CLONING, EXPRESSION, AND CHARACTERIZATION OF MOUSE-TISSUE FACTOR PATHWAY INHIBITOR (TFPI)

Tissue factor pathway inhibitor (TFPI) acts to regulate the initiation of coagulation by first inhibiting factor Xa. The complex of factor X a/TFPI then inhibits the factor VIIa/tissue factor complex. The cDNA s equences of TFPI from several different species have been previously r eported. A high level of similarity is present among TFPIs at the mole cular level (DNA and protein sequences) as well as in biochemical func tion (inhibition of factor Xa, VIIa/tissue factor). In this report, we used a PCR-based screening method to clone cDNA for full length TFPI from a mouse macrophage cDNA library. Both cDNA and predicted protein sequences show significant homology to the other reported TFPI sequenc es, especially to that of rat. Mouse TFPI has a signal peptide of 28 a mino acid residues followed by the mature protein (in which the signal peptide is removed) which has 278 amino acid residues. Mouse TFPI, li ke that of other species, consists of three tandem Kunitz type domains . Recombinant mouse TFPI was expressed in the human kidney cell line 2 93 and purified for functional assays. When using human clotting facto rs to investigate the inhibition spectrum of mouse TFPI, it was shown that, in addition to human factor Xa, mouse TFPI inhibits human factor s VIIa, IXa, as well as factor XIa. Cloning and expression of the mous e TFPI gene will offer useful information and material for coagulation studies performed in a mouse model system.