thromboplastin and Leukemia--Myelomonocytic--Acute

Ionizing radiation (IR) is associated with thrombotic vascular occlusion predicting a poor clinical outcome. Our study examined whether IR induced tissue factor (TF) expression and procoagulability. We further investigated coordinated gene alterations associated with TF upregulation in the myelomonocytic leukemia THP-1 cells.. TF expression was determined by quantitative Reverse Transcriptase (TaqMan) PCR, TF ELISA and TF activity by a two stage chromogenic assay in the time course of days 1, 3, 7, 10, and 17 post IR. To detect IR-induced alterations in gene expression, Affymetrix HG U133 Plus 2.0 microarrays were used. RESULTS IR induced a significant increase in TF/GAPDH mRNA ratios and cellular TF protein on days 3 and 7 post IR (20 Gy [p>or=0.01] and 40 Gy [p or=0.001] vs. control respectively), suggesting IR immediately alters the cellular thrombogenicity. TF upregulation post IR was confirmed in PBMNCs. Gene expression profiling showed IR increased the expression of inflammatory and apoptosis-related pathways known to be involved in the regulation of TF expression.. TF upregulation together with inflammation and apoptosis may increase the thrombogenicity of tissues. The demonstrated upregulation of TF might play a pivotal role in radiation associated thrombosis.

Topics: Apoptosis; Blood Coagulation Factors; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Factor Xa; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Humans; Inflammation; Leukemia, Myelomonocytic, Acute; Neoplasm Proteins; NF-kappa B; Nitriles; Oligonucleotide Array Sequence Analysis; Particle Accelerators; Radiation, Ionizing; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Sulfones; Thrombophilia; Thromboplastin

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 studied tissue factor gene expression in the leukocytes of 22 patients with acute myeloblastic leukemia (AML). Total RNA from peripheral blood or bone marrow cells depleted of monocytes was analyzed by Northern blot analysis using a 32P-labeled tissue factor cDNA probe. Cells from 10 cases expressed tissue factor mRNA and positive cases were distributed among the myeloblastic, myelomonocytic, and monocytic subtypes of AML. Tissue factor transcripts were not detected in cells derived from normal bone marrow. The expression of this gene product on the surface of leukemic cells could contribute to the excessive thrombin generation that has been observed in some individuals with this disorder.

Topics: Blood Coagulation Factors; Bone Marrow; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid, Acute; Leukemia, Myelomonocytic, Acute; Leukocytes, Mononuclear; Neoplasm Proteins; RNA, Messenger; RNA, Neoplasm; Thromboplastin

The human myelomonocytic cell line RC2a expressed procoagulant activity following stimulation with human lymphokine (LK) prepared by stimulating peripheral blood mononuclear cells with either mitogens (Concanavalin A, phytohaemagglutinin or antigen (tuberculin). Induction was rapid, with optimal activity observed between 6 and 8 h, was decreased in cultures containing serum and was not inhibited by actinomycin D or cycloheximide. The LK activity was not inhibited by an anti-interferon gamma (IFN gamma) antibody. IFN gamma and phorbol myristate acetate (PMA) had no activity but acted in synergy to induce procoagulant expression; interferon alpha plus PMA were without effect. In contrast to the LK-induced response, procoagulant induced by IFN gamma/PMA was not detected for up to 8 h and steadily rose over 24-48 h culture and was dependent on new protein and RNA synthesis. Bacterial lipopolysaccharide, a potent inducer of thromboplastin on normal human monocytes, failed, either alone or in combination with LK, IFN gamma, PMA or IFN gamma plus PMA, to activate procoagulant expression on RC2a cells. Both LK and IFN gamma/PMA-induced procoagulant had properties of thromboplastin expressed both intracellularly and on intact, viable cells. This study shows that RC2a cells are responsive to factors produced as the result of an activated cell-mediated immune response which may therefore contribute to the coagulopathies common to this form of malignancy.

Topics: Blood Coagulation Factors; Cell Line; Humans; Interferon-gamma; Leukemia, Myelomonocytic, Acute; Lymphokines; Monocytes; Recombinant Proteins; Tetradecanoylphorbol Acetate; Thromboplastin