Maintaining extraembryonic expression allows generation of mice with severe tissue factor pathway inhibitor deficiency

Abstract

Abstract Tissue factor pathway inhibitor (TFPI) is a serine protease with multiple anticoagulant activities. The Kunitz1 (K1) domain of TFPI binds the active site of factor VIIa and is required for inhibition of tissue factor (TF)/factor VIIa catalytic activity. Mice lacking TFPI K1 domain die in utero. TFPI is highly expressed on trophoblast cells of the placenta. We used genetic strategies to selectively ablate exon 4 encoding TFPI K1 domain in the embryo, while maintaining expression in trophoblast cells. This approach resulted in expected Mendelian frequency of TFPI K1 domain–deficient mice. Real-time polymerase chain reaction confirmed 95% to 99% genetic deletion and a similar reduction in transcript expression. Western blotting confirmed the presence of a truncated protein instead of full-length TFPI. Mice with severe TFPI K1 deficiency exhibited elevated thrombin-antithrombin (TAT) levels, frequent fibrin deposition in renal medulla, and increased susceptibility to TF-induced pulmonary embolism. They were fertile, and most lived normal life spans without any overt thrombotic events. Of 43 mice observed, 2 displayed extensive brain ischemia and infarction. We conclude that in contrast to complete absence of TFPI K1 domain, severe deficiency is compatible with in utero development, adult survival, and reproductive functions in mice. Inhibition of TFPI activity is being evaluated as a means of boosting thrombin generation in hemophilia patients. Our results show that in mice severe reduction of TFPI K1 activity is associated with a prothrombotic state without overt developmental outcomes. We note fibrin deposits in the kidney and rare cases of brain ischemia.