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Thromb Haemost 2012; 108(02): 266-276
DOI: 10.1160/TH11-12-0902
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

The Kunitz 1 and Kunitz 3 domains of tissue factor pathway inhibitor are required for efficient inhibition of factor Xa

Sameera Peraramelli
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, the Netherlands
,
Dennis P. L. Suylen
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, the Netherlands
,
Jan Rosing
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, the Netherlands
,
Tilman M. Hackeng
1   Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht, the Netherlands
› Author Affiliations
Further Information

Publication History

Received: 02 January 2012

Accepted after major revision: 01 May 2012

Publication Date:
25 November 2017 (online)

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Summary

Tissue factor pathway inhibitor (TFPI) is a slow tight-binding inhibitor that inhibits factor (F)Xa in a biphasic fashion: a rapid formation of loose FXa•TFPI encounter complex is followed by slow rearrangement into a tight FXa•TFPI* complex in which the Kunitz-2 (K2) domain of TFPI binds and inhibits FXa. In the current study, full-length TFPI (TFPIfl) and various truncated TFPI constructs were used to assess the importance of TFPI domains other than K2 in the inhibition of FXa. In the absence of Ca2+ ions, FXa was more effectively inhibited by TFPIfl than Gladomain less FXa. In turn, Ca2+ ions impaired FXa inhibition by TFPIfl but not by TFPI constructs that lack the C-terminus. This suggests that, in absence of Ca2+ ions, interactions between the C-terminus of TFPI and the Gla-domain of FXa promote FXa-inhibition. TFPIfl and K2K3 had similar efficiencies for encounter complex formation. However, K2K3 showed monophasic inhibition instead of biphasic inhibition, indicating absence of rearrangement into a tight complex. K1K2 and TFPI1–161 showed biphasic inhibition, but had less efficient encounter complex formation than TFPIfl. Finally, K2K3 was a 10-fold more efficient FXainhibitor than K2. These results indicate that K3-C-terminus enhances the formation of encounter complex and that K1 is required for isomerisation of the encounter- into tight complex. Since TFPIfl has a 10-fold higher Ki than K2K3-C-terminus, we propose that K1 is not only required for the transition of the loose to the tight FXa•TFPI* complex, but also inhibits FXa•TFPI encounter complex formation. This inhibitory activity is counteracted by K3 and C-terminus.

Keywords

Anticoagulant - TFPI - protein S - factor Xa
 

  • References

  • 1 Butenas S, Mann KG. Blood coagulation. Biochemistry (Mosc) 2002; 67: 3-12.
    PubMedGoogle Scholar
  • 2 Wilcox JN, Smith KM, Schwartz SM. et al. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci USA 1989; 86: 2839-2843.
    CrossrefPubMedGoogle Scholar
  • 3 Morrissey JH, Macik BG, Neuenschwander PF. et al. Quantitation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation. Blood 1993; 81: 734-744.
    PubMedGoogle Scholar
  • 4 Nemerson Y. The reaction between bovine brain tissue factor and factors VII and X. Biochemistry 1966; 05: 601-608.
    CrossrefPubMedGoogle Scholar
  • 5 Osterud B, Rapaport SI. Activation of factor IX by the reaction product of tissue factor and factor VII: additional pathway for initiating blood coagulation. Proc Natl Acad Sci USA 1977; 74: 5260-5264.
    CrossrefPubMedGoogle Scholar
  • 6 Girard TJ, Warren LA, Novotny WF. et al. Functional significance of the Kunitztype inhibitory domains of lipoprotein-associated coagulation inhibitor. Nature 1989; 338: 518-520.
    CrossrefPubMedGoogle Scholar
  • 7 Broze Jr GJ. Tissue factor pathway inhibitor. Thromb Haemost 1995; 74: 90-93.
    PubMedGoogle Scholar
  • 8 van 't Veer C, Golden NJ, Kalafatis M. et al. Inhibitory mechanism of the protein C pathway on tissue factor-induced thrombin generation. Synergistic effect in combination with tissue factor pathway inhibitor. J Biol Chem 1997; 272: 7983-7994.
    CrossrefPubMedGoogle Scholar
  • 9 Wun TC, Kretzmer KK, Girard TJ. et al. 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: 6001-6004.
    PubMedGoogle Scholar
  • 10 Wesselschmidt R, Likert K, Huang Z. et al. Structural requirements for tissue factor pathway inhibitor interactions with factor Xa and heparin. Blood Coagul Fibrinolysis 1993; 04: 661-669.
    CrossrefPubMedGoogle Scholar
  • 11 Ndonwi M, Tuley EA, Broze Jr GJ. The Kunitz-3 domain of TFPI -alpha is required for protein S-dependent enhancement of factor Xa inhibition. Blood 2010; 116: 1344-1351.
    CrossrefPubMedGoogle Scholar
  • 12 Hackeng TM, Sere KM, Tans G. et al. Protein S stimulates inhibition of the tissue factor pathway by tissue factor pathway inhibitor. Proc Natl Acad Sci USA 2006; 103: 3106-3111.
    CrossrefPubMedGoogle Scholar
  • 13 Broze Jr GJ, Warren LA, Novotny WF. et al. 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: 335-343.
    PubMedGoogle Scholar
  • 14 Broze Jr GJ, Girard TJ, Novotny WF. Regulation of coagulation by a multivalent Kunitztype inhibitor. Biochemistry 1990; 29: 7539-7546.
    CrossrefPubMedGoogle Scholar
  • 15 Huang ZF, Wun TC, Broze Jr GJ. Kinetics of factor Xa inhibition by tissue factor pathway inhibitor. J Biol Chem 1993; 268: 26950-26955.
    PubMedGoogle Scholar
  • 16 Morrison JF, Walsh CT. The behavior and significance of slow-binding enzyme inhibitors. Adv Enzymol Relat Areas Mol Biol 1988; 61: 201-301.
    PubMedGoogle Scholar
  • 17 Petersen LC, Bjorn SE, Olsen OH. et al. Inhibitory properties of separate recombinant Kunitz-type-protease-inhibitor domains from tissue-factor-pathway inhibitor. Eur J Biochem 1996; 235: 310-316.
    CrossrefPubMedGoogle Scholar
  • 18 Higuchi DA, Wun TC, Likert KM. et al. The effect of leukocyte elastase on tissue factor pathway inhibitor. Blood 1992; 79: 1712-1719.
    PubMedGoogle Scholar
  • 19 Sere KM, Rosing J, Hackeng TM. Inhibition of thrombin generation by protein S at low procoagulant stimuli: implications for maintenance of the hemostatic balance. Blood 2004; 104: 3624-3630.
    CrossrefPubMedGoogle Scholar
  • 20 Diaz-Collier JA, Palmier MO, Kretzmer KK. et al. Refold and characterization of recombinant tissue factor pathway inhibitor expressed in Escherichia coli. Thromb Haemost 1994; 71: 339-346.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 21 Petersen JG, Meyn G, Rasmussen JS. et al. Characterization of human tissue factor pathway inhibitor variants expressed in Saccharomyces cerevisiae. J Biol Chem 1993; 268: 13344-13351.
    PubMedGoogle Scholar
  • 22 Esnouf MP, Lloyd PH, Jesty J. A method for the simultaneous isolation of factor X and prothrombin from bovine plasma. Biochem J 1973; 131: 781-789.
    CrossrefPubMedGoogle Scholar
  • 23 Schnolzer M, Alewood P, Jones A. et al. In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences. Int J Pept Protein Res 1992; 40: 180-193.
    PubMedGoogle Scholar
  • 24 Hackeng TM, Griffin JH, Dawson PE. Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology. Proc Natl Acad Sci USA 1999; 96: 10068-10073.
    CrossrefPubMedGoogle Scholar
  • 25 Girard TJ, Broze Jr GJ. Tissue factor pathway inhibitor. Methods Enzymol 1993; 222: 195-209.
    PubMedGoogle Scholar
  • 26 Dawson PE, Muir TW, Clark-Lewis I. et al. Synthesis of proteins by native chemical ligation. Science 1994; 266: 776-779.
    CrossrefPubMedGoogle Scholar
  • 27 Ndonwi M, Tuley EA, Broze Jr GJ. The Kunitz-3 domain of TFPI -alpha is required for protein S-dependent enhancement of factor Xa inhibition. Blood 2010; 116: 1344-1351.
    CrossrefPubMedGoogle Scholar
  • 28 Lindhout T, Franssen J, Willems G. Kinetics of the inhibition of tissue factor-factor VIIa by tissue factor pathway inhibitor. Thromb Haemost 1995; 74: 910-915.
    Article in Thieme ConnectPubMedGoogle Scholar