Co-Inheritance of FV Leiden and High Levels of FIX Results in Novel Models for Thrombophilia and Spontaneous Fetal Loss.
Authors: Freguia Christian Furlan , Schuettrumpf Joerg , Baila Stefano , Liu Jianhua , Bunte Ralph , Camire Rodney M. , Arruda Valder R. 
Published: October 2019

Abstract
High levels of FIX is emerging as a risk factor for spontaneous venous thrombosis, affecting ~20% of unselect patients and increasing rates of recurrent thromboembolic events. We have generated mice expressing supraphysiological levels of human FIX (up to 400% of normal) but these animals did not develop thrombosis. Therefore, we decided to test whether an additional risk for thrombosis would trigger a prothrombotic phenotype in these mice. Because the FVL mutation is also present in 20% of thrombophilia subjects, we chose FVL mice as model. Breedings included both parents being heterozygous FVL(+/−) but only one was transgenic for FIX (tFIX: 4–8μg/ml plus the endogenous murine FIX). All mice were on C57Bl/6 background. More than 200 newborns were obtained but surprisingly no animal of FVL(+/+)/tFIX genotype was identified. When pregnancies were interrupted at embryonic age of E9.5 to E16.5, no deviation from the expected/observed embryos genotypes was observed. However, the number of reabsorbed embryos increased significantly from 13% (E9.5) to 33% (E.16.5), p<0.02. All FVL(+/+)/tFIX embryos exhibited extensive bruises and hemorrhagic areas which colocalize with markedly fibrin deposition, findings consistent with disseminated intravascular coagulation. Placental analysis (n=18) of FVL(+/+)/tFIX embryos harvested at E12.5 or later demonstrated abnormalities such as excessive fibrin deposition and innumerous apoptotic cells, determined by TUNEL assay, mainly in the labyrinthine trophoblast zone. The maternal genotype clearly influenced the pregnancy outcome since the number of newborns per litter was 2-fold lower when the mother carried tFIX compared with father tFIX (average 4.3 vs. 8.3, respectively; p<0.02). No mice of other genotypes analyzed (n=112) presented similar abnormalities neither in embryos nor in placental tissue. Mice that achieve adulthood developed normally. However, FVL(+/−)tFIX mice (>7 months of age) presented high rate of thrombotic episodes (3/40; 7.5%) followed by 5.2% FVL(−/ −)/tFIX mice, whereas no other age-matched mice (n=74) developed such complication. Monitoring for coagulation activation by serial aPTT, PT, thrombin-antithrombin (TAT) and D-Dimers demonstrated genotype-dependent procoagulant activity. In FVL(+/−)/tFIX or FVL(−/ −)/tFIX mice, levels of TAT (70ng/ml, n=28/group) and D-dimers (230ng/ml, n=17/group) were ~ 2-fold higher (P<0.04), when compared to mice (TAT: 30ng/ml and D-Dimer: 100ng/ml) of other genotypes. Furthermore, FVL(+/−)/tFIX mice presented the highest fibrin deposition determined by Western blot analysis of tissues harvested, followed by FVL(−/ −)/tFIX mice when compared to the other animals (P<0.05). To exclude the possibility of abnormal interaction of human (h) FIX with murine antithrombin (AT), we compared the rate of inactivation of hFIXa by murine or human AT using purified proteins. The results showed that murine AT efficiently inactivated hFIXa in a similar fashion of that determined for human AT. Moreover, early work showed that hFIX efficiently corrected the phenotype of FIX-deficient mice. Therefore, these data revealed that gene-dosage dependent interaction of FVL and high levels of FIX in mice increase rates of spontaneous fetal loss and adulthood thrombosis. These models provide opportunities to better understand and to test therapeutics for thrombophilia-related complications due to common risk factors identified in humans.