Combined Tissue Factor Pathway Inhibitor (TFPI) and Thrombomodulin (TM) Deficiency Produces an Augmented Hypercoagulable State with Tissue Specific Fibrin Deposition.

Background: TFPI and TM are endothelial associated anticoagulant proteins thought to control hemostasis in specific vascular beds. Mice lacking either TFPI or TM have an intrauterine lethal phenotype. A prothrombotic phenotype has not previously been described in TFPI^+/− mice. TM^pro mice have a Glu to Pro mutation between the fourth and fifth epidermal growth factor domains. This mutation reduces TM expression and disrupts TM dependent activation of protein C. TM^pro/pro mice are viable and fertile and do not exhibit spontaneous thrombosis on the C57/Bl6 background. TFPI^+/−/TM^pro/pro mice were generated to examine the consequences of TFPI deficiency in the presence of a compounding procoagulant state caused by reduced TM function. TFPI and TM are located 37 centimorgans apart of chromosome 2 resulting in allele segregation in about 40% of meioses. Therefore, breeding studies were performed without defining haplotypes of individual mice.

Methods and Results: TFPI^+/−/TM^pro/pro mice were generated in three crosses in which the mother had a different prothrombotic phenotype. Embryonic lethality was observed in two of the crosses. TFPI^+/−/TM^pro/pro mice are born at less than expected frequency in either TFPI^+/−/TM^pro/+ female with TM^pro/pro male crosses (7/151 or 4.6%) or TM^pro/pro female with TFPI^+/−/TM^pro/+ male crosses (11/129 or 8.5%). Timed matings revealed similar reduced numbers at E18.5, but not E13.5, demonstrating that the mice die in utero during embryonic development. TM^pro/+ are the least prothrombotic mothers that can produce TFPI^+/−/TM^pro/pro mice. In contrast to the other crosses, TFPI^+/−/TM^pro/pro mice are born at near the expected frequency in TM^pro/+ mothers crossed with TFPI^+/−/TM^pro/pro fathers (15/75 or 20%). Our results show that both maternal and fetal genotypes contribute to embryonic survival. Adult TFPI^+/−/TM^pro/pro mice have elevated thrombin-antithrombin complex in plasma (11.4±8.12 ng/ml for TFPI^+/−/TM^pro/pro n=16; 4.82±3.05 ng/ml for TM^pro/pro n=10, p<0.03; wild type and TFPI^+/− had no detectable TAT complex) and increased thrombus volume in an electrical injury model of venous thrombosis (mean thrombus volume of wild type is 0.042±0.03 mm³; of TFPI^+/−/TM^pro/pro 0.125±0.063 mm³ p<0.005). Interestingly, TFPI^+/− mice also have increased thrombus volume in this model (0.092±0.045 p<0.02), demonstrating for the first time a thrombotic phenotype in these mice. A fluorescently labeled cyclic peptide (CGLIIQKNEC) that specifically binds to fibrin-fibronectin complexes was used to detect tissue fibrin deposition (

Conclusions: TFPI^+/−/TM^pro/pro mice display a prothrombotic phenotype with tissue specific fibrin deposition in embryonic development and as adults. They exhibit partial intrauterine lethality when carried by mothers with an underlying prothrombotic state, demonstrating for the first time that TFPI-dependent control of hemostasis in the vascular bed of the placenta fulfills a critical role for successful pregnancy outcome. In addition to the placenta, partial TFPI deficiency interacts with decreased TM function in an organ selective manner within adult mice to produce fibrin deposition in a second specific vascular bed, the brain microcirculation.