MOLECULAR-CLONING, EXPRESSION, AND PARTIAL CHARACTERIZATION OF A 2ND HUMAN TISSUE-FACTOR-PATHWAY INHIBITOR

Previous studies have shown that tissue-factor-pathway inhibitor (TFPI ) is an important regulator of the extrinsic pathway of blood coagulat ion through its ability to inhibit factor Xa and factor VIIa-tissue fa ctor activity. We describe the molecular cloning and expression of a f ull-length cDNA that encodes a molecule, designated TFPI-2, that has a similar overall domain organization and considerable primary amino ac id sequence homology to TFPI. After a 22-residue signal peptide, the m ature protein contains 213 amino acids with 18 cysteines and two canon ical N-linked glycosylation sites. The deduced sequence of mature TFPI -2 revealed a short acidic amino-terminal region, three tandem Kunitz- type domains, and a carboxyl-terminal tail highly enriched in basic am ino acids. Northern analysis indicates that TFPI-2 is transcribed in u mbilical vein endothelial cells, liver, and placenta. TFPI-2 was expre ssed in baby hamster kidney cells and purified from the serum-free con ditioned medium by a combination of heparin-agarose chromatography, Mo no Q FPLC, Mono S FPLC, and Superose 12 FPLC. Purified TFPI-2 migrated as a single band in SDS/PAGE and exhibited a molecular mass of 32 kDa in the presence and absence of reducing agent. The amino-terminal seq uence of recombinant TFPI-2 was identical to that predicted from the c DNA. Despite its structural similarity to TFPI, the purified recombina nt TFPI-2 failed to react with polyclonal anti-TFPI IgG. Preliminary s tudies indicated that purified recombinant TFPI-2 strongly inhibited t he amidolytic activities of trypsin and the factor VIIa-tissue factor complex. In addition, the inhibition of factor VIIa-tissue factor amid olytic activity by recombinant TFPI-2 was markedly enhanced in the pre sence of heparin. TFPI-2 at high concentrations weakly inhibited the a midolytic activity of human factor Xa, but had no measurable effect on the amidolytic activity of human thrombin.