Tissue factor pathway inhibitor (TFPI) from different cell lines shows up to 15-fold differences in the ratio of anticoagulant to chromogenic activity. The anticoagulant activity was dependent on the purification procedure used and it was possible to isolate two fractions of recombinant TFPI. Only one of these fractions showed anticoagulant activity comparable with TFPI from normal human plasma, and Western blotting showed that the low-activity fraction did not react with an antibody raised against a peptide of TFPI located near the C-terminal. Analysis by mass spectroscopy of peptides from V8 protease digests showed that C-terminal amino acids could only be identified from the high-activity form, while heterologous fragmentation had taken place in the form with low anticoagulant activity. Previously published studies on TFPI have been performed using material of low anticoagulant activity compared with plasma TFPI, and we suggest that these studies have been performed with material degraded in the C-terminus.

Tissue-factor-pathway inhibitor (TFPT) is a multivalent inhibitor with three tandemly arranged Kunitztype-protease-inhibitor (KPI) domains. Previous studies [Girard, Y. .I., Warren, L. A., Novotny, W, F., Likert, K. M., Brown, S. G., Miletich, J. P. & Broze, G. J. (1989) Nature 338, 518-5201 by means of site-directed mutagenesis indicated that KPI domain 1 interacts with factor VI12,, that KPT domain 2 interacts with factor X,, and that KPJ 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 TFPT-KPT domains 1, 2 and 3 showed that KPI domain 1 inhibited factor VII,/tissue factor (K, = 250 nM), KPI domain 2 inhibited factor X;, ( K , = 90 nM), and that KPT 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 (Ka = 0.1 nM) and chymotrypsin ( K , = 0.75 nM), whereas KPI domain 1 inhibited plasmin (Ki = 26 nM) and cathepsin G (K, = 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;, inhibition by intact TFPI (K, = 0.02 nM) suggested that regions other than the contact area of the KPT domain, interacted strongly with factor X,. Secondary-site interactions were crucial for TFPI inhibition of factor X;, but was of little or no importance for its inhibition of trypsin.Keywords: tissue-factor-pathway inhibitor; Kunitz-type protease inhibitor; factor X ; factor VTT.Injury and vascular-wall damage causes exposure of tissue factor (TF) to the blood stream, and the appearance of this cellbound receptor results in dramatic upregulation of the coagulation cascade (see [I] for review). The initial event in this process is binding of factor VII to T F and its conversion to active factor VII, by factor IX, or X , 121. Auto-activation of factor VTT by factor VII, may also play a significant role [3, 41. TF-bound factor VH&, is also capable of activating factors X and 1X 15, 61. This reciprocal zymogen-activation cycle is central to the triggering of the TF-dependent-coagulation pathway, and once initiated the cycle generates factor X;, which converts prothrombin to thrombin. Ultimately this leads to fibrin-clot formation.Initiation of the TF pathway is under strict control by an inhibitor [7 -91 now designated as tissue-factor-pathway inhibitor (TFPI). Based on the primary structure predicted from cDNA sequence analysis [I 01 and expression in transfected mammalian-cell systems [ l l , 121 detailed knowledge about the inhibitor has been obtained [X, 131. TFPI is a multivalent inhibitor (molecular mass of 40 kDa) with three tandeinly arranged Kunitztype-protease-inhibitor (KPI) domai...

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SummaryAn increasing amount of evidence suggests that coagulation factors VIII and IX play a role not only in the intrinsic but also in the extrinsic pathway of coagulation. In this context the influence of the Extrinsic Pathway Inhibitor (EPI) on the coagulation time of hemophilia plasma lacking FVIII or FIX has been investigated. The coagulation time was measured in a dilute thromboplastin assay. Addition of recombinant EPI (rEPI) prolonged the coagulation time of normal plasma while the addition of an inhibitory antibody against EPI shortened the coagulation time. At low concentrations of thromboplastin the coagulation time of hemophilia plasma was prolonged and at all dilutions of thromboplastin, addition of anti-EPI IgG normalized the coagulation time of a hemophilia plasma. Analysis of 10 individual donor plasma samples and 8 individual hemophilia samples showed that addition of anti-EPI IgG shortened the coagulation time more in hemophilia plasma than in normal plasma. This illustrates the importance of a powerful extrinsic FVII dependent pathway to achieve hemostasis in the case of FVIII or FIX deficiency (hemophilia A and B).

SummaryThe effect of human neutrophil elastase and cathepsin G on recombinant tissue factor pathway inhibitor (TFPI) was investigated. A weak inhibition by TFPI of both elastase (Kx = 0.4 μM) and cathepsin G (Kx = 0.1 μM) was observed. Neutrophil elastase rapidly cleaved TFPI at the Thr87-Thr88 bond situated at the link between Kunitz domains I and II. Cleavage of TFPI by cathepsin G was also observed, but the reaction was much slower and resulted in a number of fragments. Proteolytic cleavage by both elastase and cathepsin G resulted in destruction of inhibitor function with respect to TFPI’s inhibition of factor Xa. Cleavage by neutrophil elastase was capable of restoring factor Xa amidoly-tic activity after its initial inhibition by TFPI. Inhibition of cathepsin G by TFPI was strongly augmented by stoichiometric amounts of factor Xa. However, the augmentation was temporary, presumably due to concomitant cleavage of TFPI by cathepsin G. These observations may have implications for the putative effect of neutrophil leukocyte stimulation on the regulation of the tissue factor-mediated coagulation pathway. Conversely, formation of a factor Xa/TFPI complex may reduce or modulate the proteolytic potential of stimulated leukocytes by temporary inhibition of cathepsin G.