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Thromb Haemost 1998; 79(02): 306-309
DOI: 10.1055/s-0037-1614983
Letters to the Editor
Schattauer GmbH

Cloning, Expression, and Characterization of Mouse Tissue Factor Pathway Inhibitor (TFPI)

Jen-Yea Chang
1   From the Center for Thrombosis and Hemostasis, University of North Carolina at Chapel Hill, Chapel Hill, USA
,
Dougald M. Monroe
1   From the Center for Thrombosis and Hemostasis, University of North Carolina at Chapel Hill, Chapel Hill, USA
,
Julie A. Oliver
1   From the Center for Thrombosis and Hemostasis, University of North Carolina at Chapel Hill, Chapel Hill, USA
,
Darla K. Liles
1   From the Center for Thrombosis and Hemostasis, University of North Carolina at Chapel Hill, Chapel Hill, USA
,
Harold R. Roberts
1   From the Center for Thrombosis and Hemostasis, University of North Carolina at Chapel Hill, Chapel Hill, USA
› Author Affiliations
Further Information

Publication History

Received 18 June 1997

Accepted after resubmission 10 September 1997

Publication Date:
08 December 2017 (online)

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Summary

Tissue factor pathway inhibitor (TFPI) acts to regulate the initiation of coagulation by first inhibiting factor Xa. The complex of factor Xa/ TFPI then inhibits the factor VIIa/tissue factor complex. The cDNA sequences of TFPI from several different species have been previously reported. A high level of similarity is present among TFPIs at the molecular level (DNA and protein sequences) as well as in biochemical function (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 sequences, especially to that of rat. Mouse TFPI has a signal peptide of 28 amino acid residues followed by the mature protein (in which the signal peptide is removed) which has 278 amino acid residues. Mouse TFPI, like that of other species, consists of three tandem Kunitz type domains. Recombinant mouse TFPI was expressed in the human kidney cell line 293 and purified for functional assays. When using human clotting factors to investigate the inhibition spectrum of mouse TFPI, it was shown that, in addition to human factor Xa, mouse TFPI inhibits human factors VIIa, IXa, as well as factor XIa. Cloning and expression of the mouse TFPI gene will offer useful information and material for coagulation studies performed in a mouse model system.

 

  • References

  • 1 Girard TJ, Warren LA, Novotny WF, Likert KM, Brown SG, Miletich JP, Broze GJ. Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor. Nature 1989; 338: 518-20.
    CrossrefPubMedGoogle Scholar
  • 2 Wesselschmidt W, Likert K, Huang Z, MacPhail L, Broze GJ. Structural requirements for tissue factor pathway inhibitor interactions with factor Xa and heparin. Blood Coag Fibrin 1993; 4: 661-9.
    CrossrefPubMedGoogle Scholar
  • 3 Enjyoji K-i, Miyata T, Kamikubo Y-i, Kato H. Effect of heparin on the inhibition of factor Xa by tissue factor pathway inhibitor: A segment, Gly212-Phe243, of the third Kunitz domain is a heparin-binding site. Biochemistry 1995; 34: 5725-35.
    CrossrefPubMedGoogle Scholar
  • 4 Broze GJ, Warren LA, Novotny WF, Higuchi DA, Girard JJ, Miletich JP. The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: Insight into Its possible mechanism of action. Blood 1988; 71: 335-43.
    PubMedGoogle Scholar
  • 5 Wun T-Z, Kretzmer K, Girard T, Miletich J, Broze JG. Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem kunitz-type inhibitory domains. J Biol Chem 1988; 263: 6001-4.
    PubMedGoogle Scholar
  • 6 Kamei S, Kamikubo Y-i, Hamuro T, Fujimoto H, Ishihara M, Yonemura H, Miyamoto S, Funatsu A, Enjyoji K-i, Abumiya T, Miyata T, Kato H. Amino acid sequence and inhibitory activity of rhesus monkey tissue factor pathway inhibitor (TFPI): comparison with human TFPI. J Biochem 1994; 115: 708-14.
    CrossrefPubMedGoogle Scholar
  • 7 Girard T, Gailani D, Broze JG. Complementary DNA sequencing of canine tissue factor pathway inhibitor reveals a unique nanomeric repetitive sequence between the second and third kunitz domains. Biochem J 1994; 303: 923-8.
    CrossrefPubMedGoogle Scholar
  • 8 Wesselschmidt R, Girard T, Broze JG. cDNA sequence of rabbit lipoprotein-associated coagulation inhibitor. Nucleic Acids Res 1990; 18: 6440.
    CrossrefPubMedGoogle Scholar
  • 9 Enjyoji K-i, Emi M, Mukai T, Kato H. cDNA Cloning and expression of rat tissue factor pathway inhibitor (TFPI). J Biochem 1992; 111: 681-7.
    CrossrefPubMedGoogle Scholar
  • 10 McCord DM, Monroe DM, Smith KJ, Roberts HR. Characterization of the functional defect in factor IXAlabama evidence for a conformational change due to high-affinity calcium binding in the first epidermal growth factor domain. J Biol Chem 1990; 265: 10250-4.
    PubMedGoogle Scholar
  • 11 Lundblad R. A rapid method for the purification of bovine thrombin and the inhibition of purofied enzyme with phenylmethylsulfonyl fluoride. Biochemistry 1971; 10: 2501-6.
    CrossrefPubMedGoogle Scholar
  • 12 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156-9.
    PubMedGoogle Scholar
  • 13 Amaravadi L, King M. A rapid and efficient, nonradioactive method for screening recombinant DNA libraries. BioTechniques 1994; 16: 98-103.
    PubMedGoogle Scholar
  • 14 Sanger F, Nicklen SL, Coulson AR. DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 1977; 74: 5463-7.
    CrossrefPubMedGoogle Scholar
  • 15 Chang J-Y, Stafford DW, Straight DL. The roles of factor VII’s structural domains in tissue factor binding. Biochemistry 1995; 34: 12227-32.
    CrossrefPubMedGoogle Scholar
  • 16 Pedersen A, Nordfang O, Norris F, Wiberg F, Christensen P, Moeller K, Meidahl-Pedersen J, Beck T, Norris K, Hedner U, Kisiel W. Recombinant human extrinsic pathway inhibitor. Production, isolation, and characterization of its inhibitory activity on tissue factor-initiated coagulation reactions. J Biol Chem 1990; 265: 16786-93.
    PubMedGoogle Scholar
  • 17 Wesselschmidt R, Likert K, Girard T, Wun T-C, Broze GJ. Tissue factor pathway inhibitor: The carboxy-terminus is required for optimal inhibition of factor Xa. Blood 1992; 79: 2004-10.
    PubMedGoogle Scholar
  • 18 Sprecher C, Kisiel W, Mathewes S, Foster D. Molecular cloning, expression, and partial characterization of a second human tissue-factor-pathway inhibitor. Proc Natl Acad Sci USA 1994; 91: 3353-7.
    CrossrefPubMedGoogle Scholar
  • 19 Broze GJ, Warren L, Novotny W, Roesch K, Miletich J. Properties of the lipoprotein associated coagulation inhibitor produced by HepG2 cells. Blood 1987; 70: 385a.
    PubMedGoogle Scholar