Inhibitory properties of full-length and truncated recombinant tissue factor pathway inhibitor (TFPI). Evidence that the third Kunitz-type domain of TFPI is not essential for the inhibition of factor VIIa-tissue factor complexes on cell surfaces

Human tissue factor pathway inhibitor (TFPI) is a plasma protease inhibitor that consists of three tandem Kunitz-type inhibitor domains flanked by a negatively charged NH2 terminus and a positively charged COOH-terminal tail. Previous studies have shown that the first and second Kunitz-type domains in TFPI are involved in the inhibition of factor VIIa and factor Xa activity, respectively. In the present study, we have compared the inhibitory properties of full-length recombinant TFPI and a truncated form of TFPI lacking the third Kunitz-type domain and COOH-terminal tail (TFPI1-161) with respect to inhibition of factor VIIa-tissue factor complexes on the surface of a human bladder carcinoma cell line J82. Full-length TFPI and TFPI1-161 were kinetically indistinguishable with respect to neutralization of the proteolytic activity of preformed complexes of factor VIIa-tissue factor on the J82 cell surface in the absence of factor Xa. Equimolar amounts of factor Xa augmented the anticoagulant activity of both preparations of TFPI to the same extent, and both preparations of TFPI were equally effective in inhibiting factor VIIa-tissue factor amidolytic activity in solution phase. In addition, plasma concentrations of both forms of TFPI, in stoichiometric complex with factor Xa, inhibited cell surface factor VIIa-tissue factor proteolytic activity markedly faster than plasma levels of antithrombin III, even in the presence of 1 unit/ml heparin. The results of displacement studies suggested slight differences in the affinity of the two TFPI molecules for the cell surface in that approximately 5% of a VIIa.TF.Xa.TFPI1-161 quaternary complex on J82 cells was displaceable from the cell surface by high concentrations of factor VIIa (10-100 nM), whereas only 1-2% of a VIIa.TF.Xa.TFPI complex was displaceable under comparable conditions. Pretreatment of the cells with TFPI/Xa alone or together with R152E factor VII, followed by factor VIIa treatment, revealed significant differences in the two TFPI forms with respect to the degree with which offered factor VIIa could restore factor X activation on the cell surface. These differences notwithstanding, our collective findings indicate that the third Kunitz-type domain and/or COOH-terminal tail of TFPI is not essential for the inhibition of cell surface factor VIIa-tissue factor complexes and suggests that TFPI1-161 may be a useful therapeutic agent in the treatment of thromboembolic episodes.