title: Structural requirements for tissue factor pathway inhibitor interactions with factor Xa and heparin.
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 gamma-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.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Division of Hematology/Oncology, Jewish Hospital, Washington University Medical Center, St Louis, MO 63110.
authors.forename: R
authors.initials: R
authors.lastname: Wesselschmidt
authors.forename: K
authors.initials: K
authors.lastname: Likert
authors.forename: Z
authors.initials: Z
authors.lastname: Huang
authors.forename: L
authors.initials: L
authors.lastname: MacPhail
authors.forename: G J
authors.initials: GJ
authors.lastname: Broze
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D015943:Factor Va / Q000494:pharmacology; D065427:Factor Xa Inhibitors; D017353:Gene Deletion; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010743:Phospholipids / Q000494:pharmacology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000494:pharmacology
chemical_list: D065427:Factor Xa Inhibitors; D008074:Lipoproteins; D010743:Phospholipids; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D015943:Factor Va; D006493:Heparin; D002118:Calcium