title: Molecular cloning, expression, and partial characterization of a second human tissue-factor-pathway inhibitor.
abstract: Previous studies have shown that tissue-factor-pathway inhibitor (TFPI) is an important regulator of the extrinsic pathway of blood coagulation through its ability to inhibit factor Xa and factor VIIa-tissue factor activity. We describe the molecular cloning and expression of a full-length cDNA that encodes a molecule, designated TFPI-2, that has a similar overall domain organization and considerable primary amino acid sequence homology to TFPI. After a 22-residue signal peptide, the mature protein contains 213 amino acids with 18 cysteines and two canonical 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 amino acids. Northern analysis indicates that TFPI-2 is transcribed in umbilical vein endothelial cells, liver, and placenta. TFPI-2 was expressed in baby hamster kidney cells and purified from the serum-free conditioned medium by a combination of heparin-agarose chromatography, Mono 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 sequence of recombinant TFPI-2 was identical to that predicted from the cDNA. Despite its structural similarity to TFPI, the purified recombinant TFPI-2 failed to react with polyclonal anti-TFPI IgG. Preliminary studies indicated that purified recombinant TFPI-2 strongly inhibited the amidolytic activities of trypsin and the factor VIIa-tissue factor complex. In addition, the inhibition of factor VIIa-tissue factor amidolytic activity by recombinant TFPI-2 was markedly enhanced in the presence of heparin. TFPI-2 at high concentrations weakly inhibited the amidolytic activity of human factor Xa, but had no measurable effect on the amidolytic activity of human thrombin.
authors.affiliation: ZymoGenetics, Inc., Seattle, WA 98105.
authors.forename: C A
authors.initials: CA
authors.lastname: Sprecher
authors.forename: W
authors.initials: W
authors.lastname: Kisiel
authors.forename: S
authors.initials: S
authors.lastname: Mathewes
authors.forename: D C
authors.initials: DC
authors.lastname: Foster
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015942:Factor VIIa / Q000037:antagonists & inhibitors; D005796:Genes; D006023:Glycoproteins / Q000235:genetics* / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008074:Lipoproteins / Q000235:genetics* / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011257:Pregnancy Proteins / Q000235:genetics* / Q000494:pharmacology; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D018076:DNA, Complementary; D006023:Glycoproteins; D008074:Lipoproteins; D011257:Pregnancy Proteins; C051928:lipoprotein-associated coagulation inhibitor; C021240:tissue-factor-pathway inhibitor 2; D006493:Heparin; D015942:Factor VIIa