Tissue Factor-Like Activity Of The Human Monocytic Tumor Cell Line U937

1981 ◽  
Author(s):  
D Hudig ◽  
S I Rapaport ◽  
S P Bajaj
Keyword(s):  
Cell Line ◽  
Tissue Factor ◽  
Tumor Cell Line ◽  
Procoagulant Activity ◽  
Factor Vii ◽  
U937 Cells ◽  
Factor X ◽  
Blood Monocytes ◽  
Monocytic Cell ◽  
Coagulant Activity

Cells of the human monocytic cell line U937, derived from a patient with histiocytic lymphoma (Sundstrom and Nilsson, Int. J. Cancer 17:565, 1976) have procoagulant activity similar to that of activated peripheral blood monocytes, although about 10-fold more U937 cells than monocytes are required for equivalent activity. Procoagulant activity of the cells is Ca2+ dependent and is not demonstrable in factor VII deficient or factor X deficient plasma. Culture with E. coli 0127:B8 1ipopolysaccharide increases the procoagulant activity of washed U937 cells two-fold. Exposure of U937 cells to lymphokines from normal lymphocytes does not induce further coagulant activity. The slope of the log/log plot of cells vs. clotting time parallels that of human brain thromboplastin. Other cell lines of myeloid or lymphoid origin, e.g., K562 cells, WI-L2 cells, do not have procoagulant activity. Thus, U937 cells have constitutive factor VII-dependent coagulant activity similar to the tissue factor activity induced by activation of normal monocytes.In further experiments, U937 cells were incubated with purified human factor VII in the presence or absence of Ca2+ and then repeatedly washed. When subsamples of the cells were then added to recalcified factor VII deficient plasma in the absence of added tissue factor, the following clotting times were obtained: for cells incubated with factor VII in the presence of Ca2+,45"; for cells incubated with factor VII in the absence of Ca2+, 150". These data suggest that U937 cells can bind factor VII in a reaction requiring Ca2+, which then enables the cells to express their tissue factor-like activity in factor VII deficient plasma.

Stimulation Of Human Platelet Coagulant Activity By Endotoxin: A New Leukocyte-Mediated Pathway

1981 ◽  
Author(s):  
N Semeraro ◽  
D Locati ◽  
M Colucci
Keyword(s):  
Mononuclear Cells ◽  
Human Platelet ◽  
White Blood Cells ◽  
Coagulation Factor ◽  
Procoagulant Activity ◽  
Factor Vii ◽  
Factor X ◽  
Normal Plasma ◽  
Coagulant Activity ◽  
Recalcification Time

Despite the frequent occurrence of thrombocytopenia in patients with Gram-negative sepsis, a direct human platelet- endotoxin interaction in vitro can be hardly demonstrated. We report here that human platelets develop strong coagulant activity after simultaneous incubation with endotoxin and leucocytes in plasma. Whole blood or platelet-rich plasma (PRP) enriched of leukocytes were incubated at 37°C for 4 hours with different endotoxins (E.coli LPS, S. Minnesota LPS and a purified Lipid A, 10 ug/ml final concentration); leukocyte-free washed platelet suspensions isolated from these samples were found to markedly shorten the recalcification time of normal plasma (77 ± 11 sec, as compared to 359 ± 47 sec, obtained with platelets isolated from similar samples incubated with sterile saline, n=15). When platelets were challenged with endotoxin in the absence of white blood cells (i.e. in PRP) the subsequentely isolated and washed platelets had no procoagulant activity suggesting that leukocytes are essential mediators in the development of platelet coagulant activity induced by endotoxin. Experiments in which PRP was enriched with mononuclear cells or granulocytes revealed that the former were responsible for this property. “Stimulated” platelets shortened the recalcification time of plasmas deficient in any of the factors of the intrinsic pathway or of factor VII-deficient plasma to the same extent as in normal plasma. The rate of clotting was much lower in factor X-deficient plasma. These data indicate that, in contrast to leukocytes, whose procoagulant activity has well-known tissue factor-like properties, platelets act on coagulation factor X independently of both the intrinsic and extrinsic clotting pathways. Our findings add a new function to circulating mononuclear cells and may be relevant to understanding the postulated role of platelets in the activation of blood coagulation during endotoxemia in man.

Reduced Tissue Factor Expression in Rat Prostate Tumor Cells Limits Tumor Development in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2978-2978
Author(s):  
Hui Song ◽  
Zhong Liang ◽  
William Wolter ◽  
Mark A. Suckow ◽  
Elliot D. Rosen
Keyword(s):  
Cell Line ◽  
Tissue Factor ◽  
Factor Viia ◽  
Tumor Development ◽  
Coagulation Factor ◽  
Tumor Cell Line ◽  
Prostate Tumor ◽  
Factor X ◽  
Post Transplantation

Abstract Many tumors express procoagulant activities that contribute to the hemostatic complications associated with cancer. Tumor coagulant activities include tissue factor (TF) and cancer procoagulant (CP), a cysteine protease that activates Factor X. In current models of hemostasis, TF is critical in the initiation of coagulation following vascular injury as TF expressed on cells within the vessel wall complexes with blood-borne coagulation Factor VIIa to activate FX and FIX. In addition, TF transported to a growing thrombus by blood-borne microparticles binds FVIIa and contributes to thrombus propagation and stabilization. Recently, TF as well as other coagulation factors have been implicated in a variety of non-hemostatic processes including inflammation, angiogenesis, vascular development and cancer. To study the role of TF in tumor development we down-regulated TF expression in a prostate tumor cell line developed from the Lobund-Wistar (LW) rat. The LW rat combines histologic features of prostate cancer with the clinical features resembling clinical human disease; androgen-modulated growth, age-dependent spontaneous onset, and metastatic potential. Autochthonous tumors develop spontaneously or can be induced by treatment with MNU and testosterone. In addition, a cloned cell line from a spontaneous LW prostate tumor, PA-3, provides a transplantable tumor model. PA-3 cells were transfected with a series of plasmids expressing hairpin siRNAs designed to interfere with TF expression. The plasmids contained a U6-hairpin siRNA expression cassette, a neoR gene and EGFP. Cloned stable transformants of PA-3 cells expressing a siRNA corresponding to positions 260–278 of rat TF mRNA, PA-3[797], reliably reduced TF expression by 65% compared to control clonal PA-3 transformants, PA-3[776], expressing neoR and EGFP but no siRNA. To test the consequences of reduced TF on tumor development in vivo, 4 independently isolated PA-3[797] cell lines and 4 independently isolated PA-3[776] control lines were injected subcutaneously into LW rats (3 rats per cell line). Ten of the 12 rats injected with control cells developed detectable tumors 4 weeks post transplantation (tumor mass = 2.2g +/− 1.8g). In contrast, only 1 of the 12 rats receiving the low TF expressing PA-3[797] cells developed a small tumor (0.07 gm) suggesting TF expression is required for tumorigenesis.

Antigenic and Functional Expression of Tissue Factor in Endotoxin Stimulated U937 Cells: Regulation of Activity by Calcium Ionophore A23187

1995 ◽  
Vol 74 (03) ◽  
pp. 904-909 ◽  
Author(s):  
R Consonni ◽  
R M Bertina
Keyword(s):  
Tissue Factor ◽  
Specific Activity ◽  
Calcium Ionophore ◽  
Catalytic Efficiency ◽  
Calcium Ionophore A23187 ◽  
Factor Vii ◽  
Ionophore A23187 ◽  
U937 Cells ◽  
Factor X ◽  
Monocytic Leukemia

SummaryTissue Factor (TF) is a transmembrane glycoprotein that serves as cofactor for Factor VII (FVII) in the initiation of blood coagulation and that is differentially expressed in a number of cell types, being constitutively expressed in some and inducible in others. We studied the localization and the functional activity of TF in monocytic leukemia U937 cells at different time intervals after lipopolysaccharides (LPS) stimulation, and the effect of calcium ionophore on the surface expressed TF.Exposure of U937 cells to 10 µg/ml LPS resulted in a time dependent increase of TF expression that reached a maximum at 12 h for TF antigen and at 24 h for TF activity. Blocking of surface TF with inhibitory anti-TF antibody abolished >93% of the activity of lysed cells stimulated for 24 h, while it blocked only 80% of the activity in lysed cells stimulated for 12 h suggesting that at that time about 20% of TF is not accessible for the antibody. Even at 24 h when the specific activity of surface expressed TF is 5.5 times higher than at 12 h, this specific activity is still 10 fold lower than that of TF in lysed cells. Addition of Ca++ ionophore A23187 to LPS stimulated cells resulted in a fast increase of TF activity that was dependent on the dose of ionophore, on the extracellular Ca++ concentration and on the time that the cells had been incubated with LPS. Kinetic analysis of Factor X hydrolysis by the TF/FVII complex demonstrated that the addition of ionophore resulted in an increase of both the Vmax and the apparent Km, however, without affecting the catalytic efficiency of the reaction.These data suggest that in U937 cells part of the TF induced by LPS is cryptic, that the specific activity of surface expressed tissue factor increases with time and that intracellular changes in Ca++ concentration can be important in the regulation of the expression of TF activity on the cell surface.

Biological Control of Factor VII

1976 ◽  
Vol 35 (01) ◽  
pp. 096-100 ◽  
Author(s):  
Yale Nemerson
Keyword(s):  
Biological Control ◽  
Tissue Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Activate Factor ◽  
Chain Molecule ◽  
Factor X ◽  
Hageman Factor ◽  
Coagulant Activity ◽  
Tissue Factor Pathway

SummaryThe tissue factor pathway is initiated by factor VII in the presence of tissue factor. The first proteolytic reaction involves cleavage of factor X by factor VII. Activated factor X, the product of this reaction, can activate factor VII by cleavage of a specific bond. The apparent coagulant activity of factor VII then rises about 60-fold. Activated factor X can also inactivate factor VII by catalyzing the cleavage of a second bond which results in a three chain molecule. Fragments of Hageman factor and perhaps kallikrein can also activate factor VII. Hageman factor, however, does not catalyze the inactivating cleavage of factor VII at a significant rate.Recent data showing that the tissue factor pathway can activate the intrinsic system are discussed. We have shown that activated factor X, which can be generated by the tissue factor pathway, can feed back and activate factor IX in a calcium and phospholipid requiring reaction.

Mechanism of Resveratrol-mediated Suppression of Tissue Factor Gene Expression

2002 ◽  
Vol 87 (01) ◽  
pp. 155-162 ◽  
Author(s):  
Usha Pendurthi ◽  
Feng Meng ◽  
N. Mackman ◽  
L. Vijaya Rao
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Tissue Factor ◽  
Mononuclear Cells ◽  
Cell Surface Receptor ◽  
Coagulation Cascade ◽  
Factor Vii ◽  
Dependent Manner ◽  
Monocytic Cell ◽  
Monocytic Cell Line

SummaryTissue factor (TF) is a cell surface receptor for factor VII(a), and the binding of factor VII(a) to TF initiates the coagulation cascade. Inappropriate in vivo expression of TF in vascular cells has been shown to be responsible for thrombotic disorders associated with a variety of pathological conditions, including gram-negative sepsis, cancer and atherosclerosis. A number of epidemiological studies suggest that moderate consumption of red wine provides protective effects against coronary heart disease mortality. Recently, we have shown that resveratrol, a polyphenolic compound found in wine, inhibited the induction of TF expression in endothelial cells and mononuclear cells (Pendurthi UR, Williams JT, Rao LVM. Arterioscler Thromb Vasc Biol 1999; 19: 419-426). In the present study, we examined the mechanism by which resveratrol inhibits the expression of TF in monocytes by using a monocytic cell line, THP-1, as a model cell. Northern blot analysis, gel mobility shift assays and transfection studies with various TF promoter constructs, as well as other transcription regulatory constructs, were used to elucidate the inhibitory mechanism of resveratrol. The data show that resveratrol inhibited lipopolysaccharide (LPS)-induced expression of TF in human monocytes and monocytic cell line, THP-1 in a dose dependent manner. Resveratrol did not significantly alter the binding of various transcription factors involved in TF gene expression to DNA. However, resveratrol suppressed the transcription of cloned human TF promoter. Further experiments revealed that resveratrol reduced κB- but not AP-1-driven transcriptional activity. Additional experiments showed that resveratrol suppressed the phosphorylation of p65 and its transactivation. In summary, our results indicate that resveratrol does not inhibit the activation or translocation of NF-κB/Rel proteins but inhibits NF-κB/Rel-dependent transcription by impairing the transactivation potential of p65.

Self-Damping Mechanism in Blood Coagulation

1974 ◽  
Vol 32 (01) ◽  
pp. 057-064 ◽  
Author(s):  
Y Nemerson ◽  
S.A Silverberg ◽  
J Jesty
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Calcium Ions ◽  
Control Mechanism ◽  
Factor Vii ◽  
Damping Factor ◽  
Factor V ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Two Systems

SummaryTwo reactions of the extrinsic pathway of coagulation, the activations of Factor X and prothrombin, have been studied in purified systems and shown to be self-damping. Factor X was activated by the tissue factor - Factor VII complex, and prothrombin by two systems: the coagulant protein of Taipan venom, and the physiological complex of activated Factor X, Factor V, lipid, and calcium ions. In each case the yield of enzyme, activated Factor X or thrombin, is a function of the concentration of activator. These and other observations are considered as a basis for a control mechanism in coagulation.

Factors Affecting the lnteraction of Tissue Factor/Factor Vll with Factor X in a Heterogeneous Tubular Reactor

1991 ◽  
Vol 65 (02) ◽  
pp. 139-143 ◽  
Author(s):  
Cynthia H Gemmell ◽  
Vincet T Turitto ◽  
Yale Nemerson
Keyword(s):  
Steady State ◽  
Tissue Factor ◽  
Factor Viia ◽  
Transmembrane Protein ◽  
Strong Association ◽  
Tubular Reactor ◽  
Factor Xa ◽  
Phospholipid Bilayer ◽  
Factor Vii ◽  
Factor X

SummaryA novel reactor recently described for studying phospholipiddependent blood coagulation reactions under flow conditions similar to those occurring in the vasculature has been further charactenzed. The reactor is a capitlary whose inner wall is coated with a stable phospholipid bilayer (or two bilayers) containing tissue factor, a transmembrane protein that is required for the enzymatic activation of factor X by factor VIIa. Perfusion of the capillary at wall shear rates ranging from 25 s−1 to 1,200 s−1 with purified bovine factors X and VIIa led to steady state factor Xa levels at the outlet. Assay were performed using a chromogenic substrate, SpectrozymeTMFXa, or by using a radiometric technique. In the absence of Ca2+ or factor VIIa there was no product formation. No difference was noted in the levels of factor Xa achieved when non-activated factor VII was perfused. Once steady state was achieved further factor Xa production continued in the absence of factor VIIa implying a very strong association of factor VIIa with the tissue factor in the phospholipid membrane. In agreement with static vesicle-type studies the reactor was sensitive to wall tissue factor concentration, temperature and the presence of phosphatidylserine in the bilayer.

scholarly journals Circulating Tissue Factor Procoagulant Activity and Thrombin Generation in Patients with Type 2 Diabetes: Effects of Insulin and Glucose

2007 ◽  
Vol 92 (11) ◽  
pp. 4352-4358 ◽  
Author(s):  
Guenther Boden ◽  
Vijender R. Vaidyula ◽  
Carol Homko ◽  
Peter Cheung ◽  
A. Koneti Rao
Keyword(s):  
Type 2 Diabetes ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Coagulation Factor ◽  
Procoagulant Activity ◽  
Factor Vii ◽  
Coagulation Factor Vii ◽  
Glucose Levels ◽  
Study Protocols

Abstract Context: Type 2 diabetes mellitus (T2DM) is a hypercoagulable state. Tissue factor (TF) is the principal initiator of blood coagulation. Objective: Our objective was to examine the effects of hyperglycemia and hyperinsulinemia on the TF pathway of blood coagulation in T2DM. Design: Three study protocols were used: 1) acute correction of hyperglycemia (with iv insulin) followed by 24 h of euglycemia, 2) 24 h of selective hyperinsulinemia, and 3) 24 h of combined hyperinsulinemia and hyperglycemia. Setting: The study took place at a clinical research center. Study Participants: Participants included 18 T2DM patients and 22 nondiabetic controls. Results: Basal TF-procoagulant activity (TF-PCA), monocyte TF mRNA, plasma coagulation factor VII (FVIIc), and thrombin-anti-thrombin complexes were higher in T2DM than in nondiabetic controls, indicating a chronic procoagulant state. Acutely normalizing hyperglycemia over 2–4 h resulted in a small (∼7%) but significant decline in TF-PCA with no further decline over 24 h. Raising insulin levels alone raised TF-PCA by 30%, whereas raising insulin and glucose levels together increased TF-PCA (by 80%), thrombin-anti-thrombin complexes, and prothrombin fragment 1.2. Plasma FVIIa and FVIIc declined with increases in TF-PCA. Conclusion: We conclude that the combination of hyperglycemia and hyperinsulinemia, common in poorly controlled patients with T2DM, contributes to a procoagulant state that may predispose these patients to acute cardiovascular events.

scholarly journals Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 204-212
Author(s):  
NL Sanders ◽  
SP Bajaj ◽  
A Zivelin ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Additional Material ◽  
Plasma Reaction ◽  
Progress Curve ◽  
Peptide Release ◽  
Release Data

A study was carried out to explore requirements for the inhibition of tissue factor-factor VIIa enzymatic activity in plasma. Reaction mixtures contained plasma, 3H-factor IX or 3H-factor X, tissue factor (vol/vol 2.4% to 24%), and calcium. Tissue factor-factor VIIa activity was evaluated from progress curves of activation of factor IX or factor X, plotted from tritiated activation peptide release data. With normal plasma, progress curves exhibited initial limited activation followed by a plateau indicative of loss of tissue factor-factor VIIa activity. With hereditary factor X-deficient plasma treated with factor X antibodies, progress curves revealed full factor IX activation. Adding only 0.4 micrograms/mL factor X (final concentration) could restore inhibition. Inhibition was not observed in purified systems containing 6% to 24% tissue factor, factor VII, 0.5 micrograms/mL, factor IX, 13 micrograms/mL, and factor X up to 0.8 micrograms/mL, but could be induced by adding barium-absorbed plasma to the reaction mixture. Thus, both factor X and an additional material in plasma were required for inhibition. The amount of factor X needed appeared related to the concentration of tissue factor; adding more tissue factor at the plateau of a progress curve induced further activation. These results also indicate that inhibited reaction mixtures contained active free factor VII(a). Preliminary data suggest that inhibition may stem from loss of activity of the tissue factor component of the tissue factor- factor VII(a) complex.

scholarly journals Evidence for the presence of tissue factor activity on subendothelium

Blood ◽  
1989 ◽  
Vol 73 (4) ◽  
pp. 968-975
Author(s):  
HJ Weiss ◽  
VT Turitto ◽  
HR Baumgartner ◽  
Y Nemerson ◽  
T Hoffmann
Keyword(s):  
Tissue Factor ◽  
Umbilical Artery ◽  
Factor Viia ◽  
Rabbit Aorta ◽  
Coagulation Factors ◽  
Factor Vii ◽  
Factor X ◽  
Factor Activity ◽  
Fibrin Deposition ◽  
Tissue Factor Activity

By a variety of methods, tissue factor activity was demonstrated in the subendothelium of rabbit aorta and human umbilical artery. In one method, everted segments of de-endothelialized vessels were mounted in an annular perfusion chamber and the subendothelial surface was exposed to nonanticoagulated human blood under controlled flow. Procoagulant activity was assessed by measuring fibrin deposition on subendothelium and fibrinopeptide A (FPA) levels in post chamber blood. Both fibrin deposition and FPA were decreased with rabbit vessel segments exposed (at a shear rate of 650 seconds-1) to blood from patients with factor VII deficiency and with umbilical artery segments (at shear rates of 90 to 180 seconds-1) that had been pretreated with a monoclonal antibody to human tissue factor. In a second method, everted umbilical artery segments were mounted on a stir bar and the subendothelial surface was exposed, with stirring, to plasma or purified coagulation factors. The capacity of the surface to clot plasma on addition of calcium was inhibited by the antibody to tissue factor. The surface also activated purified 3H-factor X in the presence of factor VIIa, but not in its absence, and this surface property was almost entirely eliminated by pretreating the vessel segments with antitissue factor. Tissue factor activity in subendothelium could play a role in both the arrest of bleeding and in promoting the formation of thrombi at sites of vascular injury.

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