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We have recently shown that a complex formation of tissue factor pathway inhibitor (TFPI) and factor Xa (Xa) promotes a clearance of proteoglycans-associated TFPI. In the current studies, the interaction between 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 assay for the complex of h-rTFPI with Xa. We further investigated the effect of antithrombin III on the complex formation between h-rTFPI and Xa. We found that the h-rTFPI/Xa complex formed in a time-dependent manner: the second-order rate constant (K1) for the complex formation was 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 antithrombin III's inhibition of Xa and resulted in complete abrogation of the complex formation between h-rTFPI and Xa in the absence or presence of acidic phospholipids. Furthermore, antithrombin III induced dissociation of the preformed h-rTFPI/Xa complex in the presence of heparin. These results suggest that in the presence of heparin, antithrombin III interferes with the catabolism of TFPI mediated via Xa.

In our current study, we examined the antithrombotic effect of Chinese hamster ovary cell-derived human recombinant tissue factor pathway inhibitor (h-rTFPI) by intravenous injection of h-rTFPI with or without antithrombin into endotoxin-treated rats. An injection of h-rTFPI at a high dose (4 mg/kg of h-rTFPI or three doses of 1 mg/kg) significantly prevented the decrease of fibrinogen and factor VIII and the increase of fibrin/fibrinogen degradation products and glutamic-pyruvic transaminase in rats, while a single injection of 1 mg/kg of h-rTFPI or 250 U/kg of antithrombin did not significantly prevent intravascular coagulation. However, a simultaneous injection of 1 mg/kg of h-rTFPI and 250 U/kg of antithrombin did significantly prevent intravascular coagulation. From the studies on the clearance rate and immunohistochemical staining of injected h-rTFPI into normal rats, we found that most of the intravenously-injected h-rTFPI was localized on the central vein and sinusoids in the liver and catabolized via the proteoglycan-mediated pathway with a half-life of 48 min. These results suggest that h-rTFPI and antithrombin prevented endotoxin-induced intravascular coagulation in concert by binding to the vascular walls of the liver and by inhibiting fibrin formation on Kupffer cells in hepatic sinusoids.