent of anticoagulation in a murine high-risk pregnancy model

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 demonstrate an anticoagulation-independent role of LMWH in protecting pregnancies and provide 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.

Figure 1

Thbd^Pro/Pro embryos survive pregnancies of FV^Q/QThbd^Pro/+ females if the mother is treated with LMWH or is deficient in Par3. Thbd^Pro/Pro (B,D) and littermate Thbd^Pro/+ (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. Thbd^Pro/Pro embryos are growth restricted in pregnancies of Par3^−/− FV^Q/QThbd^Pro/+ 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 Thbd^Pro/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. Scale bars represent 5 mm in (A-D) and 1 mm in (E-F). 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.

Figure 2

Thbd^Pro/+ embryos and placentae are smaller than littermate Thbd^+/+ controls in pregnancies of FV^Q/QThbd^Pro/+ females mated to Thbd^Pro/+ males. The size distribution of Thbd^Pro/+ and littermate Thbd^+/+ embryos and placentae measured at 15.5 dpc is shown. Lengthwise cross-sectional area (A), length of the embryo (B), and placental cross-sectional area (C) were measured from digital photographs taken at a known magnification and resolution. P values were computed in comparison with Thbd^+/+ controls. No live Thbd^Pro/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 Thbd^Pro/Proembryos, but these are growth retarded in utero and smaller at birth. Pregnancies of Par3^−/−FV^Q/QThbd^Pro/+ females mated to Thbd^Pro/+ males were analyzed at 15.5 dpc. Lengthwise cross-sectional area (A), length of the embryo (B), and placental cross-sectional area (C) were measured from digital photographs taken at a known magnification and resolution. Neonatal weight was taken within 18 hours of birth (D). P values were computed in comparison with Thbd^+/+ controls.