CARBOHYDRATE COMPOSITION AND LECTIN BINDING AFFINITIES OF HUMAN PLACENTAL TISSUE FACTOR

1987 ◽  
Author(s):  
H Scheefers ◽  
A Kobus ◽  
R Geyer
Keyword(s):  
Tissue Factor ◽  
Lectin Binding ◽  
Placental Tissue ◽  
Bovine Brain ◽  
Factor Ix ◽  
Gas Liquid Chromatography ◽  
Binding Affinities ◽  
Binding Studies ◽  
Con A ◽  
Human Placental Tissue

Tissue factor (TF) is a widely distibuted membrane glycoprotein and the most potent trigger of bloodcoagulation. It serves as an essential cofactor for the activation of Factor IX and X by Factor Vll/VIIa.TF is a lipoprotein composed of a phospholipid portion and a glycosylated apoprotein (apo-TF). The procoagulant activity of bovine brain TF is inhibited bythe lectin Con A indicating that the carbohydrates of TF might play a functional role in its interactionwith Factor Vll/VIIa.In the present study apo-TF was purified from human placenta by repeated SDS-PAGE to a purity of 95%. The carbohydrates of apo-TF wereanalyzed by capillary gas- liquid-chromatography andmass-fragmentography. This analysis revealed that apo-TF contains about 16% (w/w) carbohydrate consistingof 50.4 mole% N-acetylglucosamine, 22.2 mole% mannose, 21.0 mole% galactose, 3.2 mole% fucose and 3.2 mole% N-acetylgalactosamine. Further information on the structure of the carbohydrate moieties of the apoTF was achieved by determining the binding affinities of the apo-TF to ten different lectins. For this purpose a semiquantitative spot lectino sorbent assaywas developed. This assay is based on the detection of peroxidase-labeled lectins after being bound to the carbohydrate moieties of apo-TF adsorbed onto a nitrocellulose membrane. Human placental apo-TF showed the strongest affinity to wheat germ agglutinin which specifically binds to N-acetylglucosamine and sialic acid residues.In contrast to bovine brain apo-TF, human placental apo-TF only weakly interacted with Con A, which is known to recognize mannosyl residues in mannose-rich, hybrid- and biantennary glycans,but not in tri- or tetraantennary oligosaccharides of the complex type. From the carbohydrate constituent analysis and from the lectin binding studies it can be concluded that human placental apo-TF carriesabout four N-linked higher branched oligosaccharide chains.

Lectin Binding Studies on RNA Tumor Virus-Infected Cells

1975 ◽  
Vol 33 ◽  
pp. 326-327
Author(s):  
D. C. Hixson
Keyword(s):  
Binding Sites ◽  
Lectin Binding ◽  
Culture Conditions ◽  
3T3 Cells ◽  
Binding Studies ◽  
Con A ◽  
Microscopic Studies ◽  
Tumor Virus ◽  
Infected Cells ◽  
Cell Culture Conditions

The abilities of plant lectins to preferentially agglutinate malignant cells and to bind to specific monosaccharide or oligosaccharide sequences of glycoproteins and glycolipids make them a new and important biochemical probe for investigating alterations in plasma membrane structure which may result from malignant transformation. Electron and light microscopic studies have demonstrated clustered binding sites on surfaces of SV40-infected or tryp- sinized 3T3 cells when labeled with concanavalin A (con A). No clustering of con A binding sites was observed in normal 3T3 cells. It has been proposed that topological rearrangement of lectin binding sites into clusters enables con A to agglutinate SV40-infected or trypsinized 3T3 cells (1). However, observations by other investigators have not been consistent with this proposal (2) perhaps due to differences in reagents used, cell culture conditions, or labeling techniques. The present work was undertaken to study the lectin binding properties of normal and RNA tumor virus-infected cells and their associated viruses using lectins and ferritin-conjugated lectins of five different specificities.

scholarly journals Influence of mutations in tissue factor on the fine specificity of macromolecular substrate activation

1997 ◽  
Vol 321 (3) ◽  
pp. 787-794 ◽  
Author(s):  
Sabine DITTMAR ◽  
Wolfram RUF ◽  
Thomas S. EDGINGTON
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Factor Ix ◽  
Factor X ◽  
Wild Type ◽  
Binding Studies ◽  
Fine Specificity ◽  
Fibronectin Type Iii

The C-terminal fibronectin-type-III-like module of the tissue factor (TF) extracellular domain plays a requisite role in the activation of macromolecular substrates by factor VIIa (VIIa) in complex with TF. Unlike the mutations Lys165→Ala, Lys166→Ala in TF, which prevent efficient proteolysis of factor X, we found that the coagulant defect of a site-specific Trp158→Arg, Ser160→Gly replacement mutant of TF is largely attributable to the inability of TF to efficiently support the activation of the bound zymogen VII to the active protease VIIa. Binding studies demonstrated comparable affinity of binding of VIIa or VII by wild-type TF and TFR158G160. In comparison with wild-type TF, the catalytic efficiency of factor X activation was reduced 56-fold with TFA165A166 as the cofactor, but only 3.5-fold with TFR158G160. The activation of VII bound to TF by factor Xa or VIIa was reduced 2-fold in the presence of TFR158G160 and 7Ő8-fold with TFA165A166. This suggests that the molecular recognition of VII in complex with TF by the enzymes TFŐVIIa and factor Xa are similar. Generation of factor IXa by TFR158G160ŐVIIa was unaltered, but reduced 2-fold with TFA165A166. In addition, the mutations affected the cleavage of the two scissile bonds of factor IX differently, providing further support for the idea that the cofactor, TF, influences the fine specificity of activation of macromolecular substrates by the TFŐVIIa complex.

Coagulation factor III (tissue factor) interaction with phospholipid vesicles induced by cadmium: characterization of the reconstituted protein-membrane complex

1981 ◽  
Vol 1 (3) ◽  
pp. 197-205 ◽  
Author(s):  
Steven D. Carson ◽  
William H. Konigsberg
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Gel Filtration ◽  
Coagulation Factor ◽  
Bovine Brain ◽  
Factor Ix ◽  
Phospholipid Vesicles ◽  
Factor X ◽  
Lipid Complex ◽  
Proteolytic Activation

Coagulation factor III (tissue factor) is a membrane glycoprotein which serves as a cofactor in the proteolytic activation of factor X and factor IX by factor VIIa. Mixing of human placental factor III apoprotein with vesicles of bovine brain phospholipids does not produce significant reconstitution of factor III activity, but, when the mixture of apoprotein and vesicles is made 5 mM with CdCI2, the apoprotein is incorporated into the vesicles. Ultracentrifugation on sucrose density gradients demonstrated that the active factor III-lipid complex formed by reconstitution with vesicles had a density indistinguishable from that of the complex formed by detergent dialysis. Vesicles isolated after centrifugation were shown to range in diameter from 20 nm to over 100 nm using the electron microscope. Gel filtration showed that factor-III activity was associated with all size-classes of vesicles. The presence of factor III activity in the smaltest vesicles argues for a specific cadmium-mediated reconstitution of the apoprotein with phospholipid vesicles.

scholarly journals Studies on the cell surface of zoospores and cysts of the fungus Phytophthora cinnamomi: nature of the surface saccharides as determined by quantitative lectin binding studies.

1985 ◽  
Vol 33 (5) ◽  
pp. 384-388 ◽  
Author(s):  
A Bacic ◽  
M L Williams ◽  
A E Clarke
Keyword(s):  
Cell Surface ◽  
Concanavalin A ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Phytophthora Cinnamomi ◽  
Lectin Binding ◽  
Soybean Agglutinin ◽  
Binding Studies ◽  
Con A

The nature of the surface saccharides of zoospores, "partially encysted zoospores" and cysts of the root-rotting fungus Phytophthora cinnamomi, has been examined by quantitative lectin binding studies. Zoospores bound concanavalin A (Con A), but did not bind any of a variety of other lectins tested. In contrast, both cysts and "partially encysted zoospores" bound soybean agglutinin (SBA) as well as Con A. This indicates that accessible alpha-D-glucosyl/alpha-D-mannosyl-containing glycoconjugates predominate at the zoospore surface, whereas both alpha-D-glucosyl/alpha-D-mannosyl and galactosyl and/or N-acetyl-D-galactosaminosyl residues are accessible at the surface of cysts and "partially encysted zoospores." Neither Ulex europeus lectin nor wheat germ agglutinin (WGA) bound to any of the three cell preparations, indicating the absence of accessible alpha-L-fucosyl and N-acetyl-D-glucosaminosyl residues.

The Antithrombotic Properties of Human Prothrombin Fragment 1.2 in Mice

1990 ◽  
Vol 63 (03) ◽  
pp. 413-416 ◽  
Author(s):  
Amy Chung ◽  
Frederick A Ofosu ◽  
Morris A Blajchman
Keyword(s):  
Tissue Factor ◽  
Placental Tissue ◽  
Lethal Effect ◽  
Dependent Manner ◽  
Minimum Concentration ◽  
Prothrombin Fragment ◽  
Human Placental Tissue ◽  
Dose Dependent

SummaryWe have investigated the antithrombotic properties of prothrombin fragment 1.2 (F1.2) in this study. To do this, we established the minimum concentration of human placental tissue factor or human a-thrombin that was lethal in mice within 5 min after intravenous injection. Prothrombin F1.2 protected the mice from the lethal effect of tissue factor or α-thrombin in a dose dependent manner, with 500 μg (14 nmoles) of prothrombin F1.2 per mouse being the minimum amount required to protect all mice from the lethal effect of either thrombogenic stimulus. The minimum dose of heparin which protected mice from the lethal effect of thrombin or tissue factor was 6 units or ∼3.3 nmoles. The observation that prothrombin F1.2 has antithrombotic properties suggests prothrombin F1.2 can modulate coagulation in vivo, as it has previously been shown to do in vitro.

Human placental tissue factor: Protease susceptibility of extracellular and cytoplasmic domains

1995 ◽  
Vol 79 (5-6) ◽  
pp. 451-459 ◽  
Author(s):  
Sara M. Whittle ◽  
S.Christine Yoder ◽  
Steven D. Carson
Keyword(s):  
Tissue Factor ◽  
Placental Tissue ◽  
Cytoplasmic Domains ◽  
Human Placental Tissue

Studies of the Mechanism for Enhanced Cell Surface Factor VIIa/Tissue Factor Activation of Factor X on Fibroblast Monolayers after Their Exposure to N-Ethylmaleimide

1994 ◽  
Vol 72 (06) ◽  
pp. 848-855 ◽  
Author(s):  
Dzung The Le ◽  
Samuel I Rapaport ◽  
L Vijaya Mohan Rao
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Factor Viia ◽  
Cell Damage ◽  
Specific Binding ◽  
Surface Membrane ◽  
Annexin V ◽  
Factor X ◽  
Binding Studies ◽  
Anionic Phospholipid

SummaryFibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.

Endotoxin-Induced Tissue Factor in Human Monocytes is Dependent upon Protein Kinase C Activation

1993 ◽  
Vol 70 (05) ◽  
pp. 800-806 ◽  
Author(s):  
C Ternisien ◽  
M Ramani ◽  
V Ollivier ◽  
F Khechai ◽  
T Vu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tissue Factor ◽  
Factor Ix ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Human Monocytes ◽  
Kinase C ◽  
Pkc Activation

SummaryTissue factor (TF) is a transmembrane receptor which, in association with factors VII and Vila, activates factor IX and X, thereby activating the coagulation protease cascades. In response to bacterial lipopolysaccharide (LPS) monocytes transcribe, synthesize and express TF on their surface. We investigated whether LPS-induced TF in human monocytes is mediated by protein kinase C (PKC) activation. The PKC agonists phorbol 12- myristate 13-acetate (PMA) and phorbol 12, 13 dibutyrate (PdBu) were both potent inducers of TF in human monocytes, whereas 4 alpha-12, 13 didecanoate (4 a-Pdd) had no such effect. Both LPS- and PMA-induced TF activity were inhibited, in a concentration dependent manner, by three different PKC inhibitors: H7, staurosporine and calphostin C. TF antigen determination confirmed that LPS-induced cell-surface TF protein levels decreased in parallel to TF functional activity under staurosporine treatment. Moreover, Northern blot analysis of total RNA from LPS- or PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to H7 and staurosporine. The decay rate of LPS-induced TF mRNA evaluated after the arrest of transcription by actinomycin D was not affected by the addition of staurosporine, suggesting that its inhibitory effect occurred at a transcriptional level. We conclude that LPS-induced production of TF and its mRNA by human monocytes are dependent on PKC activation.

A Preliminary Study on Human Placental Tissue Impaired by Gestational Diabetes: A Comparison of Gel-Based versus Gel-Free Proteomics Approaches

2016 ◽  
Vol 22 (2) ◽  
pp. 71-82 ◽  
Author(s):  
Marco Roverso ◽  
Maura Brioschi ◽  
Cristina Banfi ◽  
Silvia Visentin ◽  
Silvia Burlina ◽  
...  
Keyword(s):  
Gestational Diabetes ◽  
Placental Tissue ◽  
Human Placental Tissue ◽  
Preliminary Study
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