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

Abstract

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. and Broze, G.J. (1989) Nature 338, 518-520] by means of site-directed mutagenesis indicated that KPI domain 1 interacts with factor VII(a), 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 VII(a)/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) ~ 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.