A kinetic analysis of the interaction of human recombinant tissue factor pathway inhibitor with factor Xa utilizing and immunoassay and the effect of antithrombin III/heparin on the complex formation.
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.
Kamikubo
Y
Y
The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan. VYG07346@niftyserve.or.jp
Hamuro
T
T
Takemoto
S
S
Nakahara
Y
Y
Kamei
S
S
Nakagaki
T
T
Miyamoto
S
S
Funatsu
A
A
Kato
H
H
D000990:Antithrombin III / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D015951:Factor Xa / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008074:Lipoproteins / Q000378:metabolism*; D046911:Macromolecular Substances; D010743:Phospholipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D008074:Lipoproteins; D046911:Macromolecular Substances; D010743:Phospholipids; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D000990:Antithrombin III; D006493:Heparin; D015951:Factor Xa