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

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.