A KINETIC-ANALYSIS OF THE INTERACTION OF HUMAN RECOMBINANT TISSUE FACTOR PATHWAY INHIBITOR WITH FACTOR XA UTILIZING AN IMMUNOASSAY AND THE EFFECT OF ANTITHROMBIN-III HEPARIN ON THE COMPLEX-FORMATION

We have recently shown that a complex formation of tissue factor pathw ay inhibitor (TFPI) and factor Xa (Xa) promotes a clearance of proteog lycans-associated TFPI. In the current studies, the interaction betwee n human recombinant TFPI (h-rTFPI) and Xa were kinetically analyzed by utilizing both a protease inhibitor, p-(amidophenyl) methanesulfonyl fluoride hydrochloride, and a specific enzyme-linked immunosorbent ass ay for the complex of h-rTFPI with Xa, We further investigated the eff ect of antithrombin III on the complex formation between h-rTFPI and X a. We found that the h-rTFPI/Xa complex formed in a time-dependent man ner: the second-order rate constant (K-1) for the complex formation wa s calculated to be 0.86x10(6) M(-1)s(-1). The addition of antithrombin III to the h-rTFPI solution modestly reduced the rate of the complex formation between h-rTFPI and Xa. Heparin strikingly enhanced antithro mbin III's inhibition of Xa and resulted in complete abrogation of the complex formation between h-rTFPI and Xa in the absence or presence o f acidic phospholipids. Furthermore, antithrombin III induced dissocia tion of the preformed h-rTFPI/Xa complex in the presence of heparin. T hese results suggest that in the presence of heparin, antithrombin III interferes with the catabolism of TFPI mediated via Xa. (C) 1998 Else vier Science Ltd.