scholarly journals Knockdown and Knockout of Tissue Factor Pathway Inhibitor in Zebrafish

2021 ◽  
Author(s):  
Revathi Raman ◽  
Weam Fallatah ◽  
Ayah Al Qaryoute ◽  
Mia Ryon ◽  
Pudur Jagadeeswaran
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Homozygous Deletion ◽  
Negative Regulator ◽  
Coagulation Activity ◽  
Genome Wide ◽  
Alternate Model ◽  
Tissue Factor Pathway ◽  
Knockout Model

Tissue Factor Pathway Inhibitor (TFPI) is an anticoagulant that inhibits factor VIIa and Xa in the blood coagulation pathways. TFPI contains three Kunitz domains, K1, K2, and K3. K1 and K2 inhibit factor VIIa and Xa, respectively. However, the regulation of TFPI is poorly studied. Since zebrafish has become an alternate model to discover novel actors in hemostasis, we hypothesized that TFPI regulation could be studied using this model. As a first step, we confirmed the presence of tfpia in zebrafish using RT-PCR. We then performed piggyback knockdowns of tfpia and found increased coagulation activity in tfpia knockdown. We then created a deletion mutation in tfpia locus using CRISPR/Cas9 method. The tfpia homozygous deletion mutants showed increased coagulation activities similar to that found in tfpia knockdown. Taken together, our data suggest that tfpia is a negative regulator for zebrafish coagulation, and silencing it leads to thrombotic phenotype. Also, the zebrafish tfpia knockout model could be used for reversing this thrombotic phenotype to identify antithrombotic novel factors by the genome-wide piggyback knockdown method.

scholarly journals Caveolin-1 Enhances Tissue Factor Pathway Inhibitor Exposure and Function on the Cell Surface

2005 ◽  
Vol 280 (23) ◽  
pp. 22308-22317 ◽  
Author(s):  
Cristina Lupu ◽  
Xiaohong Hu ◽  
Florea Lupu
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Protease Production ◽  
Extrinsic Pathway ◽  
Caveolin 1 ◽  
Tissue Factor Pathway ◽  
Quaternary Complex ◽  
And Function

Tissue factor pathway inhibitor (TFPI) blocks tissue factor-factor VIIa (TF-FVIIa) activation of factors X and IX through the formation of the TF-FVIIa-FXa-TFPI complex. Most TFPI in vivo associates with caveolae in endothelial cells (EC). The mechanism of this association and the anticoagulant role of caveolar TFPI are not yet known. Here we show that expression of caveolin-1 (Cav-1) in 293 cells keeps TFPI exposed on the plasmalemma surface, decreases the membrane lateral mobility of TFPI, and increases the TFPI-dependent inhibition of TF-FVIIa. Caveolae-associated TFPI supports the co-localization of the quaternary complex with caveolae. To investigate the significance of these observations for EC we used RNA interference to deplete the cells of Cav-1. Functional assays and fluorescence microscopy revealed that the inhibitory properties of TFPI were diminished in EC lacking Cav-1, apparently through deficient assembly of the quaternary complex. These findings demonstrate that caveolae regulate the inhibition by cell-bound TFPI of the active protease production by the extrinsic pathway of coagulation.

scholarly journals Heparanase induces tissue factor pathway inhibitor expression and extracellular accumulation in endothelial and tumor cells

2008 ◽  
Vol 99 (01) ◽  
pp. 133-141 ◽  
Author(s):  
Yona Nadir ◽  
Benjamin Brenner ◽  
Sveta Gingis-Velitski ◽  
Flonia Levy-Adam ◽  
Neta Ilan ◽  
...  
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Umbilical Vein ◽  
Human Tumor ◽  
Fold Increase ◽  
Coagulation Activity ◽  
Over Expression ◽  
Exogenous Addition ◽  
Tissue Factor Pathway

SummaryHeparanase activity is implicated in cell invasion, tumor metastasis and angiogenesis. Recently, we have reported that heparanase stimulates tissue factor (TF) expression in endothelial and cancer cells, resulting in elevation of coagulation activity. We hypothesized that heparanase regulates other coagulation modulators, and examined the expression and localization of tissue factor pathway inhibitor (TFPI) following heparanase over-expression or exogenous addition. Primary human umbilical vein endothelial cells (HUVEC) and human tumor-derived cell lines were incubated with heparanase, or were stably transfected with heparanase gene-constructs, and TFPI expression and secretion were examined. Heparanase over-expression or exogenous addition stimulated TFPI expression by 2–3 folds. TFPI accumulation in the cell culture medium exceeded in magnitude the observed induction ofTFPI gene transcription reaching 5– to 6-fold increase. Extracellular accumulation of TFPI was evident already 60 min following heparanase addition, prior toTFPI protein induction, and correlated with increased coagulation activity. This effect was found to be independent of heparanase enzymatic activity and interaction with heparan-sulfate, and correlated with reduced TFPI levels on the cell surface. Data were verified in heparanase transgenic mice tissues and plasma. Interaction between heparanase and TFPI was evident by co-immunoprecipitation. Interaction of heparanase with TFPI resulted in its displacement from the surface of the vascular endothelium and in increased pro-coagulant activity. Thus, heparanase facilitates blood coagulation on the cell surface by two independent mechanisms:dissociation ofTFPI from the vascular surface short after local elevation of heparanase levels, and subsequent induction of TF expression.

Recombinant Tissue Factor Pathway Inhibitor Prevents Lipopolysaccharide-induced Systemic Hypotension in Rats by Inhibiting Excessive Production of Nitric Oxide

2001 ◽  
Vol 86 (12) ◽  
pp. 1573-1577 ◽  
Author(s):  
Perenlei Enkhbaatar ◽  
Mitsuhiro Uchiba ◽  
Hirotaka Isobe ◽  
Hiroaki Okabe ◽  
Kenji Okajima
Keyword(s):  
Nitric Oxide ◽  
Tissue Factor ◽  
Plasma Levels ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Inos Mrna ◽  
Induced Hypotension ◽  
Inos Activity ◽  
Tnf Α ◽  
Tissue Factor Pathway

SummaryExcessive production of nitric oxide (NO) by the inducible form of NO synthase (iNOS) plays a key role in the development of endotoxin shock. Tumor necrosis factor-α (TNF-α) induces iNOS, thereby contributing to the development of shock. We recently reported that recombinant tissue factor pathway inhibitor (r-TFPI), an important inhibitor of the extrinsic pathway of the coagulation system, inhibits TNF-α production by monocytes. In this study, we investigated whether r-TFPI could ameliorate hypotension by inhibiting excessive production of NO in rats given lipopolysaccharide (LPS). Pretreatment of animals with r-TFPI prevented LPS-induced hypotension. Recombinant TFPI significantly inhibited the increases in both the plasma levels of NO2 -/NO3 - and lung iNOS activity 3 h after LPS administration. Expression of iNOS mRNA in the lung was also inhibited by intravenous administration of r-TFPI. However, neither DX-9065a, a selective inhibitor of factor Xa, nor an inactive derivative of factor VIIa (DEGR-F.VIIa) that selectively inhibits factor VIIa activity, had any effect on LPS-induced hypotension despite their potent anticoagulant effects. Moreover, neither the plasma levels of NO2 -/NO3 - nor lung iNOS activity were affected by administration of DX-9065a and DEGR-F.VIIa. These results suggested that r-TFPI ameliorates LPS-induced hypotension by reducing excessive production of NO in rats given LPS and this effect was not attributable to its anticoagulant effects, but to the inhibition of TNF-α production.

scholarly journals Small Peptides Blocking Inhibition of Factor Xa and Tissue Factor-Factor VIIa by Tissue Factor Pathway Inhibitor (TFPI)

2013 ◽  
Vol 289 (3) ◽  
pp. 1732-1741 ◽  
Author(s):  
Michael Dockal ◽  
Rudolf Hartmann ◽  
Markus Fries ◽  
M. Christella L. G. D. Thomassen ◽  
Alexandra Heinzmann ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Anticoagulant Activity ◽  
Tight Binding ◽  
Factor Xa ◽  
Factor X ◽  
Intermediate Segment ◽  
Tissue Factor Pathway ◽  
Α Helix

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits activated factor X (FXa) via a slow-tight binding mechanism and tissue factor-activated FVII (TF-FVIIa) via formation of a quaternary FXa-TFPI-TF-FVIIa complex. Inhibition of TFPI enhances coagulation in hemophilia models. Using a library approach, we selected and subsequently optimized peptides that bind TFPI and block its anticoagulant activity. One peptide (termed compound 3), bound with high affinity to the Kunitz-1 (K1) domain of TFPI (Kd ∼1 nm). We solved the crystal structure of this peptide in complex with the K1 of TFPI at 2.55-Å resolution. The structure of compound 3 can be segmented into a N-terminal anchor; an Ω-shaped loop; an intermediate segment; a tight glycine-loop; and a C-terminal α-helix that is anchored to K1 at its reactive center loop and two-stranded β-sheet. The contact surface has an overall hydrophobic character with some charged hot spots. In a model system, compound 3 blocked FXa inhibition by TFPI (EC50 = 11 nm) and inhibition of TF-FVIIa-catalyzed FX activation by TFPI (EC50 = 2 nm). The peptide prevented transition from the loose to the tight FXa-TFPI complex, but did not affect formation of the loose FXa-TFPI complex. The K1 domain of TFPI binds and inhibits FVIIa and the K2 domain similarly inhibits FXa. Because compound 3 binds to K1, our data show that K1 is not only important for FVIIa inhibition but also for FXa inhibition, i.e. for the transition of the loose to the tight FXa-TFPI complex. This mode of action translates into normalization of coagulation of hemophilia plasmas. Compound 3 thus bears potential to prevent bleeding in hemophilia patients.

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.

Genetic determinants of tissue factor pathway inhibitor plasma levels

2015 ◽  
Vol 114 (08) ◽  
pp. 245-257 ◽  
Author(s):  
Jessica Dennis ◽  
Irfahan Kassam ◽  
Pierre-Emmanuel Morange ◽  
David-Alexandre Trégouët ◽  
France Gagnon
Keyword(s):  
Risk Factors ◽  
Tissue Factor ◽  
Genetic Variants ◽  
Plasma Levels ◽  
Tissue Factor Pathway Inhibitor ◽  
Future Research ◽  
Genetic Determinants ◽  
Genome Wide ◽  
A Genome ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) impedes early stages of the blood coagulation response, and low TFPI plasma levels increase the risk of thrombosis. TFPI plasma levels are heritable, but specific genetic determinants are unclear. We conducted a comprehensive review of genetic risk factors for TFPI plasma levels and identified 26 studies. We included 16 studies, as well as results from two unpublished genome-wide studies, in random effects meta-analyses of four commonly reported genetic variants in TFPI and its promoter (rs5940, rs7586970/rs8176592, rs10931292, and rs10153820) and 10 studies were summarised narratively. rs5940 was associated with all measures of TFPI (free, total, and activity), and rs7586970 was associated with total TFPI. Neither rs10931292 nor rs10153820 showed evidence of association. The narrative summary included 6 genes and genetic variants (P151L mutation in TFPI, PROS1, F5, APOE, GLA, and V617F mutation in JAK2) as well as a genome-wide linkage study, and suggested future research directions. A limitation of the systematic review was the heterogeneous measurement of TFPI. Nonetheless, our review found robust evidence that rs5940 and rs7586970 moderate TFPI plasma levels and are candidate risk factors for thrombosis, and that the regulation of TFPI plasma levels involves genetic factors beyond the TFPI gene.

Tissue Factor Pathway Inhibitor Inhibits Aortic Smooth Muscle Cell Migration Induced by Tissue Factor/Factor VIIa Complex

1997 ◽  
Vol 78 (03) ◽  
pp. 1138-1141 ◽  
Author(s):  
Yuichiro Sato ◽  
Yujiro Asada ◽  
Kousuke Marutsuka ◽  
Kinta Hatakeyama ◽  
Yuichi Kamikubo ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Protease Inhibitor ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Dependent Manner ◽  
Factor X ◽  
Migration Ability ◽  
Tissue Factor Pathway

SummaryTissue factor (TF), a transmembrane glycoprotein, forms a high affinity complex with factor Vll/VIIa (FVIIa) and thereby initiates blood coagulation. Tissue factor pathway inhibitor (TFPI) is an endogenous protease inhibitor of TF/FVIIa-initiated coagulation. We previously reported that TF was a strong chemotactic factor for cultured vascular smooth muscle cells (SMCs). In this study, we examined the contribution of FVIIa and the effect of TFPI to TF-induced cultured SMC migration. TF/FVIIa complex showed a strong migration ability, however, neither TF alone nor FVIIa induced SMC migration. TF/FVIIa treated by a serine protease inhibitor and the complex of TF and inactivated FVIIa (DEGR-FVIIa) did not stimulate SMC migration. Pretreatment with hirudin and the antibodies to a-thrombin and factor X had no effect on TF/FVIIa-induced SMC migration, although a-thrombin and factor Xa also induced SMC migration respectively. TFPI markedly inhibited TF/FVIIa-induced SMC migration in a concentration-dependent manner, but did not affect the SMC migration induced by platelet-derived growth factor (PDGF)-BB, basic fibro blast-growth factor (bFGF), or a-thrombin. These results indicate that the catalytic activity of TF/FVIIa complex is important on SMC migration, and TFPI can reduce SMC migration as well as thrombosis.

Penthalaris, a novel recombinant five-Kunitz tissue factor pathway inhibitor (TFPI) from the salivary gland of the tick vector of Lyme disease, Ixodes scapularis

2004 ◽  
Vol 91 (05) ◽  
pp. 886-898 ◽  
Author(s):  
Thomas Mather ◽  
José Ribeiro ◽  
Ivo Francischetti
Keyword(s):  
Salivary Gland ◽  
Lyme Disease ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Ixodes Scapularis ◽  
Gel Filtration ◽  
Factor X ◽  
Tick Saliva ◽  
Tissue Factor Pathway

SummaryTick saliva is a rich source of molecules with antiinflammatory, antihemostatic and immunosupressive properties. In this paper, a novel tick salivary gland cDNA with sequence homology to tissue factor pathway inhibitor (TFPI) and coding for a protein called Penthalaris has been characterized from the Lyme disease vector, Ixodes scapularis. Penthalaris is structurally unique and distinct from TFPI or TFPI-like molecules described so far, including Ixolaris, NAPc2, TFPI-1 and TFPI-2. Penthalaris is a 308-amino-acid protein (35 kDa, pI 8.58) with 12 cysteine bridges and 5 tandem Kunitz domains. Recombinant Penthalaris was expressed in insect cells and shown to inhibit factor VIIa (FVIIa)/tissue factor(TF)-induced factor X (FX) activation with an IC50 of ∼ 100 pM. Penthalaris tightly binds both zymogen FX and enzyme FXa (exosite), but not FVIIa, as demonstrated by column gel-filtration chromatography. At high concentrations, Penthalaris attenuates FVIIa/TF-induced chromogenic substrate (S2288) hydrolysis and FIX activation. In the presence of DEGR-FX or DEGR-FXa, but not des-Gla-DEGR-FXa as scaffolds, tight and stoichiometric inhibition of FVIIa/TF was achieved. In addition, Penthalaris blocks cell surface-mediated FXa generation by monomer (de-encrypted), but not dimer (encrypted) TF in HL-60 cells. Penthalaris may act in concert with Ixolaris and other salivary anti-hemostatics in order to help ticks to successfully feed on blood. Penthalaris is a novel anticoagulant and a tool to study FVIIa/TF-initiated biologic processes.

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