thromboplastin and Leukemia--Monocytic--Acute

Our previous study has demonstrated that the Toll-like receptor 4 (TLR4) signaling pathways contribute to the induction of tissue factor (TF) expression by anti-β(2)-glycoprotein I/β(2)-glycoprotein I (anti-β(2)GPI/β(2)GPI) in human acute monocytic leukemia cell line THP-1. In this study, we focused on the identification of the downstream targets of the TLR4 pathways. When THP-1 cells were treated with anti-β(2)GPI/β(2)GPI complex, enhanced TF expression was observed, along with induced phosphorylation of p38, ERK1/2 and JNK1/2 MAPKs. When the activity of MAPKs was blocked by their corresponding inhibitors (SB203580: p38; U0126: ERK; SP600125: JNK), the expression of TF was reduced significantly. Furthermore, the anti-β(2)GPI/β(2)GPI-induced phosphorylation of p38, ERK1/2 and JNK1/2 was inhibited significantly by TAK-242, a blocker of the signaling transduction mediated by the intracellular domain of TLR4; sc-204013, a specific inhibitor of IRAKs, was also able to partially inhibit the phosphorylation of the MAPKs. Our results demonstrated that MAPKs (p38, ERK1/2 and JNK1/2) were the crucial downstream targets of the anti-β(2)GPI/β(2)GPI-triggered TLR4 signaling pathways in THP-1 cells. This essential role of MAPKs may also promote better understanding of the pathogenesis of antiphospholipid syndrome (APS).

Topics: Antigen-Antibody Complex; Antiphospholipid Syndrome; beta 2-Glycoprotein I; Cell Line, Tumor; Enzyme Activation; Gene Expression Regulation, Leukemic; Humans; Interleukin-1 Receptor-Associated Kinases; Leukemia, Monocytic, Acute; MAP Kinase Signaling System; Sulfonamides; Thromboplastin; Toll-Like Receptor 4

To examine the role of Fibrinogen-like protein 2 (fgl2)/fibroleukin in tumor development. Fgl2 has been reported to play a vital role in the pathogenesis in MHV-3 (mouse hepatitis virus) induced fulminant and severe hepatitis, spontaneous abortion, allo- and xeno- graft rejection by mediating "immune coagulation".. Tumor tissues from 133 patients with six types of distinct cancers and the animal tumor tissues from human hepatocellular carcinoma (HCC) model on nude mice (established from high metastasis HCC cell line MHCC97LM6) were obtained.. Hfgl2 was detected in tumor tissues from 127 out of 133 patients as well as tumor tissues collected from human HCC nude mice. Hfgl2 was highly expressed both in cancer cells and interstitial inflammatory cells including macrophages, NK cells, and CD8(+) T lymphocytes and vascular endothelial cells. Hfgl2 mRNA was localized in cells that expressed hfgl2 protein. Fibrin (nogen) co-localization with hfgl2 expression was determined by dual immunohistochemical staining. In vitro, IL-2 and IFN-gamma increased hfgl2 mRNA by 10-100 folds and protein expression in both THP-1 and HUVEC cell lines. One-stage clotting assays demonstrated that THP-1 and HUVEC cells expressing hfgl2 had increased procoagulant activity following cytokines stimulation.. The hfg12 contributes to the hypercoagulability in cancer and may induce tumor angiogenesis and metastasis via cytokine induction.

Topics: Adult; Aged; Animals; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Disease Models, Animal; Endothelial Cells; Female; Fibrinogen; Humans; Interferon-gamma; Interleukin-2; Leukemia, Monocytic, Acute; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; RNA, Messenger; Thrombophilia; Thromboplastin

Verotoxin (VT)-producing Escherichia coli (E. coli) O157:H7 infections are frequently complicated by thrombotic angiopathy, hemolytic uremic syndrome (HUS) and neurological symptoms. The present data demonstrate that VT-1 (Shiga toxin) stimulation of macrophage-like THP-1 cells up-regulates the activity, antigen and mRNA levels of tissue factor (TF), a key cofactor of the coagulation-inflammation-thrombosis circuit. This up-regulation is accompanied by phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase), IkappaB kinase beta (IKKbeta) and extracellular signal-regulated kinase 2 (ERK2). Changes in TF mRNA levels were in parallel with the activation of NF-kappaB/Rel and Egr-1 activation, but not with AP-1. Inhibition of PI3-kinase attenuated VT-1-induced phosphorylation of IKKbeta and ERK2, and the up-regulation of TF mRNA levels. VT-1 stimulation rapidly activated c-Yes tyrosine kinase, a member of the Src family. Treatment of the cells with c-Yes antisense oligos attenuated the VT-1-induced phosphorylation of PI3-kinase, IKKbeta and ERK2, activations of NF-kappaB/Rel and Egr-1, and up-regulation of TF mRNA levels. These results suggest that VT-1-induced macrophage stimulation activates c-Yes, which then up-regulates TF expression through activation of the IKKbeta/proteasome/NF-kappaB/Rel and MEK/ERK2/Egr-1 pathways via activation of PI3-kinase. Induction of macrophage TF expression by VT-1 may play an important role in the acceleration of the coagulation-inflammation-thrombosis circuit during infections by VT-producing E. coli.

Topics: Cell Differentiation; Cell Line, Tumor; Humans; Leukemia, Monocytic, Acute; Macrophages; Proto-Oncogene Proteins c-yes; RNA, Messenger; Shiga Toxin 1; Signal Transduction; Thromboplastin; Transcription Factors; Transcription, Genetic; Up-Regulation

This study was aimed to investigate the influence of PPARalpha agonist on the expression of TF (tissue factor) in THP-1 cells. THP-1 cells were pretreated with different concentrations of PPARalpha agonist (fenofibrate) for definite time. Lipopolysaccharide (LPS)-induced TF mRNA and protein levels were detected by RT-PCR and Western blot respectively. The results showed that fenofibrate decreased tissue factor protein and mRNA expression in supernatants of LPS-stimulated human monocytes in a concentration-dependent manner (P < 0.05 - 0.01, n = 5). It is concluded that fenofibrate inhibit TF expression induced by LPS in THP-1 cells, which may be involved in the anti-atherosclerotic effects of PPARalpha agonist.

Topics: Depression, Chemical; Fenofibrate; Humans; Leukemia, Monocytic, Acute; Lipopolysaccharides; PPAR alpha; RNA, Messenger; Thromboplastin; Tumor Cells, Cultured

AML patients may suffer from a disseminated coagulopathy, which can aggravate a pre-existing bleeding tendency due to thrombocytopenia and platelet dysfunction. The cellular and molecular mechanisms underlying this coagulopathy, however, are not completely understood. Indeed, the broad and increasing therapeutic use of cytotoxic drugs and growth factors is likely to contribute to the complexity of hemostatic abnormalities encountered in this hematologic malignancy. The nature of coagulation activation in AML was therefore investigated in vitro using the human leukemic cell line, HL60. Tissue factor (TF) was almost entirely located on the cell surface and bound factor VIIa, but only 15-25% of this TF was primarily functionally active. Treatment with increasing concentrations of daunorubicin or cytosine-beta-D-arabinofuranoside, two cytotoxic drugs commonly used in AML therapy, induced apoptosis and secondary necrosis of HL60 cells and resulted in marked decryption of TF PCA independent of de novo protein synthesis. This PCA-modulating effect was concomitant with and functionally dependent on the exposure of phosphatidylserine on the outer membrane leaflet. Similar observations were made in analogous ex vivo studies on patient-derived myeloblasts. Incubation of HL60 cells with GM-CSF, a cytokine expressed in the bone marrow microenvironment and used as an adjunct to AML treatment, evoked a cellular response, which included both enhanced TF production and release of VEGF-A and uPA into the culture medium. We conclude that both decryption of pre-formed TF PCA by chemotherapeutic drugs and de novo induction of TF by cytokines such as GM-CSF can regulate the pro-coagulant phenotype of HL60 cells in vitro.

Topics: Antineoplastic Agents; Blood Cells; Blood Coagulation; Cell Death; Cells, Cultured; Cytarabine; Daunorubicin; Granulocyte Precursor Cells; Granulocyte-Macrophage Colony-Stimulating Factor; HL-60 Cells; Humans; Leukemia, Monocytic, Acute; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Acute; Thromboplastin

Tissue factor (TF) antigen and activity were measured in leukemic cell homogenates. In leukemic cell homogenate, especially that of acute promyelocytic leukemia (APL), both TF antigen and activity were significantly higher than these levels in the mononuclear cells obtained from healthy volunteers. Both TF antigen and activity were significantly higher in myelocytic leukemia than in lymphocytic leukemia cells. In leukemic cell homogenates, there was a close correlation between TF antigen and TF activity. The TF activity/TF antigen ratio was significantly higher in myelocytic leukemia than in lymphocytic leukemia cells. As the TF activity was not increased in lymphocytic leukemia cell homogenates to which were added phospholipids, the decrease in TF activity in lymphocytic leukemia might not be due to phospholipid in the leukemic cell membrane. Values for TF activity, TF antigen, and the TF activity/TF antigen ratio in leukemic cell homogenate from patients with disseminated intravascular coagulation (DIC) were significantly higher than those in patients without DIC. Therefore, the measurement of TF antigen and activity in leukemic cells could be useful for the prediction of DIC.

Topics: Antigens, Neoplasm; Disseminated Intravascular Coagulation; Humans; Leukemia; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Monocytic, Acute; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myelomonocytic, Acute; Leukemia, Promyelocytic, Acute; Leukemia, T-Cell; Leukocytes, Mononuclear; Neoplasm Proteins; Thromboplastin

We have recently found that retinoic acids (RAs) evoke an anticoagulant effect by upregulating thrombomodulin (TM) and downregulating expression of tissue factor (TF) in acute promyelocytic leukemia (APL) and monoblastic leukemia cells. Two classes of nuclear RA receptors, termed retinoic acid receptors (RARs) and retinoid X receptors, have already been identified. Each receptor class consists of three subtypes. We have used several synthetic retinoids to find which receptor subtypes are involved in the regulation of TM and TF expression in APL cells NB4, monoblastic leukemia cells U937, and human umbilical vein endothelial cells (HUVECs). Am80, which does not have a binding affinity to RARgamma; Ch55, which does not bind to cytoplasmic retinoic acid-binding protein (CRABP); and a specific RARalpha agonist, Ro40-6055, have been shown to upregulate TM and downregulate TF in NB4 and U937 cells similar to all-trans RA (ATRA). A specific RARalpha antagonist, Ro41-5253, efficiently suppressed the upregulation of TM by ATRA and Am80 in NB4 cells, U937 cells and HUVECs. In contrast, only when both RARalpha and RARbeta antagonists were preincubated, downregulation of TF by the retinoids was suppressed in NB4 cells. Furthermore, 1,25(OH)2D3 has been shown to have anticoagulant effects on several monocytic leukemia cells and monocytes similar to RAs. These results indicate the mechanically distinct transactivation and transrepression functions of RARs, the major role of RARalpha in TM upregulation by retinoids in leukemic cells and HUVECs, and the cooperative role of RARalpha and RARbeta in TF downregulation by retinoids. It is also implied that synthetic retinoids and vitamin D derivatives will provide very useful means to control distinct targets--TM and TF genes--at the level of transcription. Synthetic retinoids and vitamin D derivatives may develop as new types of antithrombotic and antiatherosclerotic agents which change the character of cells as well as malignant cell differentiation inducers.

Topics: Anticoagulants; Calcitriol; Cell Line; Down-Regulation; Humans; Leukemia, Monocytic, Acute; Leukemia, Promyelocytic, Acute; Receptors, Retinoic Acid; Retinoid X Receptors; Retinoids; Signal Transduction; Thrombomodulin; Thromboplastin; Transcription Factors; Up-Regulation

We have recently found that all-trans retinoic acid (ATRA) upregulates thrombomodulin (TM) and downregulates tissue factor (TF) expression in acute myelogenous leukemia (AML) M3 cells (NB4) and acute monoblastic leukemia cells (U937) (Koyama et al, Blood 84:3001, 1994). We have further investigated the effects of ATRA on leukemic cells freshly isolated from patients at diagnosis. Increase of TM antigen was documented in all AML cells: M0 (n = 1), M2 (n = 5), M3 (n = 3), M4 (n = 3), M5 (n = 3), and M6 (n = 1). Decrease of TF antigen was observed in 4 M2, 1 M4, and all M3 and M5 patients. However, no TM and TF antigens were detected in all chronic lymphocytic leukemia cells (n = 3) with or without ATRA treatment. Changes of TM and TF antigen levels were associated with those of TM and TF cofactor levels on the cell surface. A stereoisomer of RA, 9-cis RA, is a high-affinity ligand for the RA receptors (RARs) and the retinoid X receptors, although ATRA and another isomer, 13-cis RA, solely bind to RARs. We have also studied the effects of 9-cis RA and 13-cis RA on the expressions of TM and TF in NB4 and U937 cells. A relatively wide range of 9-cis RA concentrations (0.01 to 1 mumol/L) compared with ATRA was optimal for prolongation of normal plasma-based recalcification time (reduction of cell surface TF activity), decrease of TF antigen, and increase of TM antigen on the surface and in the lysates of NB4 and U937 cells. Western blot analysis under nonreducing conditions showed that both ATRA and 9-cis RA markedly induced the prominent band at 75 kD of TM and reduced the band at 45 kD of TF. Northern blot analysis has shown similar changes of mRNA levels, which indicates that RAs regulate TM and TF expression in leukemic cells at transcriptional levels. Anticoagulant effects of ATRA, ie, upregulation of TM expression and downregulation of TF expression, are applied not only to established cell lines of specific subtypes (M3 and M5) but also to more universal AML (most cases of M3 and M5 and a part of the other types of AML) cells freshly isolated from patients. 9-cis RA may be more effective than ATRA as an inducer of differentiation of AML M3 cells and as an anticoagulant agent for patients with certain types of AML as well.

Topics: Anticoagulants; Base Sequence; Cell Separation; Cysteine Endopeptidases; Flow Cytometry; Gene Expression Regulation, Leukemic; Humans; Isotretinoin; Leukemia; Leukemia, Monocytic, Acute; Leukemia, Promyelocytic, Acute; Lymphoma, Large B-Cell, Diffuse; Molecular Sequence Data; Neoplasm Proteins; Neoplastic Stem Cells; Receptors, Retinoic Acid; Thrombomodulin; Thromboplastin; Tretinoin; Tumor Cells, Cultured

Stimulation of monocytic cells by inflammatory agents such as bacterial lipopolysaccharide or tumour necrosis factor-alpha leads to the rapid and transient expression of tissue factor, the major cellular initiator of the extrinsic coagulation cascade in both haemostasis and tissue inflammation. In this study we investigated whether the synthetic anti-inflammatory glucocorticoid, dexamethasone, would inhibit agonist induction of tissue factor expression in both monocytes and endothelial cells. Surprisingly, dexamethasone significantly enhanced the induction of tissue factor expression by peripheral blood mononuclear cells and an established monocytic cell line, THP-1, in response to lipopolysaccharide or tumour necrosis factor-alpha. However, unlike monocytic cells, dexamethasone did not enhance agonist induction of tissue factor in endothelial cells. Synergistic enhancement of tissue factor expression by dexamethasone was also reflected in tissue factor mRNA levels in THP-1 cells, but was not the result of improved TF mRNA stability. Synergism between bacterial lipopolysaccharide and glucocorticoid in the induction of monocyte effector function is extremely unusual and may help to explain the variable outcome of glucocorticoid treatment of septic shock.

Topics: Cells, Cultured; Dexamethasone; Drug Synergism; Endothelium, Vascular; Gene Expression Regulation; Gene Expression Regulation, Leukemic; Humans; Interleukins; Leukemia, Monocytic, Acute; Leukocytes, Mononuclear; Lipopolysaccharides; Monocytes; Neoplasm Proteins; RNA, Messenger; RNA, Neoplasm; Thromboplastin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Tissue factor-like activity was measured in the plasma of 30 patients with disseminated intravascular coagulation(DIC) and 22 patients without DIC using a chromogenic substrate. Twenty-three of the 30 patients with DIC (77%) exhibited tissue factor-like activity levels above normal range (greater than 3.0 U/L), and in eleven of these patients, the levels were more than 10 U/L. Of the 22 patients without DIC, seven patients had elevated levels (3-10 U/L), and had a possibility to be developing DIC. So, we considered them to be in a pre-DIC state. No correlation was found between tissue factor-like activity and alpha 2 plasmin inhibitor-plasmin complex or FDP-D dimer. In a patient with acute monocytic leukemia, the elevated tissue factor-like activity (84.4 U/L) rapidly decreased after the initiation of chemotherapy, whereas in a patient with pancreatic cancer, the level remained elevated (67.4-79.2 U/L). These results suggested that the plasma tissue factor-like activity is differ from the other parameters reflecting the process of DIC and is a useful indicator of the presence of an initiating factor of blood coagulation in some selected patients with DIC or pre-DIC.

Topics: Adult; alpha-2-Antiplasmin; Antifibrinolytic Agents; Cerebral Infarction; Disseminated Intravascular Coagulation; Female; Fibrin Fibrinogen Degradation Products; Fibrinolysin; Humans; Leukemia, Monocytic, Acute; Male; Middle Aged; Pancreatic Neoplasms; Thromboplastin

Cell lysates of the human monoblastic leukemia cell line, THP-1, have procoagulant activity (PCA) that is Ca++-dependent and not demonstrable in either Factor VII-, or Factor X-deficient plasma. The PCA of THP-1 cells was enhanced by human recombinant tumor necrosis factor-alpha (TNF-alpha) up to five fold. There was a dose-dependent increase in PCA when THP-1 cells were cultured with concentrations of TNF-alpha, up to 10 U/ml. PCA of cell lysates or whole cell preparations was measured in comparison to a rabbit brain thromboplastin standard. The effect of TNF-alpha was enhanced by recombinant human interferon-gamma (IFN-gamma). Cycloheximide inhibited the induction of PCA by THP-1 cells, which shows that the protein synthesis is essential to mediate the effect of TNF-alpha. THP-1 cells and U937 cells bound 125I-labeled TNF specifically. The numbers of receptors per cell were found to be 1,890 and 1,550 for THP-1 and U937 cells, respectively. Other lymphoid and myeloblastic leukemia cell lines examined did not have TNF receptors, indicating that the effect of TNF-alpha is mediated by the receptors on the cell surface.

Topics: Blood Coagulation Factors; Humans; Leukemia, Monocytic, Acute; Receptors, Cell Surface; Receptors, Tumor Necrosis Factor; Thromboplastin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The induction of procoagulant activity (PCA) by human recombinant tumor necrosis factor (rTNF) was studied in human monoblastic leukemia cell line U937 and human peripheral blood monocytes. Using a one-step recalcificating clotting assay, PCA in cell lysates or whole cell preparations was measured by comparison to a rabbit brain thromboplastin standard. There was a dose- and time-dependent increase in PCA when U937 cells were cultured with rTNF. The effect of rTNF was not enhanced by recombinant human interferon-gamma (rIFN gamma). Cycloheximide inhibited the expression of PCA by U937 cells, showing that protein synthesis was necessary to mediate the effects of rTNF. Whole cell preparations demonstrated that greater than 80% of the PCA was expressed on the surface of the cells. The PCA functioned as a tissue factor-like substance, since it required coagulation factor VII and factor X. rTNF also increased PCA in human monocytes in a dose- and time-dependent manner. This effect was abrogated by boiling the rTNF for ten minutes, and was not inhibited by adding polymyxin-B to the cultures, making it unlikely that endotoxin accounted for the observed effects. These results suggest that TNF-induced expression of tissue factor by mononuclear phagocytes may modulate immunologic, inflammatory, and hemostatic processes.

Topics: Blood Coagulation Factors; Cell Line; Humans; Interferon-gamma; Leukemia, Monocytic, Acute; Monocytes; Recombinant Proteins; Thromboplastin; Tumor Necrosis Factor-alpha

Topics: Acute Disease; Animals; Aprotinin; Blood Coagulation; Chromatography, Gel; Disseminated Intravascular Coagulation; Fibrinolysis; Heparin; Humans; Leukemia, Lymphoid; Leukemia, Monocytic, Acute; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Leukocytes; Rabbits; Thrombin; Thromboplastin; Tranexamic Acid; Trypsin Inhibitors