Complete TFPI Gene-Disruption Is More Severe Than TFPI Kunitz-1 Gene-Deletion in Mice

Abstract

Tissue factor (TF), an integral membrane protein, binds factor VIIa (FVIIa) and the FVIIa/TF catalytic complex is responsible for the initiation of coagulation and enhances the inflammatory response by inducing cell signaling through protease activated receptor-2. Tissue Factor Pathway Inhibitor (TFPI) is an important endogenous regulator that produces FXa-dependent feedback inhibition of the FVIIa/TF complex and directly inhibits FXa. Through alternative mRNA splicing, TFPI is expressed in several isoforms, which possess unique functional properties and are present at different locations. TFPIα contains three Kunitz-type protease inhibitory domains (K1, K2, K3) and a basic C-terminus. It is a soluble form that produces FVIIa/TF inhibition (through K1) and whose inhibition of FXa (through K2) is dramatically enhanced by protein S (PS, through K3 binding). TFPIβ is a glycosyl phosphatidyl inositol (GPI)-anchored cell surface form (containing K1 and K2) that inhibits FVIIa/TF and inhibits FXa well in the absence of PS. TFPIγ is a soluble two Kunitz domain (K1, K2) form that inhibits FVIIa/TF, but inhibits FXa weakly. It is expressed in the mouse, but not in man.Our initial TFPI gene-disruption targeted deletion of the K1 domain exon and is referred to herein as TFPIK1(-). Due to alternative mRNA splicing, this disrupted gene continues to express forms of TFPI that lack the K1 domain (TFPIΔK1) and thus do not inhibit FVIIa/TF. TFPIK1(-/-) gene-disrupted mice die in utero of intravascular coagulation and a consumptive coagulopathy. Others have shown that TFPIK1(-/-) mice can be rescued through gestation by concomitant FVII deficiency, but they succumb to perinatal consumptive coagulopathy. TFPIK1(-/-) mice are also rescued by low levels of TF, and by deletion of protease-activated receptor-4 (PAR4), a mouse platelet thrombin receptor.Referred to herein as TFPI(-), mice with complete TFPI gene-disruption and devoid of protein expression were generated by deletion of the TFPI exons encoding the signal peptide plus N-terminal peptide and the K1 domain. A western blot of plasma from WT, TFPIK1(+/-), and TFPI(+/-) mice demonstrates a ~50% reduction in normal TFPI in heterozygous animals and the presence of TFPIΔK1 protein in TFPIK1(+/-) plasma, which is absent in TFPI(+/-) mice.The rescue efficiencies of TFPIK1(-/-) and TFPI(-/-) mice at weaning by transgenes Tie2-hTFPIαor Tie2-hTFPIβ which direct endothelial cell and hematopoietic cell expression of low levels of hTFPIαor hTFPIβrespectively, and Tf-mXK1-halb, in which the transferrin promoter (Tf) directs expression of mXK1-halb in the liver are shown in the table below. mXK1-halb is a chimera of the mouse factor X light chain, mouse TFPI K1 and human albumin, designed as a species-select, extended half-life, direct FVIIa/TF inhibitor. The plasma level of mXK1-halb in the Tf-mXK1-halb rescued TFPIK1(-/-) mice is 1.6+0.3 nM, demonstrating that the mXK1-halb chimeric protein possesses significant anti-FVIIa/TF activity. The most striking finding is that the mXK1-halb rescues all of the TFPIK1(-/-) mice and none of the TFPI(-/-) mice. Further, whereas PAR4 gene-deletion reportedly rescues 40% of the TFPIK1(-/-) mice, we have found that it rescues none of the TFPI(-/-) mice.Timed matings show that 62.5% of TFPIK1(-/-) embryos survive to embryonic day 11.5 (E11.5), but that TFPI(-/-) mice do not progress beyond E10.0. Treatment of pregnant dams with rivaroxaban by gavage improved survival of TFPI(-/-) embryos at E11.5 from 0 to 46%. Experiments to define the cause of death in the TFPI(-/-) embryos are in progress.The disparity between the rescue of TFPIK1(-/-) and TFPI(-/-) mice demonstrates a functionally significant in vivo activity for the TFPI Δ K1 proteins in TFPIK1(-/-) mice. This activity is likely directed against FXa or, possibly, some other moiety that is also affected by rivaroxaban. The residual activity of the TF PIΔK 1 proteins confounds the interpretation of previous studies using mice with the TFPI K1 (-) disrupted gene. Extrapolation of results in TFPIK1(-) mice to the TFPI(-) condition is inappropriate. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.