Recombinant tissue factor pathway inhibitor induces apoptosis in cultured rat mesangial cells via its Kunitz-3 domain and C-terminal through inhibiting PI3-kinase/Akt pathway.

Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of tissue factor (TF) induced coagulation. In addition to its anticoagulation activity, TFPI has other functions such as antiproliferation and inducing apoptosis. In the present study, we investigated whether or not TFPI induced apoptosis in cultured rat mesangial cells (MsCs) and the possible signal pathway that involved in the apoptotic process. We demonstrated that recombinant TFPI (rTFPI) induced apoptosis in cultured MsCs via its Kunitz-3 domain and C-terminal in a dose- and time-dependent manner by Hoechst 33258 assay, flow cytometry, nucleosomal laddering of DNA, caspase 3 assay. Because the serine/threonine protein kinase Akt has attracted attention as a mediator of survival (anti-apoptotic) signal in MsCs, we investigated the expression of phosphospecific-Akt and its downstream signal phospho-IkappaB-alpha and some other signal molecules like Fas and bcl-2. The results indicated that the process of apoptosis triggered by rTFPI is, at least in part, actively conducted by rat MsCs possibly through PI3-Kinase-Akt signal pathway not by binding to tissue factor. Our findings suggest that rTFPI has the potential usefulness in inducing apoptosis of MsCs under inflammatory conditions.

A novel anti-tissue factor monoclonal antibody with anticoagulant potency derived from synthesized multiple antigenic peptide through blocking FX combination with TF.

Tissue factor (TF) has been implicated in the pathogenesis of various thrombotic disorders. Monoclonal antibodies (mAb) that specifically target TF may have potential as antithrombotic therapy. We designed a unique TF peptide (TFP) that was specific for the binding site to factor X (FX). This peptide was used to develop TF mAb that block the coagulation cascade by interfering with the combination of FX with the TF/FVIIa complex. Chemically synthesized TFP coupled to polylysine matrix was used as multiple antigenic peptide (TF-MAP) and this was used to immunize Balb/c mice for the preparation of hybridomas. One hybridoma cell line released an antibody, named TF4A12, which had high anticoagulant potency (by dilute prothrombin time assay). Western blotting showed that TF4A12 could bind TF-MAP and the soluble TF extracellular domain (sTF(1-219)). Results of FX activation assay and amidolytic activity assay showed that the anticoagulant ability of TF4A12 is due to blocking FX, but not FVII, binding to TF. Our study identified an efficient method of developing TF mAb that could block the coagulation cascade.

[Cloning, expression and biological characterization of hTFPI-2 gene].

OBJECTIVE: To clone the human tissue factor pathway inhibitor-2 (hTFPI-2) gene and express it by using prokaryotic expression system. METHODS: The hTFPI-2 coding region was obtained by RT-PCR from human placenta total RNA. The coding fragment was then inserted into prokaryotic expression vector pET19b and expressed in E. coli BL21 by IPTG induction. The produced inclusion bodies were dissolved by denaturalizing chemicals, purified by ion exchange chromatograph, and refolded in air to form proper disulfide bonds. Chromogenic and gelatin zymography methods were used to evaluate the inhibiting effects of hTFPI-2 on trypsin, plasmin and MMPs individually. The inhibitory activity of hTFPI-2 on fabrisarcoma was investigated by matrigel. RESULTS: The coding fragment of hTFPI-2 was cloned successfully and the protein was expressed as inclusion bodies which account for 20% - 30% of total host protein. The refolded hTFPI-2 could inhibit the invasive ability of fibrisarcoma HT-1080 as well as activity of plasmin, trypsin and MMPs. CONCLUSIONS: The activated hTFPI-2 is obtained by using prokaryotic expressed system effectively.

Molecular design and characterization of recombinant long half-life mutants of human tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) is a physiological inhibitor of extrinsic pathway of coagulation and has biological functions of anticoagulation and anti-inflammation. Although TFPI has been proved to be a good therapeutic agent of sepsis, inflammatory shock, and DIC, the clinical application and therapeutic effects of TFPI are impeded because of its short half-life in vivo. In order to prolong the half-life of TFPI, homology modeling and molecule docking were performed on a computer workstation principally in protein structural biology and binding characteristics between TFPI and its receptor LRP (low-density lipoprotein receptor related protein). Two recombinant long half-life human TFPI mutants coined TFPI-Mut1 and TFPI-Mut4 were designed and expressed in E.coli. In comparison with the wild-type TFPI, TFPI-Mut1 and TFPI-Mut4 presented a few of changes in spatial configuration and a decrease in relative Gibbs free energy of docking complex by 17.3% and 21.5%, respectively, as indicated by a computer simulation. After refolding and purification, anticoagulant activities, anti-TF/FVIIa and anti-FXa activities of the mutants were found to be the same as those of wide-type TFPI. The pharmacokinetics research indicated that alpha phase half-life (t1/2 alpha) of TFPI-Mut1 and TFPI-Mut4 were prolonged 1.33-fold and 1.96-fold respectively, beta phase half-life (t1/2 beta) of TFPI-Mut1 and TFPI-Mut4 were prolonged 1.62-fold and 4.22-fold respectively. These results suggested that TFPI-Mut1 and TFPI-Mut4 maintained the bioactivities of wild-type TFPI, prolonged half-life in vivo simultaneously and were expected for better clinical value and therapeutic effect.