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.

scholarly journals The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII

1990 ◽  
Vol 265 (2) ◽  
pp. 327-336 ◽  
Author(s):  
V J J Bom ◽  
R M Bertina
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Reaction Rate ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Fluid Phase ◽  
Fold Increase ◽  
Factor Vii ◽  
Factor X ◽  
Extrinsic Pathway

In the extrinsic pathway of blood coagulation, Factor X is activated by a complex of tissue factor, factor VII(a) and Ca2+ ions. Using purified human coagulation factors and a sensitive spectrophotometric assay for Factor Xa, we could demonstrate activation of Factor X by Factor VIIa in the absence of tissue-factor apoprotein, phospholipids and Ca2+. This finding allowed a kinetic analysis of the contribution of each of the cofactors. Ca2+ stimulated the reaction rate 10-fold at an optimum of 6 mM (Vmax. of 1.1 x 10(-3) min-1) mainly by decreasing the Km of Factor X (to 11.4 microM). In the presence of Ca2+, 25 microM-phospholipid caused a 150-fold decrease of the apparent Km and a 2-fold increase of the apparent Vmax. of the reaction; however, both kinetic parameters increased with increasing phospholipid concentration. Tissue-factor apoprotein contributed to the reaction rate mainly by an increase of the Vmax., in both the presence (40,500-fold) and absence (4900-fold) of phospholipid. The formation of a ternary complex of Factor VIIa with tissue-factor apoprotein and phospholipid was responsible for a 15 million-fold increase in the catalytic efficiency of Factor X activation. The presence of Ca2+ was absolutely required for the stimulatory effects of phospholipid and apoprotein. The data fit a general model in which the Ca2(+)-dependent conformation allows Factor VIIa to bind tissue-factor apoprotein and/or a negatively charged phospholipid surface resulting into a decreased intrinsic Km and an increased Vmax. for the activation of fluid-phase Factor X.

scholarly journals Factor VIIa-catalyzed activation of factor X independent of tissue factor: its possible significance for control of hemophilic bleeding by infused factor VIIa

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1069-1073 ◽  
Author(s):  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Activate Factor ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Hemophilic Patient ◽  
Factor X Activation ◽  
Rate Of Activation

Abstract Infusing factor VIIa (FVIIa) has been reported to control bleeding in hemophilic patients with factor VIII (FVIII) inhibitors. This is difficult to attribute to an enhanced FVIIa/tissue factor (TF) activation of factor X, since in vitro studies suggest that infusion of FVIIa should neither increase substantially the rate of formation of FVIIa/TF complexes during hemostasis (Proc Natl Acad Sci USA 85:6687, 1988) nor bypass the dampening of TF-dependent coagulation by the extrinsic pathway inhibitor (EPI) (Blood 73:359, 1989). Partial thromboplastin times have also been reported to shorten after infusion of FVIIa. The experiments reported herein establish that shortening of partial thromboplastin times after adding FVIIa to hemophilic plasma in vitro stems from an FVIIa-catalyzed activation of factor X independent of possible trace contamination of reagents with TF. Experiments in purified systems confirmed that FVIIa can slowly activate factor X in a reaction mixture containing Ca2+ and phospholipid but no source of TF. The rate of activation was sufficient to account for the shortening of partial thromboplastin times observed. EPI, which turned off continuing FVIIa/TF activation of factor X, was unable to prevent continuing FVIIa/phospholipid activation of factor X. Because circulating plasma contains only a trace, if any, free FVIIa, such a reaction could never occur physiologically. However, infusing FVIIa creates a nonphysiologic circumstance in which a continuing slow FVIIa/phospholipid catalyzed activation of factor X could conceivably proceed in vivo unimpeded by EPI. Such a mechanism of factor X activation might compensate for an impaired factor IXa/FVIIIa/phospholipid activation of factor X during hemostatis, and therefore control bleeding in a hemophilic patient.

scholarly journals A candidate activation pathway for coagulation factor VII

2019 ◽  
Vol 476 (19) ◽  
pp. 2909-2926
Author(s):  
Tina M. Misenheimer ◽  
Kraig T. Kumfer ◽  
Barbara E. Bates ◽  
Emily R. Nettesheim ◽  
Bradford S. Schwartz
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Contact Activation ◽  
Coagulation Factor Vii ◽  
Factor Viiia

Abstract The mechanism of generation of factor VIIa, considered the initiating protease in the tissue factor-initiated extrinsic limb of blood coagulation, is obscure. Decreased levels of plasma VIIa in individuals with congenital factor IX deficiency suggest that generation of VIIa is dependent on an activation product of factor IX. Factor VIIa activates IX to IXa by a two-step removal of the activation peptide with cleavages occurring after R191 and R226. Factor IXaα, however, is IX cleaved only after R226, and not after R191. We tested the hypothesis that IXaα activates VII with mutant IX that could be cleaved only at R226 and thus generate only IXaα upon activation. Factor IXaα demonstrated 1.6% the coagulant activity of IXa in a contact activation-based assay of the intrinsic activation limb and was less efficient than IXa at activating factor X in the presence of factor VIIIa. However, IXaα and IXa had indistinguishable amidolytic activity, and, strikingly, both catalyzed the cleavage required to convert VII to VIIa with indistinguishable kinetic parameters that were augmented by phospholipids, but not by factor VIIIa or tissue factor. We propose that IXa and IXaα participate in a pathway of reciprocal activation of VII and IX that does not require a protein cofactor. Since both VIIa and activated IX are equally plausible as the initiating protease for the extrinsic limb of blood coagulation, it might be appropriate to illustrate this key step of hemostasis as currently being unknown.

Activity and Regulation of Long-Acting Factor VIIa Analogs.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3141-3141 ◽  
Author(s):  
Samit Ghosh ◽  
Prosenjit Sen ◽  
Mirella Ezban ◽  
Usha R. Pendurthi ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Treatment Options ◽  
Coagulation Factor ◽  
Repeated Administration ◽  
Similar Rate ◽  
Factor X ◽  
Bleeding Episodes ◽  
Tissue Factor Pathway ◽  
Long Acting

Abstract Recombinant coagulation factor VIIa (rFVIIa) has proven to be a safe and effective drug for treatment of bleeding episodes in hemophilia patients with inhibitors. However, rFVIIa is cleared from the circulation relatively fast, with circulating half-life of about 2–4 h, requiring repeated administration of rFVIIa for the effective treatment. Therefore, development of FVIIa analogs that could remain in the circulation for a longer period of time would be of a great value for improving the treatment options of rFVIIa. e.g., by prophylaxis. PEGylation of plasma proteins was shown to extend their circulatory half-lives but the PEGylation may also disrupt macromolecular interactions. In the present study we characterized the interaction of two glycoPEGylated analogs of rFVIIa, rFVIIa-10K PEG and rFVIIa-40K PEG, with its cofactor tissue factor (TF), substrate factor X (FX) and plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT). Both the PEGylated FVIIa analogs exhibited similar amidolytic activity as of wild-type rFVIIa (wt-rFVIIa) in the absence or presence of relipidated TF. The analogs were as effective as wt-rFVIIa in activating FX in the absence of TF. No significant differences were found between the PEGylated rFVIIa analogs and wt-rFVIIa in TF-dependent FX activation at saturating concentrations of rFVIIa, however, at lower concentrations of rFVIIa (10 to 50 pM), rFVIIa-10K PEG and rFVIIa-40K PEG activated FX at a slightly lower rate, 50% and 75%, respectively, of wt-rFVIIa. Further studies revealed that both AT/heparin and TFPI inhibited the PEGylated rFVIIa-TF complexes effectively but slightly at a lower rate compared to that was noted for wt-rFVIIa-TF. TFPI-Xa inhibited the PEGylated rFVIIa-TF and wt-rFVIIa-TF at a similar rate. On unperturbed HUVEC, wt-FVIIa (10 nM) could activate FX, albeit slowly, (1.7 nM/h) and the PEGylated rFVIIa activated FX even at much lower rates (0.23 nM/h for rFVIIa-10K PEG and 0.15 nM/h for rFVIIa-40K PEG). On stimulated HUVEC expressing TF, the PEGylated rFVIIa variants were slightly less effective at lower concentrations compared to wt-rFVIIa in activating FX, but no significant differences were found among them in activating factor X at saturating concentrations of rFVIIa (80–100 nM/h). The PEGylated rFVIIa analogs bound to cell surface TF were inhibited by TFPI-Xa complex at a similar rate as that was observed for wt-rFVIIa (IC50 in nM: 0.102 ± 0.032 for wt-rFVIIa, 0.111 ± 0.024 for rFVIIa-10K PEG, and 0.096 ± 0.019 for rFVIIa-40K PEG). AT/heparin inhibited rFVIIa-10K PEG bound to endothelial cell TF at a similar rate as it inhibited wt-rFVIIa (IC50 in μg/ml: wt-rFVIIa, 3.42 ± 068; rFVIIa-10K PEG, 3.56 ± 0.073), but the inhibition rate was slightly lower for rFVIIa-40K PEG bound to TF (IC50 5.92 ± 0.44 μg/ml). Overall, our present data suggest that long-acting PEGylated FVIIa analogs retain full enzymatic activity and can interact TF and FX effectively, and are inhibited by AT/heparin and TFPI-Xa as for wt-rFVIIa. Although the pegylated rFVIIa variants exhibited somewhat lower affinity towards TF, this may not critically affect the TF-driven FXa generation. Further work is needed to fully characterize these molecules.

scholarly journals Factor VIIa-catalyzed activation of factor X independent of tissue factor: its possible significance for control of hemophilic bleeding by infused factor VIIa

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1069-1073 ◽  
Author(s):  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Activate Factor ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Hemophilic Patient ◽  
Factor X Activation ◽  
Rate Of Activation

Infusing factor VIIa (FVIIa) has been reported to control bleeding in hemophilic patients with factor VIII (FVIII) inhibitors. This is difficult to attribute to an enhanced FVIIa/tissue factor (TF) activation of factor X, since in vitro studies suggest that infusion of FVIIa should neither increase substantially the rate of formation of FVIIa/TF complexes during hemostasis (Proc Natl Acad Sci USA 85:6687, 1988) nor bypass the dampening of TF-dependent coagulation by the extrinsic pathway inhibitor (EPI) (Blood 73:359, 1989). Partial thromboplastin times have also been reported to shorten after infusion of FVIIa. The experiments reported herein establish that shortening of partial thromboplastin times after adding FVIIa to hemophilic plasma in vitro stems from an FVIIa-catalyzed activation of factor X independent of possible trace contamination of reagents with TF. Experiments in purified systems confirmed that FVIIa can slowly activate factor X in a reaction mixture containing Ca2+ and phospholipid but no source of TF. The rate of activation was sufficient to account for the shortening of partial thromboplastin times observed. EPI, which turned off continuing FVIIa/TF activation of factor X, was unable to prevent continuing FVIIa/phospholipid activation of factor X. Because circulating plasma contains only a trace, if any, free FVIIa, such a reaction could never occur physiologically. However, infusing FVIIa creates a nonphysiologic circumstance in which a continuing slow FVIIa/phospholipid catalyzed activation of factor X could conceivably proceed in vivo unimpeded by EPI. Such a mechanism of factor X activation might compensate for an impaired factor IXa/FVIIIa/phospholipid activation of factor X during hemostatis, and therefore control bleeding in a hemophilic patient.

scholarly journals Characterization of the inhibition of tissue factor in serum

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 150-155 ◽  
Author(s):  
GJ Jr Broze ◽  
JP Miletich
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Factor Vii ◽  
Factor X ◽  
Human Hepatoma Cells ◽  
Proteolytic Activation

Tissue factor (TF) is a lipoprotein cofactor that markedly enhances the proteolytic activation of factors IX and X by factor VIIa. The functional activity of TF is inhibited by serum in a time- and temperature-dependent fashion. The inhibitory effect is also dependent on the presence of calcium ions and can be reversed by calcium chelation (EDTA) and dilution, thus excluding direct proteolytic destruction of TF as the mechanism for inhibition. Using crude TF, serum immunodepleted of factor VII, and serum depleted of the vitamin K- dependent coagulation factors by BaSO4 absorption, it is shown that TF factor inhibition requires the presence of VII(a), X(a), and an additional moiety contained in barium-absorbed serum. When each of the other required components were at saturating concentrations, half- maximal inhibition of TF occurred in reaction mixtures containing 2% (vol/vol) of TF at a factor VII(a) concentration of 4 ng/mL (80 pmol/L), a factor X concentration of 50 ng/mL (850 pmol/L), and a concentration of barium-absorbed serum of 2.5% (vol/vol). Catalytically active factor Xa appeared to be required for the generation of optimal TF inhibition. The results are consistent with the conclusions of Hjort that barium-absorbed serum contains a moiety that inhibits the VIIa- Ca2+-TF complex. The role of factor X(a) in the generation of the inhibitory phenomenon remains to be elucidated. The inhibitor present in serum (plasma) may in part be produced by the liver in vivo since cultured human hepatoma cells (HepG2) secrete this inhibitory activity in vitro.

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.

scholarly journals A mutated factor X activatable by thrombin corrects bleedings in vivo in a rabbit model of antibody-induced hemophilia A

Haematologica ◽  
2019 ◽  
Vol 105 (9) ◽  
pp. 2335-2340
Author(s):  
Toufik Abache ◽  
Alexandre Fontayne ◽  
Dominique Grenier ◽  
Emilie Jacque ◽  
Alain Longue ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Factor Viia ◽  
Rabbit Model ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor X ◽  
Factor Viiia

Rendering coagulation factor X sensitive to thrombin was proposed as a strategy that can bypass the need for factor VIII. In this paper, this non-replacement strategy was evaluated in vitro and in vivo in its ability to correct factor VIII but also factor IX, X and XI deficiencies. A novel modified factor X, named Actiten, was generated and produced in the HEK293F cell line. The molecule possesses the required post-translational modifications, partially keeps its ability to be activated by RVV-X, factor VIIa/tissue factor, factor VIIIa/factor IXa and acquires the ability to be activated by thrombin. The potency of the molecule was evaluated in respective deficient plasmas or hemophilia A plasmas, for some with inhibitors. Actiten corrects dose dependently all the assayed deficient plasmas. It is able to normalize the thrombin generation at 20 μg/mL showing however an increased lagtime. It was then assayed in a rabbit antibody-induced model of hemophilia A where, in contrast to recombinant factor X wild-type, it normalized the bleeding time and the loss of hemoglobin. No sign of thrombogenicity was observed and the generation of activated factor X was controlled by the anticoagulation pathway in all performed coagulation assays. This data indicates that Actiten may be considered as a possible non replacement factor to treat hemophilia's with the advantage of being a zymogen correcting bleedings only when needed.

Inhibition of Tissue Factor (TF) Pathway Blunts Hepatic Tumor Development in a Rat Colorectal Cancer Model.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 920-920
Author(s):  
Philippe Zerbib ◽  
Alexandre Grimonprez ◽  
Delphine Corseaux ◽  
Lars Petersen ◽  
Brigitte Jude
Keyword(s):  
Colorectal Cancer ◽  
Cancer Cells ◽  
Tissue Factor ◽  
Early Stage ◽  
Tumor Development ◽  
Coagulation Factor ◽  
Tumor Cell Line ◽  
Hepatic Tumor ◽  
Control Group ◽  
Hepatic Tumors

Abstract Occurrence of hepatic metastasis (HM) is the leading cause of death in colorectal cancer, making their prevention a major challenge. Tissue factor (TF), the principal initiator of coagulation after binding to its ligand, coagulation factor FVIIa, is expressed by most colorectal cancer cells, and has been demonstrated to be associated to tumor invasion and metastasis. The aim of this study was to analyze the effects of inhibition of the TF pathway in a rat model of HM of colorectal cancer. Methods DHDK12 Pro B cells (20.106 cells), a tumor cell line established from chemically induced colon carcinoma in BDIX rats, were injected in the portal vein of syngenic rats. Inhibition of TF was achieved in 19 rats (treat ment group) through intraperitoneal injection of active site-inactivated factor VIIa (FFR-FVIIa, Novo Nordisk, Denmark) (10μg/g, once a day from day 3 to day 8 after HM induction). Additionally, 18 rats underwent cancer cell infusion according to the same procedure, without FFR-FVIIa treatment (control group). Rats were sacrificed at day 14. Results In the control group, infusion of cancer cells resulted in the development of macroscopical hepatic tumors in 17 out of 18 rats. In rats treated by FFR-FVIIa, no macroscopical or histological hepatic tumors were visible on the liver surface in 16 out of 19 rats (p=0.002, versus control group). Conclusion These results suggest that blockage of TF with proteolytically inactive FFR-VIIa inhibits hepatic tumor development at an early stage of tumoral development and TF is a target for adjuvant therapy in the prevention of HM.

Sign in / Sign up

Export Citation Format

Share Document