Lethality of Mouse Tissue Factor Pathway Inhibitor Gene Disruption Is Rescued By Transgenic Human Tissue Factor Pathway Inhibitor Knock in

Background: Tissue factor pathway inhibitor (TFPI) is a key regulator of the extrinsic coagulation pathway. It inhibits FXa generation by forming a quaternary complex containing factor VIIa (FVIIa), tissue factor (TF), factor Xa (FXa), and TFPI. Two TFPI isoforms, TFPI alpha (TFPI a) and TFPI beta (TFPI b), have been identified, which differ in their C-terminal part due to alternative mRNA splicing events. TFPI a consists of three Kunitz domains (KD), while TFPI b contains two KDs and a C terminal GPI anchor linking the protein to endothelial cell surface. Deletion of the first Kunitz domain of TFPI, which is present in TFPI a and TFPI b in mice is known to be incompatible with viability due to intrauterine lethality (Huang et al., 1997). Aim: To generate transgenic humanized TFPI mice in which mouse (m)-TFPI is entirely replaced by human (hu)-TFPI, in order to facilitate analysis of specific anti hu-TFPI antagonists without interference from m-TFPI. Methods: Integration of the targeting vector, consisting of the m TFPI signal sequence, followed by the human TFPI cDNA and subsequent breeding analysis, was followed by genomic PCR. A sophisticated breeding strategy was used to entirely delete m-TFPI exon 4, which encodes KD1, in humanized transgenic mice. Expression of hu-/m-TFPI a and b mRNAs was analyzed by reverse transcription, cloning, and sequencing of the obtained DNA fragments. Protein levels of hu- and m-TFPI in plasma of transgenic and wild-type (wt) mice were analyzed using species specific ELISAs. Immunoprecipitation experiments in plasma and various mouse tissues are being performed to obtain more information on the presence and distribution of the hu-TFPI protein in transgenic mice. Results: Homozygous humanized TFPI mice were viable and exhibited no obvious abnormalities. Animals showed normal litter size with equal numbers of female and male pups. Genomic PCRs revealed proper integration of the targeting vector into the mouse chromosome and the homozygous status with the expected deletion of m-TFPI exon 4. Expression analyses of humanized TFPI mice on mRNA level demonstrated the absence of full length m-TFPI a and the presence of the humanized TFPI mRNA. Alternative spliced m-TFPI b messages lacking exons three and four were identified, likely leading to a nonfunctional protein. Full length hu-TFPI a mRNA was detected in various tissues in the humanized TFPI mice. The TFPI protein level in plasma from humanized mice was below the detection limit of the ELISA and at least ~300 fold below that for wt mice. Conclusion: Low levels of hu-TFPI may compensate the function of m-TFPI in vivo and circumvent embryonic lethality. Furthermore, we established a new mouse model which allows the regulation of physiologic and pathologic pathways to be assessed at TFPI plasma concentrations below the limit of detection. Disclosures Hoellriegl: Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment.