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

• Published in: Thrombosis research Vol. 89; no. 4; pp. 179 - 186
• Main Authors: Kamikubo, Yu-ichi, Hamuro, Tsutomu, Takemoto, Sumiyo, Nakahara, Yo, Kamei, Shintaro, Nakagaki, Tomohiro, Miyamoto, Seiji, Funatsu, Akinobu, Kato, Hisao
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Ltd 15.02.1998; Elsevier Science
• Subjects: Antithrombin III; Antithrombin III - metabolism; Biological and medical sciences; Blood coagulation. Blood cells; Enzyme-Linked Immunosorbent Assay; Factor Xa; Factor Xa - metabolism; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods, hemostasis, fibrinolysis; Heparin; Heparin - metabolism; Humans; In Vitro Techniques; Kinetics; Lipoproteins - metabolism; Macromolecular Substances; Molecular and cellular biology; Phospholipids - metabolism; Recombinant Proteins - metabolism; Tissue factor pathway inhibitor; Factor Xa; Heparin; Antithrombin III; Tissue factor pathway inhibitor; Blood coagulation; Serine endopeptidases; Enzyme; Tissue factor; Coagulation Factor Xa; Environmental factor; Molecular interaction; Metabolic inhibitor; Immunological method; Peptidases; Investigation method; Hydrolases; Recombinant protein
• ISSN: 0049-3848; 1879-2472
• DOI: 10.1016/S0049-3848(98)00003-6

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 (K 1) for the complex formation was calculated to be 0.86×10 6 M −1s −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.