INHIBITORY PROPERTIES OF FULL-LENGTH AND TRUNCATED RECOMBINANT TISSUEFACTOR PATHWAY INHIBITOR (TFPI) - EVIDENCE THAT THE 3RD 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 inhi bitor that consists of three tandem Kunitz-type inhibitor domains flan ked 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 V IIa and factor Xa activity, respectively. In the present study, we hav e compared the inhibitory properties of full-length recombinant TFPI a nd a truncated form of TFPI lacking the third Kunitz-type domain and C OOH-terminal tail (TFPI1-161) with respect to inhibition of factor VII a-tissue factor complexes on the surface of a human bladder carcinoma cell line J82. Full-length TFPI and TFPI1-161 were kinetically indisti nguishable with respect to neutralization of the proteolytic activity of preformed complexes of factor VIIa-tissue factor on the J82 cell su rface in the absence of factor Xa. Equimolar amounts of factor Xa augm ented the anticoagulant activity of both preparations of TFPI to the s ame extent, and both preparations of TFPI were equally effective in in hibiting factor VIIa-tissue factor amidolytic activity in solution pha se. In addition, plasma concentrations of both forms of TFPI, in stoic hiometric complex with factor Xa, inhibited cell surface factor VIIa-t issue factor proteolytic activity markedly faster than plasma levels o f antithrombin III, even in the presence of 1 unit/ml heparin. The res ults of displacement studies suggested slight differences in the affin ity of the two TFPI molecules for the cell surface in that approximate ly 5% of a VIIa.TF.Xa.TFPI1-161 quaternary complex on J82 cells was di splaceable 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 displ aceable under comparable conditions. Pretreatment of the cells with TF PI/Xa alone or together with R152E factor VII, followed by factor VIIa treatment, revealed significant differences in the two TFPI forms wit h respect to the degree with which offered factor VIIa could restore f actor X activation on the cell surface. These differences notwithstand ing, our collective findings indicate that the third Kunitz-type domai n and/or COOH-terminal tail of TFPI is not essential for the inhibitio n of cell surface factor VIIa-tissue factor complexes and suggests tha t TFPI1-161 may be a useful therapeutic agent in the treatment of thro mboembolic episodes.