Pregnancy Outcome in Mice with Protein S Complete Deficiency

Introduction:

Pregnancy is associated with a shift of the coagulation balance with increased concentration of procoagulant factors, decreased concentration of some anticoagulants and diminished fibrinolytic activity. These changes provoke a state of hypercoagulability that protects pregnant women from fatal hemorrhage during delivery but predispose them to thromboembolism.

Protein S (PS) is a natural anticoagulant that acts as a cofactor of activated protein C and tissue factor pathway inhibitor. PS complete deficiency leads to life threatening skin necrosis and disseminated intravascular coagulation (DIC) named purpura fulminans (PF) that leads to death if untreated. Pregnant women with partial PS deficiency are at high risk of late fetal loss. The beneficial role of thromboprophylaxis in ameliorating pregnancy outcome in patients with inherited thrombophilia is debated.

Here, we used mice with complete PS deficiency (Pros1^-/-) fully rescued from lethality by the lack of factor VIII (F8^-/-Pros1^-/-). Characterization of F8^-/-Pros1^-/- mice revealed no signs of PF or DIC. These mice were used to investigate the effect of PS complete deficiency on pregnancy.

Methods:

Timed matings were set to generate E9-E17.5 embryos from F8^-/-Pros1^-/-, F8^-/-Pros1^+/+ and F8^+/+Pros1^+/+ pregnant mice (n=3-9/genotype). Embryos viability and genotype were evaluated. At the time of sacrifice, blood was drawn from pregnant female to evaluate blood cells count profile and coagulation parameters (fibrinogen, thrombin anti-thrombin complex; TAT) were measured. Anti-fibrin immunostaining was performed in collected embryos and placentas.

F8^-/-Pros1^-/- pregnant females were treated with low-dose aspirin (200mg/kg Lysine acetylsalicylate) or LMWH (Clexane, 100μg/mL delivered by osmotic pump 0.25 μL/hour) from E7 until E20 to assess if thromboprophylaxis could be beneficial for pregnancy.

Results:

No litters were produced from F8^-/-Pros1^-/- crosses.

F8^-/-Pros1^-/- females were then mate with F8^-/-Pros1^+/- male and sacrificed at different gestational stages. A progressively high number of macerated and necrotic embryos were found after E9-E10 in comparison with pregnantcontrols: 0% (E9-10), 57% (E11-E12), 64% (E14-E15) and 67% (E16-E17). In parallel, number of embryos per gestation decreased in F8^-/-Pros1^-/- compared to F8^-/-Pros1^+/+ females (4±1 vs 9±1 at E11-E12, 3±1 vs 7±1 at E14-E15, 2±1 vs 7±2 at E16-E17, respectively). Macroscopically, the majority of alive embryos showed the presence of hemorrhages without PF. F8^-/-Pros1^-/- genotype was underrepresented among alive embryos (33% vs the expected 50%) and it disappeared from those collected after E12.5.

The evaluation of DIC parameters in F8^-/-Pros1^-/- pregnant females during the period of experienced high embryonic mortality (E12-15) showed a significative reduction of platelet counts (486±105 vs 806±134 G/L F8^-/-Pros1^+/+, P<0.03), fibrinogen (1±0.1 vs 2±0.2 g/L, respectively, P<0.001) and increased TAT (33.2±5.1, 16.6±2.8 ng/L, respectively, P<0.02).

Although these results suggest an activated state of blood coagulation, fibrin staining did not reveal fibrin deposition in lungs, liver and placenta from F8^-/-Pros1^-/- pregnant mice.

Treatment of F8^-/-Pros1^-/- pregnant mice with LMWH or aspirin had a positive pregnancy outcome (n=4/female/treatment). However, litter size was reduced in comparison to F8^-/-Pros1^+/+ control mice. In particular, the aspirin group showed the lowest litter size while the LMWH group had increased pups number (3±1 vs 6±1, P<0.03). F8^-/-Pros1^-/- genotype was rescued only among newborns from LMWH treated group (n=1 over 7). Preliminary data from pups monitoring indicated that some pups were smaller, experienced hemorrhages and died after birth.

Conclusion:

Targeting F8 did not prevent pregnancy loss in F8^-/-Pros1^-/- mice. Only few F8^-/-Pros1^+/- embryos survived until E17 indicating that PS expression in the uteroplacental unit is indispensable for embryos development. F8^-/-Pros1^-/- pregnant mice displayed coagulation activation. However, further investigations are needed if the observed DIC is due to the lack of PS anticoagulant activity or to retained dead embryos. Aspirin or better LMWH partially prevented pregnancy loss indicating that thromboprophylaxis might be beneficial for pregnancy development with very low PS plasma levels.