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Tissue-factor pathway inhibitor (TFPI) inhibits the procoagulant activity of the tissue-factor/factor Vila 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 (hrTFPI) 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 Ä" d = 526 nM but that this binding is inhibited by the addition of the synthetic C-terminal peptide, Lys 254 -Met 276 , 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.

Staphylococcal enterotoxin B (SEB), a toxin produced by Staphylococcus aureus, causes food poisoning and other fatal diseases by inducing high levels of pro-inflammatory cytokines. These cytokines are released from CD4+ T cells and major histocompatibility complex (MHC) class II antigen-presenting cells, which are activated through binding of wild-type (WT) SEB to both the MHC class II molecule and specific T-cell receptor Vbeta chains. Here, we focused on a trypsin/cathepsin cleavage site of WT SEB, which is known to be cleaved in vivo between Lys97 and Lys98, located within the loop region. To know the function of the cleavage, an SEB mutant, in which both of these Lys residues have been changed to Ser, was examined. This mutant showed prolonged tolerance to protease cleavage at a different site between Thr107 and Asp108, and structural analyses revealed no major conformational differences between WT SEB and the mutant protein. However, differential scanning calorimetric analysis showed an increase in enthalpy upon thermal denaturation of the mutant protein, which correlated with the speed of cleavage between Thr107 and Asp108. The mutant protein also had slightly increased affinity for MHC. In the in vivo experiment, the SEB mutant showed lower proliferative response in peripheral blood mononuclear cells and had lower cytokine-induction activity, compared with WT SEB. These results highlight the importance of the flexible loop region for the functional, physical and chemical properties of WT SEB, thus providing insight into the nature of WT SEB that was unrevealed previously.

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