Sumiyo Takemoto

Human recombinant tissue-factor pathway inhibitor prevents the proliferation of cultured human neonatal aortic smooth muscle cells

Tissue‐factor pathway inhibitor (TFPI) inhibits the procoagulant activity of the tissue‐factor/factor VIIa complex. It was recently reported that TFPI prevented restenosis following tissue injury in a rabbit atherosclerotic model. In order to clarify the mechanism behind this successful prevention of restenosis, we investigated the direct effect of human recombinant TFPI (h‐rTFPI) on the proliferation of cultured human neonatal aortic smooth muscle cells (hSMC). We found that h‐rTFPI exhibits inhibitory activity toward hSMC proliferation, while h‐rTFPI‐C which lacks the carboxyl (C)‐terminal region does not. Furthermore, we found that h‐rTFPI binds to hSMCs with K d=526 nM but that this binding is inhibited by the addition of the synthetic C‐terminal peptide, Lys254–Met276, to h‐rTFPI. Thus, the interaction of h‐rTFPI with hSMCs mediated via the C‐terminal region is responsible for the anti‐proliferative action of h‐rTFPI. On the basis of these results, we presume that the anti‐proliferative effect of h‐rTFPI in addition to its anticoagulant function plays a significant role in preventing restenosis following tissue injury.

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 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 IIIs 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.