Human Tissue Factor Pathway Inhibitor Research Articles

Recombinant adeno‐associated virus (rAAV) expressing TFPI‐2 inhibits invasion, angiogenesis and tumor growth in a human glioblastoma cell line

Recombinant adeno-associated viruses (rAAV) have become the vector of choice for many gene therapy protocols. rAAVs have a number of attractive features including long-term transgene expression and the ability to transduce both dividing and non-dividing cells. We have shown previously the anti-cancer role of tissue factor pathway inhibitor-2 (TFPI-2), a matrix-associated serine protease inhibitor, in human glioblastomas. As a result of our present study, in which 0.8-kb fragment of human TFPI-2 was cloned into the adeno-associated viral vectors (rAAA-TFPI-2), rAAV-TFPI-2 infection of SNB19 cells significantly increased TFPI-2 as determined by Western blotting. As assessed by spheroid and Matrigel assays, infection of SNB19 cells with rAAV-TFPI-2 significantly reduced migration and invasion in a dose-dependent manner. Tumor spheroids infected with rAAV-TFPI-2 and co-cultured with fetal rat brain aggregates did not invade rat brain aggregates, whereas 90-95% of the mock and AAV-CMV infected cells invaded rat brain aggregates. In vitro angiogenesis studies (tumor cells co-cultured with endothelial cells or endothelial cells seeded on matrigel) showed reduction of capillary-like structure formation in rAAV-TFPI-2-treated cells as compared to parental and mock-transfected cells. In in vivo angiogenesis results demonstrated the formation of microvessels in SNB19 parental cells and this formation was inhibited when the SNB19 cells were infected with rAAV-TFPI-2. Further, we observed a large reduction of tumor growth in SNB19 cells treated with rAAV-TFPI-2 virus injected intracerebrally when compared to controls. Our study demonstrates that rAAV-TFPI-2-mediated gene therapy offers a novel tool for the treatment of brain tumors.

Role of Tissue Factor Pathway Inhibitor-2 in Ovarian Tumor Migration and Invasion

Human tissue factor pathway inhibitor-2 (TFPI-2) is a newly discovered serine protease inhibitor, which inhibits plasmin, trypsin, matrix metalloproteinase (MMPs), but not urokinase, tissue-type plasminogen activators and thrombin. Earlier studies have shown that the production of TFPI-2 is downregulated during the progression of various cancers. The aim of this study was to elucidate the relationship between TFPI-2 expression and ovarian tumor migration and invasion. Human TFPI-2 expression vector pBos-Cite-neo/TFPI-2 was transfected into ovarian tumor cells line A2780. After the transfected cells were screened by G418, transfected and nontransfected cells were examined for TFPI-2 mRNA and protein by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. The number of transfected or nontransfected cells passing through membrane of Boyden chamber was counted as the basis assessing tumor cells migratory and invasive behaviors. Expression of mRNA and protein of TFPI-2 were confirmed in transfected cells. In invasion assay, the number of TFPI-2-expressing cells to traverse a Matrigel-coated membrane was obviously decreased compared with that of nonexpressing cells (59.3+/-6.5 versus 109.7+/-5.5, P< 0.01); while in migration assay, no significant difference was observed between transfected and nontransfected cells (114.7+/-8.6 versus 127.3+/-7.1, P >0.05). Expression of TFPI-2 may strongly inhibit the invasive ability of ovarian tumor cells in vitro, but has no effect on the migratory ability, which provides an experimental basis for treating human ovarian tumor with gene therapy.

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.

Complete Inhibition of Acute Humoral Rejection Using Regulated Expression of Membrane-tethered Anticoagulants on Xenograft Endothelium

Xenotransplantation promises an unlimited supply of organs for clinical transplantation. However, an aggressive humoral immune response continues to limit the survival of pig organs after transplantation into primates. Because intravascular thrombosis and systemic coagulopathy are prominent features of acute humoral xenograft rejection, we hypothesized that expression of anticoagulants on xenogeneic vascular endothelium might inhibit the process. Hearts from novel transgenic mice, expressing membrane-tethered fusion proteins based on human tissue factor pathway inhibitor and hirudin, respectively, were transplanted into rats. In contrast to control non-transgenic mouse hearts, which were all rejected within 3 days, 100% of the organs from both strains of transgenic mice were completely resistant to humoral rejection and survived for more than 100 days when T-cell-mediated rejection was inhibited by administration of ciclosporin A. These results demonstrate the critical role of coagulation in the pathophysiology of acute humoral rejection and the potential for inhibiting rejection by targeting the expression of anticoagulants to graft endothelial cells. This genetic strategy could be applied in a clinically relevant species such as the pig.

Expression and characterization of the first kunitz domain of human tissue factor pathway inhibitor-2

Human tissue factor pathway inhibitor-2 (hTFPI-2) has three kunitz domains whose structure and function are unclear. We expressed the first kunitz domain of hTFPI-2 (hTFPI-2/KD1) as functional form using Pichia pastoris and investigated its characterization. In the experiment, hTFPI-2/KD1 can inhibit the plasmin and trypsin activity and the Ki of hTFPI-2/KD1 towards plasmin (30 nM) and trypsin (50 nM) was determined as 10 and 7 nM by chromogenic assay, respectively. hTFPI-2/KD1 can also inhibit MMP-2 and MMP-9 in zymography assay. Furthermore, the inhibition of hTFPI-2/KD1 to the Matrigel invasion by HT-1080 is also described. This study provides a method to produce hTFPI-2/KD1 efficiently and some insights into the structure and function of hTFPI-2/KD1.

Inhibition of intravascular thrombosis in murine endotoxemia by targeted expression of hirudin and tissue factor pathway inhibitor analogs to activated endothelium

AbstractWe have generated transgenic mice expressing the leech anticoagulant hirudin and human tissue factor pathway inhibitor tethered to the cell surface by fusion with fragments of human CD4 and P-selectin. Expression of the transgenes is under the control of the CD31 (platelet endothelial cell adhesion molecule [PECAM]) promoter, limiting expression to endothelial cells, monocytes, and platelets. In addition, the P-selectin sequence directs expression to secretory granules. Functional cell surface expression only occurs when the cells are activated. In a mouse model of systemic lipopolysaccharide (LPS)–induced endotoxemia, we show that expression of either anticoagulant on activated endothelium inhibits the widespread intravascular thrombosis, thrombocytopenia, and consumptive coagulopathy associated with endotoxemia. Importantly, non– LPS-treated transgenic mice had normal baseline bleeding times. We speculate that targeted delivery of anticoagulants to the endothelium may be a strategy worth pursuing in clinical sepsis to improve efficacy of systemic anticoagulation while minimizing potential hemorrhagic side effects.

Structure-function analysis of the reactive site in the first Kunitz-type domain of human tissue factor pathway inhibitor-2. Vol. 279 (2004) 17500-17507

Structure-function analysis of the reactive site in the first Kunitz-type domain of human tissue factor pathway inhibitor-2. Vol. 279 (2004) 17500-17507

Structure-Function Analysis of the Reactive Site in the First Kunitz-type Domain of Human Tissue Factor Pathway Inhibitor-2

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type proteinase inhibitor that regulates a variety of serine proteinases involved in coagulation and fibrinolysis through their non-productive interaction with a P(1) residue (Arg-24) in its first Kunitz-type domain (KD1). Previous kinetic studies revealed that TFPI-2 was a more effective inhibitor of plasmin than several other serine proteinases, but the molecular basis for this specificity was unclear. In this study, we employed molecular modeling and mutagenesis strategies to produce several variants of human TFPI-2 KD1 in an effort to identify interactive site residues other than the P(1) Arg that contribute significantly to its inhibitory activity and specificity. Molecular modeling of KD1 based on the crystal structure of bovine pancreatic trypsin inhibitor revealed that KD1 formed a more energetically favorable complex with plasmin versus trypsin and/or the factor VIIa-tissue factor complex primarily due to strong ionic interactions between Asp-19 (P(6)) and Arg residues in plasmin (Arg-644, Arg-719, and Arg-767), Arg-24 (P(1)) with Asp-735 in plasmin, and Arg-29 (P(5)') with Glu-606 in plasmin. In addition, Leu-26 through Leu-28 (P(2)'-P(4)') in KD1 formed strong van der Waals contact with a hydrophobic cluster in plasmin (Phe-583, Met-585, and Phe-587). Mutagenesis of Asp-19, Tyr-20, Arg-24, Arg-29, and Leu-26 in KD1 resulted in substantial reductions in plasmin inhibitory activity relative to wild-type KD1, but the Asp-19 and Tyr-20 mutations revealed the importance of these residues in the specific inhibition of plasmin. In addition to the reactive site residues in the P(6)-P(5)' region of KD1, mutation of a highly conserved Phe at the P(18)' position revealed the importance of this residue in the inhibition of serine proteinases by KD1. Thus, together with the P(1) residue, the nature of other residues flanking the P(1) residue, particularly at P(6) and P(5)', strongly influences the inhibitory activity and specificity of human TFPI-2.

The effect of human tissue factor pathway inhibitor-2 on the growth and metastasis of fibrosarcoma tumors in athymic mice

AbstractHuman tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits the plasmin- and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion, and metastasis. To directly assess its role in tumor growth and metastasis in vivo, we stably transfected HT-1080 fibrosarcoma cells expressing either fully active wild-type human TFPI-2 (WT) or inactive R24Q TFPI-2 (QT) and examined their ability to form tumors and metastasize in athymic mice in comparison to mock-transfected cells (MT). MT and QT fibrosarcoma tumors grew 2 to 3 times larger than WT tumors. Tumor metastasis was confined to the lung and was observed in 75% of mice treated with either MT or QT cells, whereas only 42% of mice treated with WT cells developed lung metastases. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of each tumor group revealed 3- to 6-fold lower levels of murine vascular endothelial growth factor gene expression in WT tumors in relation to either MT or QT tumors. Comparative tumor gene expression analysis revealed that several human genes implicated in oncogenesis, invasion, metastasis, apoptosis, and angiogenesis had significantly altered levels of expression in WT tumors. Our collective data demonstrate that secretion of inhibitory TFPI-2 by a highly metastatic tumor cell markedly inhibits its growth and metastasis in vivo by regulating pericellular extracellular matrix (ECM) remodeling and angiogenesis. (Blood. 2004;103:1069-1077)

Molecular cloning, expression, and characterization of bovine tissue factor pathway inhibitor-2

Human tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz-type serine proteinase inhibitor that is secreted by all cells of the vasculature, and presumably plays a role in the regulation of plasmin-mediated matrix remodeling. In this report, we describe the cloning and expression of a full-length cDNA for bovine TFPI-2 that exhibits 72% sequence identity with that of human TFPI-2. Following a 22 residue signal peptide, the mature protein contains 212 amino acids with 18 cysteines, three putative N-glycosylation sites, and one putative O-glycosylation site. The deduced sequence of mature bovine TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxy-terminal tail highly enriched in basic amino acids. Recombinant bovine TFPI-2 was expressed in HEK 293 cells and resolved into two isoforms, designated as α-TFPI-2 ( M r 33 kDa) and β-TFPI-2 ( M r 31 kDa), which presumably represent differentially glycosylated forms of the inhibitor. Similar to human TFPI-2, both bovine TFPI-2 isoforms exhibited strong inhibitory activity towards trypsin and plasmin, and weak inhibitory activity towards the factor VIIa-tissue factor complex.

Human tissue factor pathway inhibitor-2 does not bind or inhibit activated matrix metalloproteinase-1.

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor associated with the extracellular matrices of vascular cells. A recent report provided in vitro evidence that TFPI-2 may be a novel inhibitor of the matrix metalloproteinases MMP-1, MMP-13, MMP-2 and MMP-9. In studies aimed at identifying the structural elements of TFPI-2 mediating the putative inhibition of the above MMPs, we re-examined the ability of native TFPI-2 to form complexes with MMP-2, MMP-9 and MMP-1, as well as assess its ability to inhibit the proteolytic activity of the interstitial collagenase, activated MMP-1. We report here that TFPI-2 failed to form complexes with MMP-2, MMP-9 and MMP-1 as revealed in immunoprecipitation and ligand blotting studies. In addition, TFPI-2 had no influence on the proteolytic activity of activated MMP-1 towards triple-helical collagen. These data provide presumptive evidence that TFPI-2 does not bind to MMP-2, MMP-9 and MMP-1, or regulate MMP-1, in the extracellular matrix.

The ERK/MAPK Pathway Regulates the Activity of the Human Tissue Factor Pathway Inhibitor-2 Promoter

Human tissue factor pathway inhibitor-2 (hTFPI-2) is a 32-kDa serine protease inhibitor that is associated with the extracellular matrix. hTFPI-2 inhibits several extracellular matrix-degrading serine proteases and may play a role in tumor invasion and metastasis. To study the signal transduction pathway that leads to the activation of the hTFPI-2, we cloned the potential promoter region of this gene adjacent to a heterologous luciferase reporter gene. Phorbol 12-myristate 13-acetate (PMA) induced the luciferase reporter gene in HEK293 cells and other epithelial cell lines, such as the human lung carcinoma A549 cells, the breast carcinoma MCF7 cells, and the cervical HeLa cells. This PMA induction was blocked with the MEK inhibitor UO126, suggesting that the PMA-induced activation of the hTFPI-2 promoter is mediated through MEK. Furthermore, epidermal growth factor induced the luciferase reporter gene in HeLa cells. Cotransfection of the luciferase construct with constitutively active components of the Ras/Raf/MEK/ERK pathway in EcR-293 cells lead to a 7- to 92-fold induction of the luciferase reporter gene, indicating that regulation of hTFPI-2 is mediated through this pathway. A series of luciferase reporter gene constructs with progressive deletions of the 5'-flanking region suggested that the minimal basal promoter activity is located between nucleotide positions -89 and -384, whereas the minimal inducible promoter activity is between -89 and -222. We have used the computer program TFSEARCH and mutagenesis to analyze potential transcription factor binding sites. We identified an AP-1 binding site at nucleotide position -156 (inducible activity) and a Sp1 site at position -134 (basal activity) as potential cis-acting elements in the promoter region of the hTFPI-2.

Characterization and functional analysis of TFPI-2 gene promoter in a human choriocarcinoma cell line

Tissue factor pathway Inhibitor-2 (TFPI-2) is associated with extracellular matrices and plays a major role in cell migration and tumor invasion. In this study, a 4.8-kb human TFPI-2 gene 5′-flanking region was isolated, cloned and sequenced. Promoter region analysis revealed a high GC-rich content without canonical TATA and CAAT boxes but three transcription initiation sites were identified. Moreover, several putative binding sites for transcription factors were identified (MyoD, LYF1, NF-Y, GATA, oct-1, AP-1, Sp1, NF1, NF-κB and egr-1). To characterize potential regulatory regions, TFPI-2/luciferase promoter constructs were then transfected in human choriocarcinoma JEG-3 cells. We first showed that the minimal TFPI-2 promoter is located between −166 and −111 from the translation start site. Luciferase activity consistently increased after stimulation of JEG-3 cells by phorbol 12-myristate 13-acetate indicating that NF1, NF-κB and egr-1/Sp1 binding sites are crucial in inducible TFPI-2 expression. Moreover, negative regulatory regions included AP-1 binding sites were identified. This study demonstrates that the TFPI-2 gene promoter exhibits typical features of a housekeeping gene.

Local expression of C-type natriuretic peptide suppresses inflammation, eliminates shear stress-induced thrombosis, and prevents neointima formation through enhanced nitric oxide production in rabbit injured carotid arteries.

We previously observed that adenovirus-mediated expression of C-type natriuretic peptide (CNP) markedly inhibits neointima formation after balloon injury in rat carotid arteries, suggesting that CNP has multiple effects over its modest inhibitory effect on cellular proliferation. We hypothesized that local expression of CNP might have antithrombotic and antiinflammatory effects. Balloon-injured rabbit carotid arteries were infected with an adenovirus expressing human CNP (AdCNP), human tissue factor pathway inhibitor (AdTFPI), or bacterial beta-galactosidase (AdLacZ) or infused with saline. Seven days later, shear stress-induced thrombosis was evaluated by cyclic flow variation (CFV), reflecting recurrent cycles of thrombus formation and dislodgment. CFV was observed in all AdLacZ-infected and saline-infused arteries but not in arteries infected with AdCNP or AdTFPI even in the presence of epinephrine. Injury increased the expressions of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and infiltration of macrophages. However, these effects were markedly reduced in AdCNP-treated arteries but not in AdTFPI-infected ones. In AdCNP-infected arteries, injury-induced expression of inducible NO synthase (iNOS) was enhanced, leading to increased NO generation. Interestingly, when the enhanced NO production was inhibited, neither inhibitory effect was observed, and suppression of neointima formation by CNP was canceled. Our study demonstrates that overexpression of CNP shows antithrombotic and antiinflammatory effects and reduces neointima formation mainly through enhanced NO production.

Anti-inflammatory effects of long-lasting locally-delivered human recombinant tissue factor pathway inhibitor after balloon angioplasty.

Tissue factor pathway inhibitor (TFPI) has anti-proliferative and anti-migratory effects on cultured smooth muscle cells (SMC) in addition to its anti-thrombotic activity. Here, we assess how long locally delivered recombinant TFPI (rTFPI) remains detectable at the delivery sites and clarify the main mechanism by which rTFPI blocks neointimal growth in vivo. The iliac arteries of 85 Japanese white rabbits were injured using a Cutting Balloon. First, to establish the efficacy of local delivery of rTFPI, 5 groups of 3 rabbits each were examined immediately or 1, 2, 4 or 7 days after delivery. They were treated locally with a total amount of 200 microg of rTFPI given at 40 microg per pulse per minute by means of a Pulse Spray catheter. Thereafter, 34 rabbits which had received 200 microg of rTFPI after cutting angioplasty were compared to the same number of controls regarding thrombosis, inhibition of neointimal proliferation and inflammation. A total of 2.6+/-1.6 ng rTFPI persisted on the injured vessel 4 days after delivery. rTFPI was still present on 48 % of the cut sites 7 days after delivery, despite its short half-life in plasma. Thrombosis in the rTFPI-treated group was significantly reduced compared to the controls. The number of macrophages present within the media and intima was significantly decreased at day 7 after delivery of rTFPI. Furthermore, the number of Ki-67-positive cells 14 days after rTFPI delivery was significantly lower than in controls although there were no significant differences between them after 2 days. Local delivery of rTFPI decreased the degree of neointimal formation 4 weeks after TFPI delivery because of anti-inflammatory and anti-proliferative effects in addition to, or rather than, via anti-thrombosis.

Assessment of renal function during the postoperative period following liver xenotransplantation from transgenic pig to baboon.

Xenotransplantation using pig organs could solve the significant increasing shortage of donor organs for allotransplantation. In the last two decades, major progress has been made in understanding the xenoimmunobiology of pig-to-nonhuman primate transplantation, and today we are close to clinical trials.The ability to genetically engineer pigs, such as human decay-accelerating factor (hDAF), CD46 (membrane cofactor protein), or α1,3-galactosyltransferase gene-knockout (GT-KO), has been a significant step toward the clinical application of xenotransplantation. Using GT-KO pigs and novel immunosuppressant agents, 2 to 6 months' survival of heterotopic heart xenotransplants has been achieved. In life-supporting kidney xenotransplantation, promising survival of close to 3 months has been achieved. However, liver and lung xenotransplantations do not have such encouraging survival as kidney and heart xenotransplantation.Although the introduction of hDAF and GT-KO pigs largely overcame hyperacute rejection, acute humoral xenograft rejection (AHXR) remains a challenge to be overcome if survival is to be increased. In several studies, when classical AHXR was prevented, thrombotic microangiopathy and coagulation dysregulation became more obvious, which make them another hurdle to be overcome.The initiating cause of failure of pig cardiac and renal xenografts may be antibody-mediated injury to the endothelium, leading to the development of microvascular thrombosis. Potential contributing factors toward the development of the thrombotic microangiopathy include: 1) the presence of preformed anti-non-Gal antibodies, 2) the development of very low levels of elicited antibodies to non-Gal antigens, 3) natural killer cell or macrophage activity, and 4) inherent coagulation dysregulation between pigs and primates.The breeding of pigs transgenic for an ‘anticoagulant’ or ‘anti-thrombotic’ gene, such as human tissue factor pathway inhibitor, hirudin, or CD39, or lacking the gene for the prothrombinase, fibrinogen-like protein-2, is anticipated to inhibit the change in the endothelium to a procoagulant state that takes place in the pig organ after transplantation.A further limitation for organ xenotransplantation is the potential for cross-species infection. As far as exogenous viruses are concerned, porcine cytomegalovirus has been detected in the tissues of recipient non-human primates, although no invasive disease was reported. Until today, no formal evidence has been presented from in vivo studies in non-human primates or from humans exposed to pig organs, tissues, or cells that porcine endogenous retroviruses infect primate cells.Xenotransplantation is a potential answer to the current organ shortage. Its future depends on; 1) further genetic modification of pigs, 2) the introduction of novel immunosuppressive agents that target the innate immune system and plasma cells, and 3) the development of clinically-applicable methods to induce donor-specific tolerance.

Lipoprotein (a) binds and inactivates tissue factor pathway inhibitor: a novel link between lipoproteins and thrombosis

AbstractLipoprotein (a) [Lp(a)] has been associated with both anti-fibrinolytic and atherogenic effects. However, no direct link currently exists between this atherogenic lipoprotein and intravascular coagulation. The current study examined the binding and functional effects of Lp(a), its lipoprotein constituents, apoliprotein (a) [apo(a)] and low-density lipoprotein (LDL), and lysine-plasminogen (L-PLG), which shares significant homology with apo(a), on tissue factor pathway inhibitor (TFPI), a major regulator of tissue factor-mediated coagulation. Results indicate that Lp(a), apo(a), and PLG but not LDL bound recombinant TFPI (rTFPI) in vitro and that apo(a) bound to a region spanning the last 37 amino acid residues of the c-terminus of TFPI. The apparent binding affinity for TFPI was much higher for Lp(a) (KD ∼150 nM) compared to PLG (KD ∼800 nM) and nanomolar concentrations of apo(a) (500 nM) inhibited PLG binding to TFPI. Lp(a) also inhibited in a concentration-dependent manner rTFPI activity and endothelial cell surface TFPI activity in vitro, whereas PLG had no such effect. Moreover physiologic concentrations of PLG (2 μM) had no effect on the concentration-dependent inhibition of TFPI activity induced by Lp(a). In human atherosclerotic plaque, apo(a) and TFPI immunostaining were shown to coexist in smooth muscle cell–rich areas of the intima. These data suggest a novel mechanism whereby Lp(a) through its apo(a) moiety may promote thrombosis by binding and inactivating TFPI.

A novel function of tissue factor pathway inhibitor-2 (TFPI-2) in human glioma invasion.

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine protease inhibitor that inhibits plasmin, trypsin, chymotrypsin, cathepsin G, and plasma kallikrein but not urokinase-type plasminogen activator, tissue plasminogen activator, or thrombin. Preliminary findings in our laboratory suggested that the expression of TFPI-2 is downregulated or lost during tumor progression in human gliomas. To investigate the role of TFPI-2 in the invasiveness of brain tumors, we stably transfected the human high-grade glioma cell line SNB19 and the human low-grade glioma cell line Hs683 with a vector capable of expressing a transcript complementary to the full-length TFPI-2 mRNA in either sense (0.7 kb) or antisense (1 kb) orientations. Parental cells and stably transfected cell lines were analysed for TFPI-2 protein by Western blotting and for TFPI-2 mRNA by Northern blotting. The levels of TFPI-2 protein and mRNA were higher in the sense clones (SNB19) and decreased in the antisense (Hs683) clones than in the corresponding parental and vector controls. In spheroid and matrigel invasion assays, the SNB19 parental cells were highly invasive, but the sense-transfected SNB-19 clones were much less invasive; the antisense-transfected Hs683 clones were more invasive than their parental and vector controls. After intracerebral injection in mice, the sense-transfected SNB19 clones were less able to form tumors than were their parental and vector controls, and the antisense-Hs683 clones but not the parental or vector controls formed small tumors. This is the first study to demonstrate that down- or upregulation of TFPI-2 plays a significant role in the invasive behavior of human gliomas.

A novel role of tissue factor pathway inhibitor-2 in apoptosis of malignant human gliomas

Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa serine protease inhibitor found at high levels in extracellular matrix. Recombinant human TFPI-2 has recently been shown to be a strong inhibitor of trypsin, plasmin, plasma kallikrein, and factor XIa amidolytic activity. Earlier studies in our laboratory showed that the expression of TFPI-2 is lost during tumor progression in human gliomas. We stably transfected this protease inhibitor in multiform glioblastoma cell line (SNB-19) and in low-grade glioma cell line (Hs683) in sense and antisense orientation respectively. This confirmed that the upregulation/down-regulation of TFPI-2 plays a significant role in the invasive behavior of human gliomas both in vitro and in vivo models. Collectively, these results suggested an idea to determine whether TFPI-2 is necessary for cell survival and inhibition of tumor formation in nude mice, due to apoptosis of intracerebrally injected SNB-19 cells. In the present study we determined p-ERK levels and found that they are decreased in TFPI-2 over-expressed clones (SNB-19) and increased in TFPI-2 down-regulated clones (Hs683). We also checked the levels of BAX/BCl-2, caspases (for e.g., 9, 7, 3, 8), PARP, cytochrome-c and Apaf-1. Moreover, the increase of apoptosis in vitro is associated with increased and decreased expression of apoptotic protein BAX in sense clones (SNB-19) and antisense clones (Hs683) respectively, when compared to controls and vice versa with Bcl-2 the anti-apoptotic protein. Caspases (9, 7 and 3), cytochrome-c, Apaf-1 and PARP levels are increased in SNB-19 and decreased in Hs683. Caspase 8 was not expressed in either cell line. Caspases 9 and 3 activity assay revealed higher activity in sense clones (SNB-19) but lesser in antisense clones (Hs683) compared to controls. This is the first report of TFPI-2 playing a novel role in cell survival in human gliomas.

Tissue factor pathway inhibitor activity in severe sepsis

To review the preclinical and clinical evidence that provides the therapeutic rationale for recombinant human tissue factor pathway inhibitor (rTFPI) as a novel treatment for human sepsis. A summary of published English-language literature regarding preclinical studies and limited information published about three phase II clinical studies for the evaluation of rTFPI safety in sepsis patients. Tissue factor pathway inhibitor, the physiologic inhibitor of the tissue factor pathway, interrupts activation of coagulation at multiple steps, including tissue factor VIIa activity, Xa activity, prothrombinase complex, and thrombin generation. Recombinant human TFPI exhibits anticoagulant and anti-inflammatory activities in animal models and humans with sepsis. These activities appear to have an important therapeutic role in protecting the microvasculature from injury and preventing multiple organ failure in sepsis. Tissue factor pathway inhibitor is a potent inhibitor of clotting in the microvasculature, which is thought to protect organs from injury. Recombinant TFPI improved survival of septic animals in multiple models. Recent phase II results suggest that rTFPI is well tolerated, and they show a trend toward reduction in 28-day all-cause mortality in rTFPI-treated patients; in addition, rTFPI demonstrated significant reduction in thrombin generation. These results suggest that a powered study is indicated to further evaluate rTFPI utility for the adjunctive management of severe sepsis.