Structure, function and biology of tissue factor pathway inhibitor-2

2005 ◽  
Vol 94 (12) ◽  
pp. 1122-1130 ◽  
Author(s):  
Hitendra S. Chand ◽  
Donald C. Foster ◽  
Walter Kisiel
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Serine Proteinase ◽  
In Vivo Studies ◽  
Amino Terminal ◽  
Tissue Factor Pathway ◽  
Kunitz Type ◽  
Carboxy Terminal

SummaryTissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa matrix-associated Kunitz-type serine proteinase inhibitor consisting of a short amino-terminal region, three tandem Kunitz-type domains and a positively charged carboxy-terminal tail. Human TFPI-2, previously designated as placental protein 5, inhibits a broad spectrum of serine proteinases almost exclusively through its first Kunitz-type domain, and is thought to play an important role in the regulation of extracellular matrix digestion and re-modeling. In this context, reduced synthesis of TFPI-2 has been related to numerous pathophysiological processes such as inflammation, angiogenesis, atherosclerosis, retinal degeneration and tumor growth/metastasis. In this review, we document current information regarding the expression of TFPI-2 by various tissues, its inhibitory activity and proteinase specificity in-vitro, and discuss possible physiological roles for this inhibitor based on in-vivo studies.

Human Tissue Factor Pathway Inhibitor-2 Induces Caspase-Mediated Apoptosis in a Human Fibrosarcoma Cell Line (HT-1080).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4165-4165
Author(s):  
Prakasha Kempaiah ◽  
Walter Kisiel
Keyword(s):  
Tissue Factor ◽  
Human Tissue ◽  
Tissue Factor Pathway Inhibitor ◽  
Serine Proteinase ◽  
Annexin V ◽  
Polymyxin B ◽  
Bax Protein ◽  
Tissue Factor Pathway ◽  
Kunitz Type

Abstract Human tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa extracellular matrix-associated Kunitz-type serine proteinase inhibitor that regulates the plasmin and trypsin- mediated activation of zymogen matrix metalloproteinases and growth factors essential for tumor growth, invasion, and metastasis. We previously demonstrated that HT-1080 human fibrosarcoma cells do not express TFPI-2, but restoration of TFPI-2 expression in these cells by stable transfection with the human TFPI-2 cDNA markedly inhibited their growth and metastasis in-vivo. In this study, 1 μM concentrations of either recombinant human TFPI-2 or its mutated first Kunitz-type domain (R24K KD1), were offered to HT-1080 cells, incubated for 48 h, and the degree of apoptosis assessed by nuclear fragmentation, ethidium bromide/acridine orange (EB/AO) staining, fluorescence-activated cell sorting (FACS) and immunoblotting. Agarose gel electrophoresis of DNA from HT-1080 cells treated with either TFPI-2 or R24K KD1 for 48h indicated DNA fragmentation with a ladder pattern typically associated with apoptosis. EB/AO staining of TFPI-2- and R24K KD1- treated cells revealed that 40–70% of the cells were apoptotic in relation to vehicle-treated cells as judged by fluoresence microscopy. Co-administration of TFPI-2 with polymyxin B produced similar results, suggesting that apoptosis was not endotoxin-dependent. Consistent with our earlier studies showing its enhanced inhibitory activity relative to TFPI-2, R24K KD1 was able to induce apoptosis in 68% of HT-1080 cells after 48h of treatment compared to 39% for the parent TFPI-2 at an equivalent concentration. Moreover, HT-1080 cells treated with a KD1 preparation lacking the reactive site arginine residue (R24Q KD1) produced only an 18% apoptosis rate, thereby linking the observed apoptosis with serine proteinase inhibition. FACS analysis, using propidium iodide and annexin-V labeling, revealed similar apoptotic rates to that seen by EB/AO staining assays. In addition, immunoblotting experiments of vehicle and TFPI-2-treated cells indicated increased caspase-3 activation in TFPI-2-treated cells, thus providing evidence that apoptosis is caspase-mediated. We also observed up-regulation of the proapoptotic Bax protein by immunoblotting following treatment of HT-1080 cells with either TFPI-2 or R24K KD1. Taken together, our results demonstrate that treatment of HT-1080 cells with exogenous TFPI-2 or R24K KD1 activates caspase-mediated, proapoptotic signaling pathways and induces apoptosis. In addition, our data provides suggestive evidence that peritumor application of either TFPI-2 or R24K KD1 may facilitate tumor apoptosis in-vivo.

Effect of Depolymerized Holothurian Glycosaminoglycan (DHG) on Tissue Factor Pathway Inhibitor: In Vitro and In Vivo Studies

1997 ◽  
Vol 78 (02) ◽  
pp. 864-870 ◽  
Author(s):  
Hideki Nagase ◽  
Kei-ichi Enjyoji ◽  
Yu-ichi Kamikubo ◽  
Keiko T Kitazato ◽  
Kenji Kitazato ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Free Form ◽  
Initial Reaction ◽  
Extrinsic Pathway ◽  
Factor Xa Inhibition ◽  
Tissue Factor Pathway

SummaryDepolymerized holothurian glycosaminoglycan (DHG) is a glycosaminoglycan extracted from the sea cucumber Stichopus japonicusSelenka. In previous studies, we demonstrated that DHG has antithrombotic and anticoagulant activities that are distinguishable from those of heparin and dermatan sulfate. In the present study, we examined the effect of DHG on the tissue factor pathway inhibitor (TFPI), which inhibits the initial reaction of the tissue factor (TF)-mediated coagulation pathway. We first examined the effect of DHG on factor Xa inhibition by TFPI and the inhibition of TF-factor Vila by TFPI-factor Xa in in vitro experiments using human purified proteins. DHG increased the rate of factor Xa inhibition by TFPI, which was abolished either with a synthetic C-terminal peptide or with a synthetic K3 domain peptide of TFPI. In contrast, DHG reduced the rate of TF-factor Vila inhibition by TFPI-factor Xa. Therefore, the effect of DHG on in vitroactivity of TFPI appears to be contradictory. We then examined the effect of DHG on TFPI in cynomolgus monkeys and compared it with that of unfractionated heparin. DHG induced an increase in the circulating level of free-form TFPI in plasma about 20-fold when administered i.v. at 1 mg/kg. The prothrombin time (PT) in monkey plasma after DHG administration was longer than that estimated from the plasma concentrations of DHG. Therefore, free-form TFPI released by DHG seems to play an additive role in the anticoagulant mechanisms of DHG through the extrinsic pathway in vivo. From the results shown in the present work and in previous studies, we conclude that DHG shows anticoagulant activity at various stages of coagulation reactions, i.e., by inhibiting the initial reaction of the extrinsic pathway, by inhibiting the intrinsic Xase, and by inhibiting thrombin.

Inhibition of restenosis by tissue factor pathway inhibitor: in vivo and in vitro evidence for suppressed monocyte chemoattraction and reduced gelatinolytic activity

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1653-1661 ◽  
Author(s):  
Christoph W. Kopp ◽  
Thomas Hölzenbein ◽  
Sabine Steiner ◽  
Rodrig Marculescu ◽  
Helga Bergmeister ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Early Stage ◽  
Thrombus Formation ◽  
Matrix Metalloproteinase 2 ◽  
Monocyte Chemoattractant Protein 1 ◽  
Markers Of Inflammation ◽  
Tissue Factor Pathway

AbstractActivation of inflammatory and procoagulant mechanisms is thought to contribute significantly to the initiation of restenosis, a common complication after balloon angioplasty of obstructed arteries. During this process, expression of tissue factor (TF) represents one of the major physiologic triggers of coagulation that results in thrombus formation and the generation of additional signals leading to vascular smooth muscle cell (VSMC) proliferation and migration. In this study, we have investigated the mechanisms by which inhibition of coagulation at an early stage through overexpression of tissue factor pathway inhibitor (TFPI), an endogenous inhibitor of TF, might reduce restenosis. In a rabbit femoral artery model, percutaneous delivery of TFPI using a recombinant adenoviral vector resulted in a significant reduction of the intimamedia ratio 21 days after injury. Investigating several markers of inflammation and coagulation, we found reduced neointimal expression of monocyte chemoattractant protein-1 (MCP-1), lesional monocyte infiltration, and expression of vascular TF, matrix metalloproteinase-2 (MMP-2), and MMP-9. Moreover, overexpression of TFPI suppressed the autocrine release of platelet-derived growth factor BB (PDGF-BB), MCP-1, and MMP-2 in response to factors VIIa and Xa from VSMCs in vitro and inhibited monocyte TF activity. These results suggest that TFPI exerts its action in vivo through not only thrombotic, but also nonthrombotic mechanisms.

scholarly journals Complementary DNA sequencing of canine tissue factor pathway inhibitor reveals a unique nanomeric repetitive sequence between the second and third Kunitz domains

1994 ◽  
Vol 303 (3) ◽  
pp. 923-928 ◽  
Author(s):  
T J Girard ◽  
D Gailani ◽  
G J Broze
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Peptide Sequence ◽  
Tissue Factor Pathway ◽  
Kunitz Type ◽  
Canine Plasma

Tissue factor pathway inhibitor (TFPI) is a factor Xa-dependent inhibitor of the factor VIIa-tissue factor complex of blood coagulation. The primary amino acid sequence of canine TFPI has been deduced from cDNA sequences obtained using the techniques of reverse transcription followed by amplification using PCR and conventional screening of a canine endothelial cell cDNA library. The open reading frame for canine TFPI encodes a signal peptide of 28 amino acids followed by a 40.7 kDa protein of 368 amino acids. Similar to human, rat and rabbit TFPI, canine TFPI contains a negatively-charged cluster of amino acids at its mature amino-terminus, followed by three Kunitz-type proteinase inhibitory domains and a cluster of positively-charged amino acids near its carboxy-terminus. In contrast to other TFPIs, following its second Kunitz-type proteinase inhibitory domain canine TFPI contains an additional amino acid insert which includes a nanomeric peptide-sequence repeated six times. Recombinant canine TFPI was expressed in both bacterial- and insect cell-expression systems for functional analysis and the generation of antibodies. The recombinant canine TFPI inhibits tissue factor-induced coagulation in an in vitro canine system. Immunoprecipitation of TFPI from canine plasma, followed by Western-blot analysis, tentatively identifies canine TFPI as an 80,000 kDa protein. Anti-peptide antibodies raised to the nanomeric peptide repeat immunoprecipitate an identical, cross-reactive, 80,000 kDa protein.

Nucleotide Sequence of the Gene Encoding Murine Tissue Factor Pathway Inhibitor-2

2000 ◽  
Vol 83 (01) ◽  
pp. 141-147 ◽  
Author(s):  
Yoshiaki Kazama ◽  
Shintaro Kamei ◽  
Joseph Kuijper ◽  
Donald Foster ◽  
Walter Kisiel
Keyword(s):  
Nucleotide Sequence ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Genomic Library ◽  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
Specific Expression ◽  
Tissue Factor Pathway ◽  
Flanking Region ◽  
Kunitz Type

SummaryTissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein 5, is a 32 kDa extracellular matrix-associated serine proteinase inhibitor consisting of three tandemly-arranged Kunitz-type domains. Two overlapping genomic clones containing sequences encoding murine TFPI-2 were isolated from a A FIXII 129 SVJ mouse genomic library, and the complete nucleotide sequence of the gene was determined. The murine TFPI-2 gene spans approximately 9.3 kilobases and consists of five exons and four introns. The nucleotide sequences surrounding all the exon-intron boundaries are highly conserved and obey the GT-AG rule. Each Kunitz-type domain is encoded by a single exon, similar to that observed for other Kunitz-type proteinase inhibitors. A total of 1,577 bp of the 3’-flanking region contains a probable polyadenylation site (ATTAAA) at +5,759 and an apparent cleavage or termination site (CATTG) at +6,170. The 5’-flanking region of the murine TFPI-2 gene contains a prototypical TATA box, a GC box and two CAAT boxes. In addition, several candidate transcription factor binding sites responsible for placenta-, endothelial cell-, and smooth muscle cell-specific expression of the TFPI-2 gene were also identified.

Tissue Factor Reduction and Tissue Factor Pathway Inhibitor Release after Heparin Administration

1999 ◽  
Vol 81 (04) ◽  
pp. 589-593 ◽  
Author(s):  
A. M. Gori ◽  
G. Pepe ◽  
M. Attanasio ◽  
M. Falciani ◽  
R. Abbate ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Plasma Levels ◽  
Tissue Factor Pathway Inhibitor ◽  
Day Treatment ◽  
Procoagulant Activity ◽  
Cytokine Gene Expression ◽  
Heparin Administration ◽  
Tissue Factor Pathway

SummaryElevated plasma levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) and large amounts of monocyte procoagulant activity (PCA) have been documented in unstable angina (UA) patients. In in vitro experiments heparin is able to blunt monocyte TF production by inhibiting TF and cytokine gene expression by stimulated cells and after in vivo administration it reduces adverse ischemic outcomes in UA patients. TF and TFPI plasma levels and monocyte PCA have been investigated in 28 refractory UA patients before and during anticoagulant subcutaneous heparin administration (thrice daily weight- and PTT-adjusted for 3 days) followed by 5000 IU × 3 for 5 days. After 2-day treatment, immediately prior to the heparin injection, TF and TFPI plasma levels [(median and range): 239 pg/ml, 130-385 pg/ ml and 120 ng/ml, 80-287 ng/ml] were lower in comparison to baseline samples (254.5 pg/ml, 134.6-380 pg/ml and 135.5 ng/ml, 74-306 ng/ml). Four h after the heparin injection TF furtherly decreased (176.5 pg/ml, 87.5-321 pg/ml; -32.5%, p<0.001) and TFPI increased (240.5 ng/ml, 140-450 ng/ml; +67%, p<0.0001).After 7-day treatment, before the injection of heparin, TF and TFPI plasma levels (200 pg/ml, 128-325 pg/ml and 115 ng/ml, 70-252 ng/ml) significantly decreased (p<0.05) in comparison to the pre-treatment values. On the morning of the 8th day, 4 h after the injection of heparin TF plasma levels and monocytes PCA significantly decreased (156.5 pg/ml, 74-259 pg/ml and from 180 U/105 monocytes, 109-582 U/105 monocytes to 86.1 U/105 monocytes, 28-320 U/105 monocytes; - 38% and -55% respectively) and TFPI increased (235.6 ng/ml, 152-423 ng/ ml; +70%, p<0.001). In conclusion, heparin treatment is associated with a decrease of high TF plasma levels and monocyte procoagulant activity in UA patients. These actions of heparin may play a role in determining the antithrombotic and antiinflammatory properties of this drug.

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