Structural requirements for tissue factor pathway inhibitor interactions with factor Xa and heparin

R. Wesselschmidt, K. Likert, Z. Huang, L. MacPhail, G. J. Broze

Research output: Contribution to journalArticlepeer-review

96 Scopus citations

Abstract

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor, which inhibits factor Xa directly and in a factor Xa dependent manner inhibits the factor VIIa/tissue factor catalytic complex. Altered forms of recombinant TFPI (rTFPI) were tested for their ability to inhibit human factor Xa and bovine γ-carboxyglutamate (Gla)-domainless factor Xa in the presence and absence of calcium ions, heparin, phospholipids, and factor Va. Sequential deletions of the positively charged C-terminus of TFPI produces proteins that have decreasing inhibitory activity against factor Xa as well as decreasing affinity for heparin-agarose. Deletion of the C-terminus distal to Leu181, which eliminates the third Kunitz-type domain, results in the loss of heparin-agarose binding at physiological ionic strength. Furthermore, the entire C-terminal polypeptide, including at least a portion of the third Kunitz-type domain, appears to be involved in heparin binding. Residues 230-241 probably form an alpha helix in which Lys231 and Arg237 within the Kunitz domain and Lys240 and Lys241 could provide a positively charged surface epitope capable of binding heparin. Heparin and Ca2+ together, but not individually, enhance the rate of factor Xa inhibition by full-length TFPI. The effect of heparin is concentration dependent and biphasic (maximal between 0.1 and 1.0 unit/ml) suggesting that the acceleration of factor Xa inhibition occurs at least in part through a 'template' mechanism. Full-length rTFPI inhibits factor Xa in the presence of Ca2+, phospholipids and factor Va more effectively than factor Xa in the presence of Ca2+ alone and is a more potent inhibitor of factor Xa under the former conditions than carboxy-terminal truncated forms of rTFPI. Additional studies show that the acidic N-terminus of TFPI is not required for Xa inhibition and suggest that in a non-physiological system lacking Ca2+ the cluster of basic amino acids near the C-terminus of TFPI may interact with factor Xa in a Gla domain dependent fashion.

Original languageEnglish
Pages (from-to)661-669
Number of pages9
JournalBlood Coagulation and Fibrinolysis
Volume4
Issue number5
DOIs
StatePublished - 1993

Keywords

  • factor Xa
  • heparin
  • tissue factor pathway inhibitor (TFPI)

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