Abstract 290: Circulating Tissue Factor Pathway Inhibitor is Predominantly Derived from Cells which Express Tie2
Background: Tissue factor pathway inhibitor (TFPI) is a proteinase inhibitor with three Kunitz-type domains and is a key regulator of hemostasis and thrombosis through its inhibition of the factor VIIa/tissue factor complex. Genetic disruption of TFPI exon 4 results in intrauterine lethality in mice. As such, the ability to study the biologic and tissue specific effects of TFPI deficiency has been limited. We aimed to define the tissue specific roles of TFPI in adult mice using the Cre-lox strategy in mice.
Methods: Using standard techniques we generated mice with flanking loxP sites surrounding TFPI exon 4 which encodes for the first Kunitz domain. These chimeric mice were used as founders for inbreeding. These mice proved to be fertile and provided germline transmission of the floxed-TFPI allele. The ability of the Cre-lox strategy to recapitulate the knockout was verified when floxed mice were crossed with CMV-Cre mice (expressing Cre ubiquitously) resulting in no mice born (of 66 live births) carrying the homozygously deleted allele. Homozygous mice with this transgenic floxed-TFPI were crossed with the Tie2 Cre+ mice and subsequently inbred to create homozygously floxed mice carrying the Cre recombinase. PCR with primers designed to detect the floxed segment revealed the desired deletion of the exon 4 segment of TFPI in DNA extracted from blood. Sequencing of these PCR products revealed the desired deletion of exon 4. Presence of the deleted mRNA product was confirmed by RT-PCR. Floxed TFPI-Tie2 Cre+ mice with deletion were phenotypically similar to floxed-TFPI littermates including weight, blood counts, and prothrom-bin time. Floxed TFPI-Tie2 Cre+ mice with deletion had a 77% reduction (p<0.05) in circulating TFPI activity as compared with floxed-TFPI littermate controls.
Conclusion: Circulating TFPI levels are derived predominantly from cells which express Tie2 including hematopoietic and endothelial cells. Deletion of TFPI from these cells is consistent with viability and fertility in mice. These mice will provide unique opportunities to study the biology of TFPI in health and disease.