Overexpression of Platelet Tfpiα Rescues TFPI Deficient Embryos from Mid-Gestational, but Not Late Gestational Lethality

Tissue Factor Pathway Inhibitor (TFPI) is an alternatively spliced Kunitz-type protease inhibitor required for murine embryonic development. On a mixed 129-C57BL/6 background, deletion of the first Kunitz (K1) domain of TFPI (Tfpi^tm1Gjb; Tfpi^-/-) results in complete intrauterine lethality that occurs in about 60% of Tfpi^-/- embryos during mid-gestation with the remaining 40% dying during late gestation. We recently discovered that this late gestation lethality is associated with severe cerebrovascular defects that are completely absent in Tfpi^-/- embryos harboring genetic deletion of Factor V (FV), indicating that regulation of FV-dependent procoagulant activity by TFPI is necessary for proper cerebral vascular development. In mice, TFPIα is the only isoform present in platelets, TFPIβ is the major isoform on endothelium, and TFPIγ is the major isoform in plasma. All isoforms inhibit the tissue factor-Factor VIIa (TF-FVIIa) catalytic complex and Factor Xa (FXa) via the K1 and K2 domains, respectively. TFPIα is distinct from other isoforms as it contains a basic C-terminus that enables inhibition of early forms of prothrombinase (FXa-FVa).

To better understand the biological activities of platelet TFPIα (pTFPIα), a transgenic mouse model expressing TFPIα under control of the platelet specific murine Gp1ba promoter was generated on the C57BL/6NCrl background. The transgene (Tg) was bred onto the Tfpi^tm1Gjb congenic C57BL/6J (B6J) background to examine how overexpression of pTFPIα alters embryonic survival of Tfpi^-/- mice.

Out of 407 offspring derived by crossing Tfpi^+/- females to Tfpi^+/- Tg⁺ males, the expected 50% Tg transmission ratio was observed in Tfpi^+/+ and Tfpi^+/- mice at wean, yet no Tfpi^-/- Tg⁺ or Tfpi^-/- Tg⁰ mice were produced. When examining Tg function in assays measuring TF-FVIIa mediated generation of FXa, it was found that Tfpi^+/+ and Tfpi^+/- Tg⁺ mice had 4- to 5-fold increased platelet TFPI inhibitory activity over Tg⁰ littermates, while plasma TFPI activity and antigen were unaltered indicating functional expression and storage of transgenic TFPIα within platelets.

To study the impact of pTFPIα overexpression on Tfpi^-/- survival, 120 late gestation embryos derived from Tfpi^+/- x Tfpi^+/- Tg⁺ backcrosses were harvested between E14.5-18. In comparison to the 5% frequency of Tfpi^-/- 129-C57BL/6 embryos observed at this time interval, the Tfpi alleles were normally distributed with 28% Tfpi^+/+, 49% Tfpi^+/- and 23% Tfpi^-/-. Of the Tfpi^-/- embryos, 86% were Tg⁺ and 14% were Tg⁰. However, only 21% of Tfpi^+/+ embryos were Tg⁺ (50% expected) suggesting that the Tg caused lethality in Tfpi^+/+ embryos, a finding not observed in the weaning age cohort. Given this, we hypothesized that underlying genetic factors were complicating survival outcomes. PCR-based DNA walking uncovered that non-independent assortment played a major role in the disproportionate embryo frequencies, as the Tg integrated into an intergenic region on chromosome 2, ~36.4 megabases proximal to, and in linkage with, the endogenous Tfpi locus. Additionally, by intercrossing Tfpi^+/- mice on the congenic B6J background, we found that 14% of 359 embryos harvested between E14.5-16.5 were Tfpi^-/- , which was higher than the 5% observed in 129-C57BL/6 embryos, indicating that strain specific genetic interactions modulate Tfpi^-/- mid-gestational lethality. After accounting for recombination rates, male breeder haplotypes, and expected frequencies at late gestation, pTFPIα overexpression completely suppressed mid-gestational lethality of Tfpi^-/- embryos. Histological analyses revealed that the Tg had no effect on development of cerebrovascular defects in Tfpi^-/- embryos during late gestation consistent with their lack of survival to wean.

In conclusion, underlying genetic factors complicated our analyses illustrating the importance of meticulously characterizing transgenic mouse models to avoid spurious interpretation of results. After accounting for these unexpected genetic effects, our analyses indicated that pTFPIα overexpression totally rescued Tfpi^-/- embryos from mid-gestational lethality. However, late gestational lethality persisted likely because overexpression of pTFPIα did not prevent the severe cerebrovascular developmental defects occurring at this stage of development in Tfpi^-/- embryos.