Inhibitory Properties of Separate Recombinant Kunitz‐Type‐Protease‐Inhibitor Domains from Tissue‐Factor‐Pathway Inhibitor

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Abstract
Tissue-factor-pathway inhibitor (TFPT) is a multivalent inhibitor with three tandemly arranged Kunitz-type-protease-inhibitor (KPI) domains. Previous studies [Girard, Y. J., Warren, L. A., Novotny, W. F., 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 KPT domain 2 interacts with factor Xa, 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 (Ki= 250 nM), KPI domain 2 inhibited factor Xa (Ki= 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 (Ki= 0.1 nM) and chymotrypsin (Ki= 0.75 nM), whereas KPI domain 1 inhibited plasmin (Ki= 26 nM) and cathepsin G (Ki= 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 Xa inhibition by intact TFPI (Ki≈ 0.02 nM) suggested that regions other than the contact area of the KPT domain, interacted strongly with factor Xa. Secondary-site interactions were crucial for TFPI inhibition of factor Xa but was of little or no importance for its inhibition of trypsin.