Inhibitory properties of separate recombinant Kunitz-type-protease-inhibitor domains from tissue-factor-pathway inhibitor

Tissue-factor-pathway inhibitor (TFPI) is a multivalent inhibitor with three tandemly arranged Kunitz-type-protease-inhibitor (KPI) domains. Previous studies [Girard, Y. J., Warren, L. A., Novotny, W. E. Likert, K. M., Brown, S. G., Miletich, J. P. & Broze, G. J. (1989) Nature 338, 518-520] by means of site-directed mutagenesis indicated that KPI domain 1 interacts with factor VIIa, that KPI domain 2 interacts with factor X(a), and that KPI domain 3 is apparently without inhibitory function. To elucidate the reaction mechanism of this complex inhibitor, we followed a different approach and studied the inhibitory properties of fragments of TFPI obtained by expression in yeast. Results obtained with TFPI(1-161)-peptide and separate recombinant TFPI-KPI domains 1, 2 and 3 showed that KPI domain 1 inhibited factor VIIa/tissue factor (K-i = 250 nM), KPI domain 2 inhibited factor X(a) (K-i = 90 nM), and that KPI domain 3 was without detectable inhibitory function. Studies with separate KPI domains also showed that KPI domain 2 was mainly responsible for inhibition of trypsin (K-i = 0.1 nM) and chymotrypsin (K-i = 0.75 nM), whereas KPI domain 1 inhibited plasmin (K-i = 26 nM) and cathepsin G (K-i = 200 nM). The structural basis for the interaction between serine proteases and KPI domains is discussed in terms of putative three-dimensional models of the proteins derived by comparative molecular-modelling methods. Studies of factor X(a) inhibition by intact TFPI (K-i approximate to 0.02 nM) suggested that regions other than the contact area of the KPI domain, interacted strongly with factor X(a). Secondary-site interactions were crucial for TFPI inhibition of factor X(a) but was of little or no importance for its inhibition of trypsin.