thromboplastin and Leukemia--Promyelocytic--Acute

Coagulopathy is a unique component of the pathology of acute promyelocytic leukaemia (APL). Though many causative factors have been elucidated, therapies to rectify the coagulopathy are far from being realised. Thrombotic and bleeding complications remain the major causes of early deaths. In this chapter, the known causes of abnormalities in haemostatic function, namely the coagulopathy and changes in the fibrinolytic system, will be reviewed. Major risk factors for these complications are identified. Current available measures for correction of the coagulopathy and their effectiveness are critically examined. Unless the coagulopathy can be effectively controlled, bleeding complications will remain an obstacle to achieving a cure for this disease. The issues that need to be addressed in next phase of investigations are also discussed.

Topics: Annexin A2; Anticoagulants; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Blood Coagulation Disorders; Blood Coagulation Tests; Carboxypeptidase B2; Disseminated Intravascular Coagulation; Fibrinolysis; Forecasting; Granulocyte Precursor Cells; Hemorrhagic Disorders; Humans; Leukemia, Promyelocytic, Acute; Oxides; Recombinant Proteins; Risk Factors; S100 Proteins; Thrombomodulin; Thrombophilia; Thromboplastin; Tretinoin; Urokinase-Type Plasminogen Activator

Acute promyelocytic leukemia (APL) has evolved from being a deadly to a highly curable disease, due to targeted molecular therapy with all-trans retinoic acid (ATRA). As a result, the incidence of early hemorrhagic deaths for which APL is notorious has reduced to 5-10% as reported in clinical trials. These results are not replicated outside of clinical trials as is evident from recent population-based registries. High incidence of early hemorrhagic deaths remains the greatest contributor to treatment failure in this otherwise curable leukemia. Additionally, thrombosis is now being increasingly recognized in APL patients and may be associated with ATRA usage.

Topics: Antifibrinolytic Agents; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Blood Coagulation Tests; Clinical Trials as Topic; Combined Modality Therapy; Cysteine Endopeptidases; Disseminated Intravascular Coagulation; Factor VIIa; Factor VIII; Fibrinogen; Fibrinolysis; Hemorrhage; Heparin; Humans; Leukemia, Promyelocytic, Acute; Multicenter Studies as Topic; Neoplasm Proteins; Oxides; Plasma; Platelet Count; Platelet Transfusion; Recombinant Proteins; Thromboplastin; Thrombosis; Treatment Failure; Tretinoin

Topics: Antineoplastic Agents; Blood Coagulation; Cysteine Endopeptidases; Cytokines; Disseminated Intravascular Coagulation; Fibrinolytic Agents; Humans; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Neoplasms; Thromboplastin; Tretinoin

Thrombotic complications in patients with hematologic malignancies are as frequent as in those with solid tumors and significantly affect morbidity and mortality. In acute leukemia, thrombosis and bleeding manifestations may occur concomitantly as a part of the same thrombo-hemorrhagic syndrome. In patients with Philadelphia chromosome-negative chronic myeloproliferative disorders (i.e., essential thrombocythemia [ET] and polycythemia vera [PV]), a thrombosis rate as high as 40% has been recorded. A hypercoagulable state is present in virtually all of these patients, even without clinical manifestations. In this review, we focus on the pathogenic mechanisms underlying the hypercoagulable state of these two hematologic malignancies. Although the pathogenesis of hypercoagulability is complex, a central role is played by the fundamental molecular changes of both the leukemic cells and of the progeny arising from the hematopoietic progenitor cells that have undergone clonal rearrangement. These cells overexpress procoagulant factors, as well as adhesion molecules and cytokines capable of inducing procoagulant changes in the vascular wall and stimulating increased cellular interactions. Recent molecular studies in experimental models of human tumors have demonstrated for the first time that oncogene- and repressor gene-mediated neoplastic transformation induces activation of blood coagulation. Similarly, in cells from patients with acute promyelocytic leukemia, the T15-17 translocation induces hyperexpression of tissue factor (TF) and renders the patient hypercoagulable. Furthermore, in blood cells from patients with PV or ET, the presence of the JAK2V617F mutation translates into activation of hemostasis, with increased expression of platelet-associated TF microparticles and the formation of increased platelet/neutrophil aggregates. Understanding the pathophysiology of hypercoagulability is critical to the design of appropriate measures for intervention in these hematologic disorders to prevent thromboembolic complications.

Topics: Hematologic Neoplasms; Hemostasis; Humans; Leukemia, Promyelocytic, Acute; Myeloproliferative Disorders; Neovascularization, Pathologic; Polycythemia Vera; Thrombocythemia, Essential; Thrombophilia; Thromboplastin; Up-Regulation

Thrombosis is the most frequent complication and the second cause of death in patients with overt malignant diseases. Increasing evidence suggests that thrombotic episodes may also precede the diagnosis of cancer by months or years thus representing a potential marker for occult malignancy. Recently, emphasis has been given to the potential risk of cancer therapy (both surgery and chemotherapy) in enhancing the risk for thromboembolic disease. Post-operative deep-vein thrombosis is indeed more frequent in patients operated for malignant diseases than for other disorders. On the other hand, both chemotherapy and hormone therapy are associated with an increased thrombotic risk, which can be prevented by low-dose oral anticoagulation. Possible contributory causes for thromboembolic disease in cancer include the capacity of tumor cells and their products to interact with platelets, clotting and fibrinolytic systems, as well as their interactions with endothelial cells and tumor-associated macrophages. In particular, procoagulant activities of tumor cells have been extensively studied; one of these, cancer procoagulant, could represent a novel marker of malignancy in both solid tumors and acute promyelocytic leukemia (APL). In solid tumors, CP, a vitamin K dependent enzyme could represent the selective target of the antimetastatic effects of warfarin treatment. In APL, CP may contribute to trigger the well known intravascular coagulation syndrome accompanying the early manifestations of the disease and is depressed by all-trans-retinoic acid, an agent capable to determine complete remission with a rapid amelioration of the bleeding syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antineoplastic Agents; Cysteine Endopeptidases; Disease Models, Animal; Disease Progression; Hormones; Humans; Leukemia, Promyelocytic, Acute; Mice; Mice, Transgenic; Neoplasm Proteins; Neoplasms; Neoplasms, Experimental; Neoplasms, Unknown Primary; Plasminogen Activator Inhibitor 1; Thrombophlebitis; Thromboplastin

Acute promyelocytic leukemia (APL) cells constitutively express a large amount of tissue factor (TF) antigen, most of which is present in the cytoplasm. Coagulopathy may persist after induction therapy. We evaluated the overall role of circulating microparticles (MPs) in coagulation activation in APL-associated coagulopathy before and during induction therapy. Eleven adult patients with ≥ World Health Organization's (WHO) grade 2 bleeding events and 11 sex- and age-matched healthy controls were selected. All patients received arsenic trioxide alone as induction therapy. MP-associated TF (MP-TF) activity and MP procoagulant activity (MP-PCA) and 12 coagulation- and anticoagulation-associated indexes were measured before, during, and after induction therapy. Correlation between MP-associated indexes and the other 12 indexes was analyzed in patients. The MP-TF activity was negligible in controls, whereas it markedly increased in patients, dropped rapidly after treatment, and returned to normal at the end of induction therapy. The MP-PCA was similar between patients and controls. The correlation analysis revealed that TF-bearing MPs in patients mainly originated from APL cells. Partially differentiated APL cells could also release TF-bearing MPs, and the higher the degree of APL cell differentiation, the lower the ability of APL cells to release TF-bearing MPs. MP-TF was the main source of active TF in plasma and an important contributor for the coagulation activation in APL-associated coagulopathy. It was MPs released by APL cells/partially differentiated APL cells that served as the vehicle to transfer the large amount of TF to plasma to activate coagulation.

Topics: Adult; Antineoplastic Agents; Arsenic Trioxide; Blood Coagulation; Cell-Derived Microparticles; Female; Hemorrhage; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Prospective Studies; Thromboplastin

The aberrant expression of tissue factor (TF) in acute promyelocytic leukemia (APL) cells has been implicated in the pathogenesis of the APL coagulopathy. In this study, we found that in APL patients receiving ATRA or As2O3 treatment, the improvement in hypercoagulobility and hyperfibrinolysis paralleled the correction of plasma fibrinogen level and amelioration of bleeding symptoms. Notably, clinical improvement was also correlated to ATRA/As2O3-induced rapid decrease of membrane procoagulant activity (PCA) and TF contents of APL blasts. Consistent with the in vivo findings, the membrane PCA, TF antigen and its mRNA level within NB4 cells were rapidly down-regulated by 1 microM ATRA or As2O3, while 0.2 microg/ml DNR increased these TF parameters prior to its effect upon apoptosis induction. The down-regulation of TF mRNA by ATRA was partially de novo protein synthesis-dependent and at least partially attributed to a mechanism of destabilizing TF mRNA. On the other hand, in addition to its modulation on mRNA, As2O3 could also induce an accelerated TF protein turnover. These distinct effects were corroborated with the properties of these agents in causing the degradation of PML-RARalpha protein. All three therapeutic agents, however, enhanced the potential of NB4 cells to stimulate the expression of TF and PCA in endothelium. Taken together, our data suggest that the rapid and distinct regulation of TF on APL cells by these therapeutic agents might at least partially contribute to their effects on APL coagulopathy.

Topics: Adolescent; Adult; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Blood Coagulation; Daunorubicin; Drug Screening Assays, Antitumor; Endothelium, Vascular; Female; Fibrinolysis; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Oxides; Thromboplastin; Tretinoin

Hemostatic molecular markers were serially monitored in a prospective fashion during remission induction therapy with all-trans retinoic acid (ATRA) in sixteen patients with acute promyelocytic leukemia (APL). One patient with leukocytosis before treatment and three patients who later developed hyperleukocytosis also received chemotherapy with behenoyl Ara-C and daunorubicin. Plasma levels of E-fragment of fibrin and fibrinogen degradation product (FDP-E), FDP-D dimer (D-D), thrombin-antithrombin complex (TAT), and plasmin-alpha 2 plasmin inhibitor complex (PIC) were markedly elevated in all but one patient before treatment, and these parameters decreased to normal or near normal ranges in most patients within the first 7 days of treatment. Interestingly, we have found that these parameters were again elevated during the later course of ATRA therapy (after day +7) in eleven patients for various reasons including cytotoxic chemotherapy (3 cases), fever (5 cases; 2 cases with apparent infection, 3 cases without known etiology), Caesarean section (1 case), and no apparent etiology (2 cases). Three patients showed bleeding complications during re-elevation of molecular markers, but none developed thrombosis. Plasma elastase-alpha 1 proteinase inhibitor complex (E-alpha 1 PI) was markedly elevated in all patients at diagnosis and did not decrease significantly during ATRA therapy. Plasma tissue factor antigen was mildly elevated in one out of four patients studied, and thrombomodulin was elevated in two out of ten patients tested. These results confirmed the rapid normalization of coagulopathy during the early phase of remission induction therapy with ATRA but suggest that re-elevation of molecular markers occurs frequently during the later course of ATRA therapy.

Topics: Adolescent; Adult; Aged; Antineoplastic Agents; Biomarkers; Blood Coagulation; Child; Female; Fibrinolysis; Follow-Up Studies; Hemostasis; Humans; Leukemia, Promyelocytic, Acute; Leukocytes; Male; Middle Aged; Pancreatic Elastase; Prospective Studies; Remission Induction; Thrombomodulin; Thromboplastin; Tretinoin

Acute promyelocytic leukemia (APL) is associated with a high risk of bleeding and thrombosis. APL patients have an activated coagulation system, hyperfibrinolysis, and thrombocytopenia. APL cells express tissue factor (TF), a receptor and cofactor for factor VII/VIIa. This study had 2 goals. Firstly, we measured biomarkers of coagulation and fibrinolysis activation as well as platelet counts and bleeding in both mouse xenograft and allograft models of APL. Secondly, we determined the effect of inhibiting TF on the activation of coagulation in these models. We observed increased levels of plasma thrombin-antithrombin complexes (TAT), D-dimer, and plasmin-antiplasmin complexes, reduced platelet counts, and increased tail bleeding in both mouse models of APL. Fibrinogen levels decreased in the xenograft model but not in the allograft model. In contrast, the red blood cell count decreased in the allograft model but not in the xenograft model. Inhibition of APL-derived human TF with an anti-human TF monoclonal antibody reduced the level of TAT, increased platelet count, and normalized tail bleeding in a xenograft model. Inhibition of all sources of TF (APL cells and host cells) in the allograft model with a rat anti-mouse TF monoclonal antibody decreased the levels of TAT but did not affect the platelet count. Our study demonstrates that TF plays a central role in the activation of coagulation in both the xenograft and allograft mouse models of APL. These APL mouse models can be used to investigate the mechanisms of coagulopathy and thrombocytopenia in APL.

Topics: Animals; Antibodies, Monoclonal; Blood Coagulation; Blood Coagulation Disorders; Hemorrhage; Humans; Leukemia, Promyelocytic, Acute; Rats; Thrombocytopenia; Thromboplastin

The characteristic hemorrhages of acute promyelocytic leukemia (APL) are caused in part by the high expression of tissue factor (TF) on leukemic cells, which also produce TNF and IL-1β, proinflammatory cytokines known to increase TF in various cell types. Exposure of NB4 cells, an APL cell line, to all-trans retinoic acid (ATRA) or arsenic trioxide (ATO) rapidly and strongly reduced TF mRNA. Both drugs also reduced TNF mRNA, but later, and moreover increased IL-1β mRNA. The effect on procoagulant activity of cells and microparticles, as measured with calibrated automated thrombography, was delayed and only partial at 24 h. TNF and IL-1β inhibition reduced TF mRNA and activity only partially. Inhibition of the inflammatory signaling intermediate p38 reduced TF mRNA by one third but increased TNF and IL-1β mRNA. NF-κB inhibition reduced, within 1 h, TF and TNF mRNA but did not change IL-1β mRNA, and rapidly and markedly reduced cell survival, with procoagulant properties still being present. In conclusion, although we provide evidence that TNF, IL-1β, and their signaling intermediates have a regulatory function on TF expression by NB4 APL cells, the effect of ATRA and ATO on TF can only partially be accounted for by their impact on these cytokines.

Topics: Antineoplastic Agents; Arsenic Trioxide; Arsenicals; CD11c Antigen; Cell Line, Tumor; Enzyme Inhibitors; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Imidazoles; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; Leukemia, Promyelocytic, Acute; NF-kappa B; Oxides; p38 Mitogen-Activated Protein Kinases; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thromboplastin; Time Factors; Tretinoin; Tumor Necrosis Factor-alpha; U937 Cells

To evaluate the role of microparticle (MP) derived from acute promyelocytic leukemia (APL) cells and tissue factor (TF) carried by the MP in hypercoagulable state, and the effect of treatment with cytotoxic chemotherapy/differentiating agents on procoagulant activity (PCA) of these MP.. Bone marrow mononuclear cells (BMMNC) were extracted from 5 APL patients and 5 sex- and age- matched patients with iron deficiency anemia as controls. The cells were cultured in vitro for 48 h, then MP-rich culture medium and MP-free culture medium were harvested and MP was further obtained from certain volume of MP-rich culture medium. Subsequently, TF expression on MP was measured by ELISA. PCA of MP-rich culture medium or MP-free culture medium was assessed with thrombin generation assay. The role of TF on MP-related PCA was evaluated using anti-human TF antibody. In addition, APL cells were treated with all-trans retinoic acid (ATRA), arsenic trioxide (ATO) or daunorubicin (DNR) for 48 h, then MP-rich culture medium were harvested and the PCA was determined.. No TF expression was found in the MP released from bone marrow MNC in control group, whereas the obvious TF expression was found in the MP originated from BMMNC of APL. MP from both APL and control BM-MNC had obvious PCA. However, compared with the MP derived from control MNC, the MP from APL BM-MNC induced significantly higher PCA. TF played a crucial role in the PCA of APL BM-MNC derived MP, while played no role in that of MP from control MNCs. DNR-treating APL BM-MNC resulted in an increase in the PCA of MP, whereas ATO or ATRA exposure lead to exactly the opposite results.. MP derived from APL BM-MNC posseses obvious PCA. TF plays a crucial role in the MP-related PCA. The PCA of MP increases after treating APL BM-MNC with chemotherapy agent DNR and decreases following exposure of APL BM-MNC to differentiating agents ATRA or ATO.

Topics: Arsenicals; Blood Coagulation Disorders; Cell-Derived Microparticles; Humans; Leukemia, Promyelocytic, Acute; Oxides; Thromboplastin; Tretinoin

Oncogenic transformation is believed to impact the vascular phenotype and microenvironment in cancer, at least in part, through mechanisms involving extracellular vesicles (EVs). We explored these questions in the context of acute promyelocytic leukemia cells (NB4) expressing oncogenic fusion protein, PML-RARa and exquisitely sensitive to its clinically used antagonist, the all-trans retinoic acid (ATRA). We report that NB4 cells produce considerable numbers of EVs, which are readily taken up by cultured endothelial cells triggering their increased survival. NB4 EVs contain PML-RARa transcript, but no detectable protein, which is also absent in endothelial cells upon the vesicle uptake, thereby precluding an active intercellular trafficking of this oncogene in this setting. ATRA treatment changes the emission profile of NB4-related EVs resulting in preponderance of smaller vesicles, an effect that occurs in parallel with the onset of cellular differentiation. ATRA also increases IL-8 mRNA and protein content in NB4 cells and their EVs, while decreasing the levels of VEGF and tissue factor (TF). Endothelial cell uptake of NB4-derived EVs renders these cells more TF-positive and procoagulant, and this effect is diminished by pre-treatment of EV donor cells with ATRA. Profiling angiogenesis-related transcripts in intact and ATRA-treated APL cells and their EVs reveals multiple differences attributable to cellular responses and EV molecular packaging. These observations point to the potential significance of changes in the angiogenic signature and activity associated with EVs released from tumor cells subjected to targeted therapy.

Topics: Blood Coagulation; Cell Line, Tumor; Extracellular Vesicles; Gene Expression Regulation, Leukemic; Human Umbilical Vein Endothelial Cells; Humans; Intercellular Signaling Peptides and Proteins; Leukemia, Promyelocytic, Acute; Neovascularization, Pathologic; Oncogene Proteins, Fusion; RNA, Messenger; Thromboplastin; Tretinoin

This test cultivated three groups of acute promyelocytic leukemia (APL) and NB4 cells in liquid in vitro, processed them with arsenic trioxide (ATO), daunorubicin (DNR), ATO+DNR respectively, and then set up blank control group. Apoptosis of cells in each group was observed using flow cytometry, procoagulant activity of APL and NB4 cells in each group was detected with recalcification time, and expressions of tissue factor (TF), thrombomodulin and annexin II of NB4 cells in each group were measured using ELISA method. The results showed that the apoptosis rate increased 4-8 times compared with blank control group after processing APL and NB4 cells with ATO and DNR; procoagulant activity decreased obviously; and expression of TF and annexin II of NB4 cells reduced significantly (P<0.05). We concluded that combination of ATO and DNR could promote APL and NB4 cell apoptosis effectively without aggravating blood coagulation disorders, which might improve coagulation function of APL by inhibiting coagulation and hyperfibrinolysis through reducing expression of TF and annexin II. This drug combination may be a safe and effective method in the treatment of APL of primary high white blood cells type.

Topics: Adult; Annexin A2; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Arsenic Trioxide; Arsenicals; Blood Coagulation; Cell Line, Tumor; Daunorubicin; Female; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Oxides; Thrombomodulin; Thromboplastin; Time Factors; Tumor Cells, Cultured; Young Adult

Patients with hematological malignancies have a 28-fold increased risk of venous thromboembolism (VTE). Among patients with acute myelogenous leukemia (AML), the 2-year cumulative incidence of VTE is 5.2%. Several studies suggest that microvesicles (MVs) harboring TF may play a role in VTE and disseminated intravascular coagulation (DIC) in acute promyelocytic leukemia (APL). The aim of this study was to assess the capacity of untreated (APL) cells to shed procoagulant MVs. APL cells (NB4 and HL-60 cell lines) and MVs were separated by filtration (0.1-0.22-0.45-0.65 μm). The procoagulant activity (PCA) was assessed by thrombin generation assay (TGA). Alternatively, MVs were incubated with anti-Tissue Factor (TF) antibodies, with annexin V to assess the contribution of TF and phospholipids (PL) to the PCA, respectively. NB4 cells had a high PCA mainly triggered by MVs of size under 0.45 μm. The PCA of MVs was related to the expression of active TF and PL. HL-60 cells had a weaker PCA since TF is mostly present in its inactive form. Moreover, HL-60 do not produce MVs<0.65 μm associated with PCA. MVs could have a predicting value for VTE and DIC in patients with acute promyelocytic leukemia and could inform physicians about the optimal use of a thromboprophylaxis.

Topics: Antibiotics, Antineoplastic; Cell Differentiation; Cell Growth Processes; Cell Line, Tumor; Cell-Derived Microparticles; Daunorubicin; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Microscopy, Electron, Transmission; Thrombophilia; Thromboplastin; Thrombosis; Tumor Cells, Cultured; Venous Thromboembolism

The coagulopathy of acute promyelocytic leukemia (APL) is mainly related to procoagulant substances and fibrinolytic activators of APL blasts, but the fate of these leukemic cells is unknown. The aim of this study was to investigate the removal of APL blasts by macrophages and endothelial cells in vitro and consequent procoagulant and fibrinolytic activity of APL cells. We found that human umbilical vein endothelial cells as well as THP-1 and monocyte-derived macrophages bound, engulfed, and subsequently degraded immortalized APL cell line NB4 and primary APL cells. Lactadherin promoted phagocytosis of APL cells in a time-dependent fashion. Furthermore, factor Xa and prothrombinase activity of phosphatidylserine-exposed target APL cells was time-dependently decreased after incubation with phagocytes (THP-1-derived macrophages or HUVECs). Thrombin production on target APL cells was reduced by 40%-45% after 2 hours of coincubation with phagocytes and 80% by a combination of lactadherin and phagocytes. Moreover, plasmin generation of target APL cells was inhibited 30% by 2 hours of phagocytosis and ∼ 50% by lactadherin-mediated engulfment. These results suggest that engulfment by macrophages and endothelial cells reduce procoagulant and fibrinolytic activity of APL blasts. Lactadherin and phagocytosis could cooperatively ameliorate the clotting disorders in APL.

Topics: Adult; Antigens, Surface; Blood Coagulation; Cell Line; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Factor Xa; Female; Fibrinolysin; Human Umbilical Vein Endothelial Cells; Humans; Leukemia, Promyelocytic, Acute; Macrophages; Male; Microscopy, Confocal; Microscopy, Electron; Middle Aged; Milk Proteins; Phagocytosis; Phosphatidylserines; Thrombin; Thromboplastin; Young Adult

To explore the effect of arsenic trioxide (ATO) and daunorubicin (DNR) on phosphatidylserine (PS) exposure and related procoagulant activity of acute promyelocytic leukemia (APL) cells.. Mononuclear cell and neutrophil isolated from whole blood of 12 healthy volunteers were used as control group while APL cells obtained from 12 newly diagnosed APL patients at First Affiliated Hospital, Harbin Medical University from March 2007 to February 2009 were used as experimental group. APL cells were treated with 1 µmol/L ATO and 1 µmol/L DNR for 24 h. PS exposure of APL cells were measured by flow cytometry and confocal microscopy. And the related procoagulant activity was detected by the assays of coagulation time and coagulation factor formation. Lactadherin was used as a probe for PS exposure and anticoagulant on the cells of 12 APL patients.. ATO induced a decrease of PS exposure on APL cells by flow cytometry and no staining with lactadherin was observed under confocal microscopy. However, DNR induced the significantly elevated PS exposure and staining green with a rim pattern on membrane of APL cells was obtained. Coagulation time was (180 ± 25) s and (220 ± 41) s before and after treatment with ATO, respectively (P < 0.05). The formation of coagulation factors decreased after treatment with ATO (P < 0.05). While coagulation time was (180 ± 25) s and (80 ± 20) s before and after treatment with DNR, respectively (P < 0.05). The formation of coagulation factors increased after treatment with DNR (all P < 0.05). Lactadherin inhibited the procoagulant activities of DNR-treated APL cells (all P < 0.05).. Procoagulant activity is positively correlated with the exposed PS of APL cells. ATO and DNR inhibited and enhanced procoagulant activity with decreased and increased PS exposure, respectively.

Topics: Adult; Arsenic Trioxide; Arsenicals; Blood Coagulation; Blood Coagulation Tests; Case-Control Studies; Daunorubicin; Female; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Oxides; Phosphatidylserines; Thromboplastin; Tumor Cells, Cultured; Young Adult

Objective of this study was to detect the level of tissue factor-positive microparticles (TF(+)MP) by flow cytometry (FCM) and to analyze its clinical significance in the haemostatic disorder. TF(+) MP was detected by FCM using antibody CD142-PE in 25 cases of acute promyelocytic leukemia (APL), 20 cases of hemostatic diseases and 20 healthy adults as controls. The differences of TF(+) MP between various groups were determined. The results showed that the level of TF(+) MP in the patients with thrombotic complications was significantly higher than that in the healthy adults (P < 0.05). The TF(+) MP level was higher in the patient with APL than that in the healthy adults, especially in course before therapy (P < 0.01), but the difference was not statistically significant in the patient with APL after therapy and the healthy adults. Among these patient with APL, the level of TF(+) MP in the 18 patients who complicated with disseminated intravascular coagulation (DIC) was also higher than that in the healthy adults (P < 0.05), but the level of TF(+) MP in the other 7 patients who did not complicate with DIC was similar before and after treatment. It is concluded that the method of TF(+) MP detection by FCM is feasible and simple, it is useful for the diagnosis of thrombotic disorder, and helps evaluation for the prognosis of APL patient.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Blood Coagulation Disorders; Case-Control Studies; Female; Flow Cytometry; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Prognosis; Thromboplastin; Young Adult

The high expression of tissue factor (TF) is related to the coagulation disorder in acute leukemia. TF in blood circulation is mainly expressed in cells, microparticles (MP) and alternatively spliced human tissue factor (asHTF). To elucidate the role of TF in the coagulation disorder of acute myeloid leukemia (AML), RT-PCR was performed on 6 common AML cell lines NB4, HL-60, Kasumi-1, U937, K562 and THP-1. The results showed that only NB4 and U937 cells expressed baseline full-length TF and asHTF which were proved by sequencing. The flow cytometric detection, TF activity and TF antigen tests in NB4 and U937 cells revealed that the asHTF was expressed in trace amount and almost had no activity, while the TF antigen and activity in microparticles were significantly higher than that in asHTF. It is concluded that asHTF may play an unimportant role in the coagulation disorder of AML. Microparticle associated tissue factor (MP-TF) is the predominant source of TF activity released from AML cells.

Topics: Alternative Splicing; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Thromboplastin; Tumor Cells, Cultured; U937 Cells

Acute promyelocytic leukemia (APL) frequently causes disseminated intravascular coagulation that can worsen with cytotoxic chemotherapy but improve with the therapeutic differentiating agents, all trans retinoic acid (ATRA) and arsenic trioxide (As(2)O(3)). APL cells display tissue factor but the relationship of tissue factor and other procoagulant activity to phosphatidylserine (PS) exposure is largely unknown.. Lactadherin, a milk protein with stereospecific binding to phosphatidyl-L-serine, was used as a probe for PS exposure on an immortalized APL cell line (NB4) and on the cells of eight patients with APL. PS exposure was evaluated with flow cytometry, confocal microscopy, coagulation assays, and purified prothrombinase and factor (F) Xase assays.. Plasma procoagulant activity of NB4 and APL cells increased approximately 15-fold after exposure to etoposide or daunorubicin and decreased 80% after treatment with ATRA or As(2)O(3). Procoagulant activity corresponded to exposed PS on viable APL cells. PS exposure decreased after treatment with ATRA or As(2)O(3) and increased after treatment with daunorubicin or etoposide. Excess lactadherin inhibited 80-85% of intrinsic FXase, FVIIa-tissue factor and prothrombinase activities on both NB4 cells and APL cells. Confocal microscopy identified membrane patches that stained with lactadherin, but not annexin V, demonstrating focal, low-level PS exposure.. PS is exposed on viable APL cells and is necessary for approximately 80% of procoagulant activity.

Topics: Adolescent; Adult; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Blood Coagulation; Blood Coagulation Tests; Cell Differentiation; Cell Line, Tumor; Cell Membrane; Cell Survival; Daunorubicin; Etoposide; Factor Xa; Female; Flow Cytometry; Humans; Leukemia, Promyelocytic, Acute; Male; Membrane Glycoproteins; Microscopy, Confocal; Milk Proteins; Oxides; Phosphatidylserines; Thrombin; Thromboplastin; Tretinoin; Young Adult

A severe coagulopathy is a life-threatening complication of acute promyelocytic leukemia (APL) and is ascribable mainly to the excessive levels of tissue factor (TF) in APL cells regulated in response to the promyelocytic leukemia/retinoic acid receptor alpha (PML/RARalpha) fusion protein. The underlying molecular mechanisms for this regulation remain ill-defined. With U937-PR9 cell lines stably expressing luciferase reporter gene under the control of different mutants of the TF promoter, both luciferase and ChIP data allowed the localization of the PML/RARalpha-responsive sequence in a previously undefined region of the TF promoter at position -230 to -242 devoid of known mammalian transcription factor binding sites. Within this sequence a GAGC motif (-235 to -238) was shown to be crucial because deletion or mutation of these nucleotides impaired both PML/RARalpha interaction and promoter transactivation. However, EMSA results showed that PML/RARalpha did not bind to DNA probes encompassing the -230 to -242 sequences, precluding a direct DNA association. Mutational experiments further suggest that the activator protein 1 (AP-1) sites of the TF promoter are dispensable for PML/RARalpha regulation. This study shows that PML/RARalpha transactivates the TF promoter through an indirect interaction with an element composed of a GAGC motif and the flanking nucleotides, independent of AP-1 binding.

Topics: Base Sequence; Cell Line, Tumor; Coagulation Protein Disorders; DNA; Gene Expression Regulation, Leukemic; Humans; Leukemia, Promyelocytic, Acute; Oncogene Proteins, Fusion; Promoter Regions, Genetic; Thromboplastin; Transcription Factor AP-1; Transcriptional Activation

Acute promyelocytic leukaemia (APL) confers an increased risk of thrombosis and bleeding. Current treatments are insufficient to inhibit these complications. We recently showed that a combined immunoinhibitory and immunostimulatory short interference (si) RNA effectively inhibited leukaemic growth and metastasis in rats with APL. We now asked if the reported anti-leukaemic effects of siRNA treatment could be explained by inhibition of hypercoagulability. We measured markers of coagulation and fibrinolysis in plasma collected from APL rats with overt leukaemia using conventional assays. Coagulopathy developed in untreated leukaemic rats evidenced by increase in several haemostatic markers. Treatment of leukaemic rats with the siRNA reduced (p < 0.05) the concentration of thrombin-anti-thrombin complex (a marker of coagulation) by 40% compared with rats treated with an inactive, control siRNA. Substantial reductions (p < 0.05) were also obtained for two markers of fibrinolysis: D-dimer (72%) and plasminogen activator inhibitor type 1 (51%). The activity of tissue factor, the main initiator of coagulation, was not increased (p > 0.05) in untreated leukaemic rats compared with healthy rats, and did not change (p > 0.05) upon treatment with the siRNA. The bifunctional siRNA reduces the hypercoagulable state in APL in addition to its direct anti-leukaemic properties, supporting testing of this small molecule in human APL.

Topics: Animals; Antithrombin III; Biomarkers; Blood Coagulation; Cells, Cultured; Dendritic Cells; Disease Models, Animal; Fibrin Fibrinogen Degradation Products; Fibrinogen; Genetic Therapy; Interleukin-10; Leukemia, Promyelocytic, Acute; Male; Peptide Hydrolases; Plasminogen Activator Inhibitor 1; Rats; Rats, Inbred BN; RNA Interference; RNA, Small Interfering; Thrombophilia; Thromboplastin; Transfection

To investigate the effect of tanshinone II A on the procoagulant activity (PCA) of human umbilical vein endothelial cells (HUVEC) induced by acute promyelocytic leukemia (APL) cell line NB4 cells.. The HUVEC were incubated for 6, 12, and 24 hours in different tanshinone II A conditioned medias (Tan II A-NB4-24h-CM, Tan II A-NB4-72h-CM, Tan II A-NB4-120h-CM). Then the HUVEC were incubated for 6, 12, 24, and 72 hours with Tan II A-NB4-120h-CM and different concentrations of Tan II A (0, 0.25, 0.5, 1.0 microg/mL). The HUVEC lysates were obtained by three repeated freezing and thrawing. Their PCA were tested using the one stage clotting assay. The activity of tissue factor (TF : act) was tested using the chromogenic substrate assay. The control groups included 0.3 microg/mL ATRA, 0.01% DMSO and RPMI 1640.. Tan II A-(72 h,120 h)-NB4-CM elevated PCA of HUVEC and six hours of incubation in the 120 h-NB4-CM had the greatest PCA. The PCA of HUVEC in the 1.0 microg/mL Tan II A-NB4-CM was the same as in the 0.3 microg/mL ATRA-NB4-CM. (2) The NB4-CM induced PCA of HUVEC decreased with 5.0 microg/mL of Tan II A, at a level similar to the decrease with 0.3 microg/mL of ATRA. Less than 5.0 microg/mL of Tan II did not reduce the NB4-CM induced PCA of HUVEC. (3) Both Tan II A 120 h-NB4-CM and ATRA 120 h-NB4-CM elevated the TF : act of HUVEC. The TF : act reached the peak after 6 hours of incubation. The Tan II A 120 h-NB4-CM maintained the peak level of TF : act at the 12th hour and fell to the base line at the 24th hour. The ATRA 120 h-NB4-CM induced TF:act dropped down with time after reaching its peak at the 6th hour. (4) The 1.0 microg/mL of Tan II A did not reduce the TF : act of HUVEC induced by the Tan II A 120 h-NB4-CM. But the 0.3 microg/mL of ATRA reduced the TF : act of HUVEC at the 6th hour.. TanIIA-NB4-CM increases PCA and TF : Act of HUVEC. TanIIA decreases PCA of HUVECs induces by TanIIA-NB4-CM.

Topics: Abietanes; Blood Coagulation Factors; Cell Line, Tumor; Endothelial Cells; Humans; Leukemia, Promyelocytic, Acute; Phenanthrenes; Thromboplastin; Tretinoin; Umbilical Veins

To investigate the effect of arsenic trioxide (As(2)O(3)) or all-trans retinoic acid (ATRA) on the mRNA and protein expression of tissue factor (TF) and thrombomodulin (TM) and procoagulant activity (PCA) in NB4 cells. The NB4 cells were cultured in vitro and treated with As(2)O(3) or ATRA, expression of TF and TM antigen, and PCA change of treated NB4 cells were detected with ELISA, TF and TM mRNA transcription on the NB4 cells was assayed with reversed transcription polymerase chain reaction (RT-PCR). The results showed that 1 micromol/L As(2)O(3) and 1 micromol/L ATRA both gradually downregulated the expression of TF antigen and mRNA on NB4 cells, a human promyelocytic leukemia cell line, in time-dependent manner, as compared with control. The levels of TF antigen expression in AS(2)O(3) group were 13.3 +/- 1.8, 8.6 +/- 1.9, 10.8 +/- 1.5, 2.0 +/- 0.6 and 2.6 +/- 0.9 ng/10(7) respectively; while the levels of TF antigen expression in ATRA group were 12.4 +/- 1.1, 11.3 +/- 1.8, 5.7 +/- 1.7, 2.8 +/- 0.8 and 2.0 +/- 0.6 ng/10(7) at 24, 48, 72, 96 and 120 hours respectively (P<0.05). The procoagulant activity (PCA) of NB4 cells was decreased, blood coagulation times were 123.5 +/- 10.5, 156.3 +/- 11.6, 179.3 +/- 15.3, 248.9 +/- 20.1, 312.0 +/- 29.8 seconds in As(2)O(3) groups, respectively; 76.4 +/- 5.6, 146.8 +/- 10.9, 198.2 +/- 15.6, 265.8 +/- 20.6 and 363.8 +/- 31.9 seconds in ATRA groups respectively at 24, 48, 72, 96 and 120 hours (P<0.05). ATRA upregulated TM antigen expression on NB4 cells. It is concluded that the As(2)O(3) and ATRA decrease mRNA transcription of TF, downregulate expression of TF and reduce procoagulant activity in NB4 cells. The TM transcription and expression upregulated by ATRA may alleviate dysfunction of coagulation in APL.

Topics: Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Humans; Leukemia, Promyelocytic, Acute; Oxides; RNA, Messenger; Thrombomodulin; Thromboplastin; Tretinoin; Tumor Cells, Cultured

Topics: Angioplasty, Balloon, Coronary; Biopsy; Blood Cell Count; Bone Marrow; Coronary Occlusion; Coronary Thrombosis; Humans; Incidental Findings; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Myocardial Infarction; Pancytopenia; Radiography; Thromboplastin

Tissue factor (TF) is a transmembrane glycoprotein that serves as the prime initiator of blood coagulation and plays a critical role in thrombosis and hemostasis. In addition, a variety of tumor cells overexpress cell-surface TF, which appears to be important for tumor angiogenesis and metastasis. To elucidate the mechanism involved in the upregulation of TF in human tumor cells, a comprehensive analysis of TF mRNA from various normal and tumor cells was performed. The results of these studies indicate that, in addition to possessing a normal full-length TF transcript and minor levels of an alternatively spliced transcript known as alternatively-spliced tissue factor (asTF), human tumor cells express additional full-length TF transcripts that are also generated by alternative splicing. Reverse transcriptase-polymerase chain reaction (RT-PCR) and 5'-rapid amplification of cDNA ends- (5'-RACE) based analyses of cytoplasmic RNA from normal and tumor cells revealed that there is alternative splicing of the first intron between exon I and exon II resulting in 2 additional TF transcripts. One of the transcripts has an extended exon I with inclusion of most of the first TF intron (955 bp), while the second transcript is formed by the insertion of a 495 bp sequence, referred to as exon IA, derived from an internal sequence of the first intron. The full length TF transcript with alternatively spliced novel exon IA, referred to as alternative exon 1A-tissue factor (TF-A), represented approximately 1% of the total TF transcripts in normal cells, but constituted 7-10% of the total TF transcript in tumor cells. Quantitative real-time RT-PCR analysis indicated that cultured human tumor cells contain 10-25-fold more copy numbers of TF-A in comparison to normal, untransformed cells. We propose that high-level expression of the novel TF-A transcript, preferentially in tumor cells, may have utility in the diagnosis and staging of a variety of solid tumors.

Topics: Adenocarcinoma; Alternative Splicing; Base Sequence; Carcinoma, Hepatocellular; Carcinoma, Transitional Cell; Cytoplasm; Exons; Humans; Introns; Leukemia, Promyelocytic, Acute; Liver Neoplasms; Molecular Sequence Data; Neoplasm Staging; Neoplasms; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Thromboplastin; Tumor Cells, Cultured; Up-Regulation

To investigate the effects of Tanshinone IIA (Tan IIA) on procoagulant activity (PCA) of human ECV304 cells induced by acute promyelocytic leukemia cell line NB4 cells.. ECV304 monolayers were respectively incubated for different hours at 37 degrees C in the conditioned media (CM) of NB4 cells treated with 0.5 microg/mL Tan IIA(Tan IIA-NB4-CM), 0.3 microg/mL all-trans retinoidic acid (ATRA)(ATRA-NB4-CM), DMSO(DMSO-NB4-CM) or the RPMI1640 medium. ECV304 lysates were tested for PCA using the one-stage clotting assay as well as for tissue factor activity (TF: Act) using the chromogenic substrate assay; ECV304 cell monolayers were incubated for different hours at 37 degrees C in a medium system including 0.5 microg/mL Tan IIA and Tan IIA-NB4-CM, and the ECV304 cell lysates were tested for PCA in the same way as above. Also they were controlled by 0.3 microg/mL ATRA, DMSO or RPMI1640 medium.. (1) The conditioned mediums from 0. 5 microg/mL Tan IIA that treated NB4 cells for 24, 72 and 120 hours respectively could elevate PCA of ECV cells, and this capability developed with the time of reaction. ATRA did the same as Tan IIA (P > 0.05). (2) 0.5 microg/mL Tan IIA down-regulated the PCA of ECV304 cells induced by Tan IIA-NB4-CM, and the inhibitory effects increased with time, reaching the highest at 120 hours. (3) Tan IIA120 h-NB4-CM up-regulated TF:Act of ECV304 cells, and the effect increased with time. (4) 0. 5 microg/mL Tan IIA down-regulated PCA and TF: Act of ECV304 cells induced by Tan IIA-NB4-CM, and the inhibitory effect increased with time; simultaneously, the test was controlled with 0.3 microg/mL ATRA, the effects on PCA and TF: Act were not significantly different (P > 0.05).. Tan IIA-NB4-CM can increase the levels of PCA and TF: Act of ECV304 cells through some unidentified factor; however, Tan IIA can obviously decrease the PCA and TF: Act levels of ECV304 cells induced by Tan IIA-NB4-CM.

Topics: Abietanes; Anticoagulants; Cell Differentiation; Cell Line; Cell Line, Tumor; Culture Media, Conditioned; Drugs, Chinese Herbal; Endothelial Cells; Humans; Leukemia, Promyelocytic, Acute; Phenanthrenes; Thromboplastin; Tretinoin; Umbilical Veins

To investigate the effect of Panax notoginseng saponin (PNS) on procoagulant activity (PCA) and differentiation induction in NB4 cells.. After NB4 cells were treated with PNS, the recalcification time, PCA and TF-mRNA expression in NB4 cells were tested by RT-PCR. The inhibitory effect of PNS on NB4 cell proliferation was analysed by MTT method, NBT assay, cell morphological observation and flow cytometry.. (1) PNS of all concentrations could significantly prolong the recalcification time and lower the PCA level in NB4 cells in time-concentration-dependent manner. Simultaneously it down-regulated the expression of TF-mRNA. (2) PNS could partially inhibit the NB4 cell proliferation. (3) PNS could raise the NBT reducing capability of NB4 cells (P < 0.05). And morphological examination showed the differentiating tendency of monocyte and macrophage.. PNS could reduce the procoagulant activity and TF-mRNA expression in NB4 cells, and partially induce the differentiation of NB4 cells, therefore, it is hopeful to be a new anti-coagulant agent.

Topics: Antineoplastic Agents; Blood Coagulation Factors; Cell Division; Cell Transformation, Neoplastic; Cysteine Endopeptidases; Humans; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Panax; Saponins; Thromboplastin; Tumor Cells, Cultured

Retinoids are involved in cell differentiation, morphogenesis, proliferation and antineoplasic processes. Thus, the retonoic acid receptor (RARalpha) agonist, AM80, regulates tissue factor (TF), thrombomodulin (TM) expression and granulocytic differentiation in promyelocytic cells, while the RARgamma-selective retinoid, CD437, inhibits in vitro cell proliferation and induces apoptosis. The mitogen-activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI3K) pathways constitute key integration points along the signal transduction cascades that link diverse extracellular stimuli to proliferation, differentiation and survival.. PI3K and MEK/ERK kinase-dependent pathways were examined as potential targets of retinoid signaling. Likewise, by using specific inhibitors, the role of those kinases in retinoid-induced granulocytic differentiation, apoptosis, and TF and TM expression n NB4 cells were analyzed.. AM80-treated NB4 cells had increased PI3K activity and phosphoinositide turnover. High steady-state pERK-1/-2 activity levels were not significantly changed by AM80. Yet, PI3K inhibitor LY294002 significantly reduced AM80-elicted ERK-1/-2 activity. Thus, the PI3K pathway might contribute to elevated ERK-1/-2 activity in NB4 cells. Inhibition of the PI3K and MEK/ERK pathways reversed AM80-induced granulocytic differentiation, TF down-regulation and TM induction. Besides, CD437 significantly reduced ERK-1/-2 activity of NB4 cells. Further ERK-1/-2 activity inhibition with the MEK inhibitor, PD98059, increased the retinoid pro-apoptotic effect with an additive effect.. Regardless the different regulation of PI3K and MAPK pathways promoted by AM80 and CD437, there is a varying degree of cross-talk between these pathways in the control of the overall response of promyelocytic cells to retinoids. Thus, disruption of targeted pathways, together with specific retinoids, might be an effective therapeutic treatment for acute promyelocytic leukemia.

Topics: Apoptosis; Benzoates; Cell Differentiation; Cell Line, Tumor; Flavonoids; Gene Expression; Humans; Leukemia, Promyelocytic, Acute; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 3; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Retinoids; Tetrahydronaphthalenes; Thrombomodulin; Thromboplastin

We studied the pathogenesis of the bleeding disorder in acute promyelocytic leukemia by measuring procoagulant, profibrinolytic, and proinflammatory mediators in peripheral blood and bone marrow cells from 25 previously untreated patients. Patients were induced with either all-trans retinoic acid (ATRA) or chemotherapy. Plasma levels of fibrinopeptide A (FPA), fibrin d-dimer, thrombin antithrombin (TAT) complex, prothrombin fragment 1.2 (F1.2), urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (t-PA) and plasminogen activator-inhibitor 1 (PAI-1) were measured before and after therapy, as was the cellular expression of the genes for tissue factor (TF) and interleukin-1 beta (IL-1 beta). The mean plasma levels of fibrin d-dimer, F1.2, TAT and FPA were markedly elevated prior to therapy and declined during the first 30 days of treatment with either ATRA or chemotherapy, but more rapidly and to a greater extent in patients treated with ATRA. ATRA treatment was associated with a significant decrease in TF gene expression in bone marrow cells during the first 30 days of treatment, whereas IL-1 beta gene expression, which decreased in the cells of six patients treated with either chemotherapy or ATRA, actually increased in the remaining six patients treated with either chemotherapy or ATRA. In patients with APL, treatment with either chemotherapy or ATRA rapidly ameliorates the coagulopathy, as indicated by an abrupt decline in markers of clotting activation. An increase in cytokine gene expression (e.g. IL-1 beta) may provide an explanation for the persistent hypercoagulability observed in some patients with APL, regardless of therapeutic approach. Our data confirms and extends earlier observations by others that ATRA is more effective than chemotherapy alone in rapidly reducing the procoagulant burden of APL tumor cells. However, our data also suggests that cytokine expression in some patients may be accelerated by either chemotherapy or ATRA. The implications of this observation for understanding the retinoic acid syndrome will require further studies.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Antithrombins; Blood Coagulation; Bone Marrow Cells; Cell Line, Tumor; Child; Child, Preschool; Cytokines; Female; Fibrin Fibrinogen Degradation Products; Fibrinolysis; Fibrinopeptide A; Humans; Infant; Interleukin-1; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Plasminogen Activator Inhibitor 1; Ribonucleases; Thrombin; Thromboplastin; Time Factors; Tissue Plasminogen Activator; Tretinoin; Urokinase-Type Plasminogen Activator

Localized large vessel thrombosis in acute leukaemia is rare, haemorrhagic complications being more common.. We present a patient with acute promyelocytic leukaemia (APL) presenting with an acutely ischaemic lower limb. Large vessel thrombosis is a rare presentation of APL. We reviewed the literature on the coagulopathy of APL and discuss the pathology and current treatment options.. Disordered haemostasis is typical of acute promyelocytic leukaemia (FAB M3) and relates to the intrinsic properties of the blast cells as well as thrombocytopenia from bone marrow involvement. Expression of procoagulants, stimulation of cytokines and alterations in endothelial cell anticoagulant properties initiate a disseminated intravascular coagulation (DIC) resulting in the typical clinical and laboratory findings in APL. The promyelocytes are characterized by the balanced reciprocal translocation between chromosomes 15 and 17. All-trans-retinoic acid (ATRA) induces differentiation in these cells, revolutionizing the treatment of APL.. Unexpected limb ischaemia in a young, apparently healthy patient might be the presenting symptom of an underlying haematological disorder such as APL. A thorough haematological investigation should be performed prior to contemplating surgery. New treatment strategies based on knowledge of the molecular biology of APL has improved the prognosis of patients suffering from APL.

Topics: Amputation, Surgical; Antineoplastic Combined Chemotherapy Protocols; Arterial Occlusive Diseases; Cysteine Endopeptidases; Cytarabine; Daunorubicin; Female; Gangrene; Humans; Intermittent Claudication; Ischemia; Leg; Leukemia, Promyelocytic, Acute; Middle Aged; Neoplasm Proteins; Neoplastic Stem Cells; Popliteal Artery; Remission Induction; Smoking; Thioguanine; Thrombophilia; Thromboplastin; Thrombosis; Toes; Tretinoin

Constitutive expression of tissue factor (TF) by acute promyelocytic leukemia (APL) cells may contribute to thrombotic complications. In this study we examined the transcriptional mechanisms of all-trans retinoic acid (ATRA)-induced down-regulation of TF in the APL cell line NB4, by analysis of stable clones expressing the luciferase gene under the control of 5' flanking regions of the TF gene. We show that the TF promoter is constitutively active in NB4 cells, and that ATRA induces rapid suppression of the promoter. Basal activity and ATRA-induced suppression of TF promoter is determined by the proximal -383 to +121 bp of the promoter. Electrophoretic mobility shift assays demonstrate the binding of Fos/Jun complexes to two TF promoter AP-1 sites in this region. Both complexes were suppressed by ATRA treatment. The ectopic expression of the APL-specific PML/RARalpha oncoprotein in U-937 cells results in induction of TF mRNA and promoter activity. Interestingly, this PML/RARalpha-mediated increase in TF promoter activity is sensitive to ATRA treatment. These data indicate that TF expression in APL cells is exacerbated by the presence of the PML/RARalpha fusion protein, and implicates the loss of Fos/Jun binding to the TF promoter in ATRA-induced suppression of TF.

Topics: Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Oncogene Proteins, Fusion; Promoter Regions, Genetic; Protein Binding; Thromboplastin; Transcription Factor AP-1; Transcription, Genetic; Tretinoin

To explore the effects of arsenic trioxide (As2O3) on tissue factor (TF), plasminogen activator inhibitor-1(PAI-I) and PAI-2 expression in NB4, HL-60 and THP-1 cells.. Inhibition of the cell proliferation of NB4, HL-60 and THP-1 cells treated with As2O3 in different concentrations was observed by use of growth curves. Apoptosis was assessed by DNA electrophoresis and flow cytometric DNA contents analysis. TF, PAI-1 and PAI-2 antigen levels in cell lysates were measured by enzyme-linked immunoassay (ELISA).. 1. After 24 hours exposure to 1 mumol/L As2O3 or 8 mumol/L As2O3, induced apoptosis of NB4 or HL-60 cells was noted. However induced apoptosis of THP-1 cells was not seen after exposure to 8 mumol/L As2O3 for 24 h. 2. The TF antigen levels were down-regulated during the As2O3-induced apoptosis of NB4 cells (P < 0.01), and the down-regulation effect of TF antigen levels was in the As2O3-concentration dependent fashion. Moreover, 2 mumol/L As2O3 increased the PAI-1 antigen levels in NB4 cells (P < 0.05). 3. The TF antigen levels were up-regulated and the PAI-2 antigen levels were down-regulated during the As2O3-induced apoptosis of HL-60 cells (P < 0.01 and P < 0.05, TF and PAI-2 respectively). 4. The TF and PAI-2 antigen levels in THP-1 cells were up-regulated after the exposure to 8 mumol/L As2O3 for 24 h (P < 0.05 and P < 0.01, TF and PAI-2 respectively).. The effects of As2O3 on TF, PAI-1 and PAI-2 antigen levels in NB4, HL-60 and THP-1 cells are distinct.

Topics: Apoptosis; Arsenic Trioxide; Arsenicals; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Oxides; Plasminogen Activator Inhibitor 1; Plasminogen Activator Inhibitor 2; Thromboplastin; Tumor Cells, Cultured

To investigate the effects of Realgar on procoagulant activity (PCA), tissue factor expression and tissue factor mRNA transcription in acute promyelocytic leukemia (APL) cell lines NB4 and MR2 cells.. NB4 and MR2 cells were treated with 300 micrograms.L-1 Realgar PCA of the treated cells was detected using one-stage clotting assay. TF antigen was detected by ELISA and TFmRNA by semi-quantitive RT-PCR.. The PCA and TF antigen level in NB4 and MR2 cells were significantly higher than that in HL-60 and K562 cells. Realgar could down-regulate the membrane PCA, TF antigen and TF mRNA transcription of NB4 and MR2 cells in a time-dependent manner.. Down-regulating TF expression and PCA of NB4 and MR2 cells by Realgar may be one of the mechanism of its improvement effect on DIC-related hemorrhage of APL patients.

Topics: Antineoplastic Agents; Arsenicals; Blood Coagulation Factors; Cysteine Endopeptidases; Drug Resistance, Neoplasm; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; K562 Cells; Leukemia, Promyelocytic, Acute; Materia Medica; Neoplasm Proteins; RNA, Messenger; Sulfides; Thromboplastin; Tretinoin

To study the effect of arsenic trioxide (As2O3) or all-trans retinoic acid (ATRA) on coagulopathy in patients with acute promyelocytic leukemia (APL), and the mechanism of hemorrhage in these patients.. Thrombomodulin (TM) or tissue factor (TF) transcription of mRNA of freshly isolated bone marrow blast from APL patients was detected by semi-quantitative RT-PCR. The parameters of coagulation and cell procoagulation activity (PCA) were assessed in plasmic levels. Bleeding symptom was observed during As2O3 or ATRA treatment.. TM expression in the APL cell surface was significantly upregulated from (14.31 +/- 1.60) ng/10(7) to (21.61 +/- 6.82) ng/10(7) cells. The levels of P-selectin, soluble fibrin monomer complex (SFMC) and D-dimer (D-D) decreased after ATRA or As2O3 treatment. Abnormal high expression of TF in APL cell was downregulated in patients treated with ATRA or As2O3. The expression level was (14.81 +/- 6.23) ng/L before treatment, but undetected after 20 days of treatment. In addition, the membrane PCA of fresh APL cells was predominantly FVII-dependent after ATRA or As2O3 treatment. Bleeding symptom was ameliorated during As2O3 or ATRA treatment.. Bleeding symptom was controlled in patients with APL after As2O3 or ATRA treatment.

Topics: Adult; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Blood Coagulation Disorders; Down-Regulation; Female; Hemorrhage; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Oxides; RNA, Messenger; Thrombomodulin; Thromboplastin; Tretinoin

To study the in vivo effect of all-trans-retinoic acid (ATRA) and arsenic trioxide (As(2)O(3)) on the expression of tissue factor (TF) and the other hemostatic disturbance, a series of parameters were measured either in bone marrow blasts or plasma from acute promyelocytic leukemia (APL) patients. The plasma parameters were measured by ELISA or chromogenic studies. The TF transcription was assessed using reverse transcription-polymerase chain reaction (RT-PCR) technique. The results indicated that the blast cell procoagulant activity (PCA), TF antigen of APL cell lysate, as well as the transcription of APL TF mRNA elevated at diagnosis, were reduced after ATRA or As(2)O(3) therapy. The plasma level of P-selectin, TF, thrombin-antithrombin complex (TAT), soluble fibrinmonomer complex, thrombomodulin (TM), tissue factor pathway inhibitor (TFPI), plasmin-antiplasmin complex, tissue plasminogen activator (t-PA) activity, urokinase plasminogen activator (u-PA) and its receptor (u-PAR), and D-dimer (D-D) significantly increased. Fibrinogen (Fg), antigen level of protein C (PC), plasminogen (PLG) activity, alpha(2)-plasminogen inhibitor activity (alpha(2)-PI), and plasminogen activator inhibitor (PAI) activity were decreased at diagnosis. The protein C activity (PC:A) and protein S (PS) remained unchanged. All the parameters were restored to normal ranges after complete remission (CR) except elevation of TF and TAT in both groups, as well as PC:A, PS, and t-PA in the ATRA group. In conclusion, there existed activation of platelets and consumption of anticoagulants as well as activation of coagulation and fibrinolytic system before treatment. Both ATRA and As(2)O(3) therapy downregulated the expression of TF mRNA, decreased the PCA and TF level in APL cells, significantly inhibited coagulation activation, corrected secondary hyperfibrinolysis and the other hemostatic abnormalities, and thus greatly improved the bleeding symptom in early stage of the treatment.

Topics: Adolescent; Adult; Arsenic Trioxide; Arsenicals; Case-Control Studies; Female; Hemorrhage; Hemostasis; Hemostatics; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Oxides; Thromboplastin; Tretinoin

Acute promyelocytic leukaemia (APL) may be associated with disseminated intravascular coagulation, as a result of increased tissue factor (TF) expression and reduced thrombomodulin (TM) expression by APL blast cells. During retinoid acid (RA)- and dibutyryl cAMP (dbcAMP)-induced differentiation of the APL cells, there is a marked up-modulation of both the protein kinase A (PKA) and C (PKC) activities. In order to further assess whether these kinases are intimately associated with both the differentiation process and the regulation of TF and TM expression, we have correlated the modulation of their respective pathways with the extent of differentiation and modulation of these cellular receptors. NB4 cells were incubated with all-trans-RA (ATRA) or dbcAMP for up to 48 h. The contribution of phospholipase C (PLC), inositol phosphate (IP), PKC and PKA in the expression of CD11b, TF and TM was studied by the use of specific inhibitors. Myo-inositol uptake and PKC activity increased in cells induced to differentiate by ATRA but the retinoid did not affect cAMP levels or PKA activity. Under treatment with dbcAMP, PKA activity was increased while inositol uptake and PKC activity remained unchanged. Our results show that the effects of ATRA and dbcAMP on promyelocytic cells are closely related, respectively, to the PLC/IP/PKC and the cAMP/PKA pathways. In cells induced to differentiate by ATRA, CD11b expression seems more closely related to inositol uptake than to PKC activity while the expression of TF and TM show the opposite pattern, which suggests cellular events regulated at a different level within a common signal transduction pathway.

Topics: Bucladesine; Cell Differentiation; Cyclic AMP-Dependent Protein Kinases; Granulocytes; Humans; Leukemia, Promyelocytic, Acute; Macrophage-1 Antigen; Protein Kinase C; Signal Transduction; Thrombomodulin; Thromboplastin; Tretinoin; Tumor Cells, Cultured

To study the effect of all-trans retinoic acid (ATRA) and arsenic troxide (As2O3) on tissue factor (TF) expression and procoagulant activity (PCA) of acute promyelocytic leukemia (APL) cells in vivo and in vitro.. PCA from freshly isolated APL blasts from APL patients treated with ATRA or As2O3 was detected using a one-stage clotting assay. TF antigen was detected by ELISA and TF mRNA by RT-PCR. The maturation sensitive (NB4) or resistant subclones (NB4-R1) of the promyelocytic NB4 cell line, as well as U937 cells infected with pMSCV-PML-RARa treated with or without ATRA or As2O3, were also examined.. Both ATRA and As2O3 can down-regulate the TF antigen, its mRNA transcription and membrane PCA of APL cells in vivo and in vitro, in a time-dependent manner. The TF antigen level in PML-RARa + U937 cells was significantly higher than that in U937 cells infected with retrovirus vector. Both ATRA and As2O3 can also down-regulate the TF antigen in U937 cells transfected with or without PML-RARa.. Tissue factor expression and PCA in APL cells may be down-regulated by ATRA and As2O3. By down-regulating TF expression, As2O3 might also be used to improve the DIC-related hemorrhage in APL. Our data indicate that elevated TF antigen in PML-RARa + U937 may be related to the fusion protein PML-RARa. The down-regulating effect of ATRA and As2O3 on TF expression in U937 cells might not involve this fusion protein.

Topics: Adolescent; Adult; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Female; Gene Expression Regulation, Leukemic; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Neoplasm Proteins; Oncogene Proteins, Fusion; Oxides; RNA, Messenger; Thromboplastin; Tretinoin; Tumor Cells, Cultured

To study the changes of tissue factor expression and hemostatic molecular markers in acute promyelocytic leukemia during all-trans retinoic acid (ATRA) or arsenic trioxide (AS(2)O(3)) treatment.. The plasma level of tissue factor (TF), tissue factor pathway inhibitor(TFPI), thrombin antithrombin complex(TAT), plasmin antiplasmin complex(PAP), urokinase type plasminogen activator(u-PA), urokinase type plasminogen activator receptor(u-PAR) and the TF level of bone marrow blasts lysate were measured by ELISA. Transcription of TF mRNA was detected by RT-PCR.. The plasma levels of TF [(98.3 +/- 19.8) ng/L, (89.6 +/- 15.2) ng/L], TFPI [(94.4 +/- 37.0) mg/L, (93.5 +/- 36.4) mg/L], TAT [(21.9 +/- 9.6) microg/L, (18.2 +/- 9.7) microg/L[, PAP [(0.73 +/- 0.26) mg/L, (0.63 +/- 0.33) mg/L], u-PA [(0.63 +/- 0.23) microg/L, (0.57 +/- 0.01) microg/L] and u-PAR [(0.41 +/- 0.14) microg/L, (0.47 +/- 0.16) microg/L], the TF of bone marrow blasts lysate [(680.24 +/- 456.61) pg/10(7), (368.02 +/- 151.2) pg/10(7)] and transcription of mRNA were all remarkably elevated at the time of diagnosis. They all decreased after ATRA and AS(2)O(3) administration.. There is over expression of TF, activation of coagulation system and hyperfibrinolysis, in patients with acute promyelocytic leukemia, these can be ameliorated with clinical improvement. All-trans retinoic acid and arsenic trioxide down-regulate the expression of TF mRNA and decrease the TF contents in APL blasts. However, there is also high plasma level of TF and TAT indicating the existence of hypercoagulability after remission.

Topics: Adult; Aged; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Female; Hemostasis; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Oxides; RNA, Messenger; Thromboplastin; Tretinoin

Topics: Annexin A2; Disseminated Intravascular Coagulation; Humans; Leukemia, Promyelocytic, Acute; Neoplasms; 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

Topics: Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Promyelocytic, Acute; Oxides; Reverse Transcriptase Polymerase Chain Reaction; Thrombomodulin; Thromboplastin; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

This study evaluated hemostatic data in 28 patients with newly diagnosed acute promyelocytic leukemia (APL) and 15 patients with relapsed APL. Activated partial thromboplastin time and prothrombin time were prolonged at initial onset of APL. Plasma level of fibrinogen was significantly decreased in patients with initial disease of APL, but it was not decreased significantly during the relapse of APL. Plasma fibrin and fibrinogen degradation products levels were significantly increased and platelet counts significantly decreased in both groups. Plasma levels of antiplasmin significantly decreased at initial onset but not during relapse. Plasma levels of antithrombin were within normal range in patients with initial disease but significantly decreased in those with relapse. Plasma levels of D-dimer, soluble fibrin monomer (sFM), plasmin-plasmin inhibitor complex (PPIC), and thrombin antithrombin complex (TAT) levels were significantly high in both groups. Plasma levels of PPIC, sFM, and D-dimer were significantly higher at initial onset of APL than during relapse. However, there was no significant difference in DIC score between patients with initial onset and those with relapse; plasma levels of tissue factor (TF) significantly increased in both groups, but they were significantly higher at initial onset of APL than during relapse. TF and tissue type plasminogen activator (t-PA) antigen levels in leukemic cell lysate were significantly increased in both groups, and they were significantly lower during relapse than at initial onset. Hemostatic abnormalities occurring in patients with relapsed APL might be the result of the decrease of TF and t-PA in leukemic cells. These findings suggest that DIC in APL patients with relapse might not be caused only by TF and t-PA and thus should be treated with different therapy from patients with initial onset of APL.

Topics: Adolescent; Adult; Antigens; Female; Fibrinogen; Hemostasis; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Recurrence; Thromboplastin; Tissue Plasminogen Activator

To study in vivo effect of all-trans-retinoic acid (ATRA) or arsenic trioxide (As2O3) on the expression of tissue factor (TF) and the hemostatic disorders, a series of parameters were measured in bone marrow blasts and plasma from acute promyelocytic leukemia (APL) patients.. The plasma variables were measured by ELISA or chromogenic study. The TF transcription was assessed using reverse transcription-polymerase chain reaction technique (RT-PCR).. The blast cell procoagulant activity (PCA), TF antigen of APL cell lysates, as well as the transcription of APL TF mRNA elevated at diagnosis, were reduced after ATRA or As2O3 therapy. The plasma level of platelet alpha-granular membrane protein-140, soluble fibrinomonomer complex, thrombomodulin, tissue plasminogen activator and D-dimer significantly increased, fibrinogen, antigen level of protein C, plasminogen, alpha 2-plasminogen inhibitor and plasminogen activator inhibitor decreased at diagnosis, were restored to normal after complete remission but protein C activity and protein S remained elevated in ATRA group.. There existed activation of platelets and consumption of anticoagulants as well as activation of coagulation and fibrinolytic system before treatment. Both ATRA and As2O3 therapy down-regulated the expression of TF mRNA, decreased the PCA and TF level in APL cells, inhibited coagulation activation, secondary hyperfibrinolysis and recorrected other hemostatic abnormalities, thus greatly improved the bleeding symptom in early stage of the treatment.

Topics: Adolescent; Adult; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Blood Coagulation Factors; Female; Hemostasis; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Oxides; Thromboplastin; Tretinoin

To investigate the effects of all-trans retinoic acid (ATRA) and arsenic trioxide (As(2)O(3)) on both tissue factor (TF) expression and procoagulant activity (PCA) of acute promyelocytic leukemia (APL) cells in vivo and in vitro.. The PCA of APL blasts freshly isolated from the APL patients treated with ATRA or As(2)O(3) was detected using a one-stage clotting assay; the TF antigen was detected by ELISA and TF mRNA by RT-PCR. The maturation sensitivity (NB4) or resistant subclones (NB4-R1) of the promyelocytic NB4 cell line as well as U937 cells transfected with pMSCV-PML-RARa treated with or without ATRA or As(2)O(3) were also examined.. Both ATRA and As(2)O(3) time-dependently down-regulated the TF antigen, its mRNA transcription and membrane PCA of APL cells in vivo and in vitro. The TF antigen level in PML-RARa(+) U937 cells was significantly higher than that in U937 cells transfected with retrovirus vector (890 pg/8 x 10(5) +/- 80 pg/8 x 10(5) cell and 728 pg/8 x 10(5) +/- 86 pg/8 x 10(5) cell, respectively). Both ATRA and As(2)O(3) down-regulated the TF antigen of the U937 cells transfected with or without PML-RARa.. Tissue factor expression and PCA of APL cells can be down-regulated by ATRA and As(2)O(3). By down-regulating the TF expression, As(2)O(3) might also be used to improve the DIC-related hemorrhage of APL. It is also suggested that elevated TF antigen in the PML-RARa(+) U937 cells may be related with the fusion protein PML-RARa, while the down-regulation effect of ATRA and As(2)O(3) on the TF expression of U937 cells might not be involved in the fusion protein.

Topics: Adolescent; Adult; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Blood Coagulation; Cells, Cultured; Female; Gene Expression; Humans; Leukemia, Promyelocytic, Acute; Leukocytes, Mononuclear; Male; Middle Aged; Neoplasm Proteins; Oncogene Proteins, Fusion; Oxides; RNA, Messenger; Thromboplastin; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; U937 Cells

To investigate molecular mechanism of tissue factor (TF) expression on acute promyelocytic leukemia cell line NB4 cells down-regulated by all-trans retinoic acid (ATRA) and arsenic trioxide (As(2)O(3)).. Cyclohexamide (CHX) inhibition test for de novo protein synthesis and actinomycin D (Act D) inhibition test for RNA synthesis were used to check the effect of ATRA on the TF expression. TF antigen of U937 cells transfected with pMSCV-PML-RARalpha treated with or without ATRA and As(2)O(3) was detected.. CHX treatment completely suppressed the down-regulation effect of ATRA on the TF mRNA expression, Act D inhibition test showed that half-life of TF mRNA in treated NB4 cells was shortened to about 30 min from that of around 60 min in untreated NB4 cells. The TF antigen contents in U937 cells transfected with pMSCV-PML-RARalpha were significantly higher than that in transfected U937 cells with retrovirus vector. Both ATRA and As(2)O(3) could down-regulate the TF antigen level in U937 cells transfected with or without PML-RARalpha.. The modulation of the TF mRNA expression in NB4 cells by ATRA might be indirect. TF mRNA destabilization was involved in the TF regulation process mediated by ATRA. Elevated TF antigen level in U937 cells transfected with pMSCV-PML-RARalpha may be related to the fusion protein PML-RARalpha. The down-regulation effect of ATRA and As(2)O(3) on the TF expression of U937 cells might not involve the fusion protein.

Topics: Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Cycloheximide; Dactinomycin; Down-Regulation; Gene Expression Regulation; Humans; Leukemia, Promyelocytic, Acute; Oxides; Thromboplastin; Tretinoin; Tumor Cells, Cultured

Tissue factor (TF) production is under strict control in mature monocytic cells. However, constitutive expression of TF can be found in myelomonocytic cells and in haematopoietic cells arrested at an early stage of differentiation. In this paper we show that TF expression is down-regulated during the monocyte/granulocyte differentiation process, using the human monoblastic U-937 and the acute promyelocytic leukaemia NB4 cell lines as models. Expression of TF mRNA, protein and procoagulant activity (PCA) was constitutively high in untreated cells. Exposure of U-937 cells to 1alpha,25-dihydroxycholecalciferol (VitD3) and all-trans retinoic acid (ATRA) resulted in down-regulation of TF expression and PCA. In NB4 cells induction by ATRA, but not VitD3, resulted in the down-regulation of TF expression and PCA. Consistent with this, induction of terminal differentiation, as confirmed by the expression of differentiation associated antigens and cell cycle arrest, was inversely correlated to TF expression in U-937 and NB4 cells. Moreover, terminally differentiated U-937 cells retained the capacity to respond to inflammatory mediators, i.e. lipopolysaccharide and interferon-gamma, by a rapid increase in TF expression. In conclusion, we show that not only ATRA but also VitD3 is a potent suppressor of monocytic TF expression and thus might have potential clinical use for the treatment of coagulopathies.

Topics: Cell Cycle; Cell Differentiation; Cholecalciferol; Gene Expression Regulation, Leukemic; Humans; Interferon-alpha; Interferon-gamma; Interleukin-1; Ionomycin; Leukemia, Promyelocytic, Acute; Lipopolysaccharides; Neoplasm Proteins; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Thromboplastin; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; U937 Cells

To investigate the effects of all-trans retinoic acid(ATRA), arsenic trioxide(As2O3) and daunorubicin(DNR) on tissue factor(TF) expression in acute promyelocytic leukemia (APL) cell line NB4 cells.. Procoagulant activity(PCA) of NB4 cells treated with 1 mumol/L ATRA, 1 mumol/L As2O3 or 0.2 microgram/ml DNR was detected using one-stage clotting assay, TF antigen by ELISA, and TF mRNA by RT-PCR.. Both ATRA and As2O3 could down-regulate the TF antigen, its mRNA transcription and membrane PCA of NB4 cells with a time-dependent manner, while DNR was shown to increase these parameters. Moreover, by dideoxy sequencing of DNA fragment derived from PCR, it was found that there was a exon 5 deletion transcript of TF in APL cells. Its biological significance remained unknown.. TF expression and PCA of APL cells may be down-regulated by ATRA and As2O3, therefore, As2O3 might also improve DIC-related hemorrhage of APL while inducing APL cells to apoptosis. The distinct regulation of TF and PCA on APL cells by As2O3 and DNR may at least partially contribute to their effects on APL coagulopathy through the influence on coagulant factors activation.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Daunorubicin; Factor VIII; Humans; Leukemia, Promyelocytic, Acute; Oxides; RNA, Messenger; Thromboplastin; Tretinoin; Tumor Cells, Cultured

All-trans-retinoic acid (ATRA) downregulates the expression of two cellular procoagulants, tissue factor (TF) and cancer procoagulant (CP), in human promyelocytic leukemia cells. To evaluate whether or not changes of the procoagulant activities (PCAs) may share mechanisms with the ATRA-induced cyto-differentiation process, we have characterized the effect of ATRA on the TF and CP expression by NB4 cells, an ATRA maturation-inducible cell line, and two NB4-derived cell lines resistant to ATRA-induced maturation, the NB4. 306 and NB4.007/6 cells. Next, we evaluated the effect on the PCAs of the NB4 parental cells of three synthetic retinoid analogues, ie: AM580 (selective for the retinoic acid receptor [RAR] alpha), capable to induce the granulocytic differentiation of NB4 cells; and CD2019 (selective for RARbeta) and CD437 (selective for RARgamma), both lacking this capability. Cells were treated with either ATRA or the analogues (10(-6) to 10(-8) mol/L) for 96 hours. The effect on cell differentiation was evaluated by morphologic changes, cell proliferation, nitro blue tetrazolium reduction assay, and flow cytometry analysis of the CD33 and CD11b surface-antigen expression. PCA was first measured in 20 mmol/L Veronal Buffer cell extracts by the one-stage clotting assay of normal and FVII-deficient plasmas. Further TF and CP have been characterized and quantified in cell-sample preparations by chromogenic and immunological assays. In the first series of experiments, ATRA downregulates both TF and CP in NB4 parental cells, as expected. However, in the differentiation-resistant cell lines, it induced a significant loss of TF but had little or no effect on CP. In a second series of experiments, in the NB4 parental cells, the RARalpha agonist (AM580) induced cell maturation and reduced 91% CP expression, whereas CD437 and CD2019 had no cyto-differentiating effects and did not affect CP levels. On the other hand, in the same cells the TF expression was reduced by ATRA and AM580, but also by the RARbeta agonist CD2019, which did not induce cell maturation. These data indicate that in NB4 cells, ATRA modulation of CP occurs in parallel with signs of cell differentiation, while the regulation of TF appears to be at least in part independent from these processes, and involves both alpha and beta nuclear retinoid receptors.

Topics: Antineoplastic Agents; Cysteine Endopeptidases; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Thromboplastin; Tretinoin; Tumor Cells, Cultured

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

In order to study the effect of all-trans retinoic acid (ATRA) or arsenic trioxide (As2O3) treatment on the expression of tissue factor (TF) in acute promyelocytic leukemia(APL).. The plasma level of soluble fibrin monomer complex(SFMC) and D-dimer(D-D), and the TF level of cell lysate were measured by ELISA, the transcription of TF mRNA was assessed by RT-PCR.. The plasma level of SFMC and D-D, the procoagulant activity(PCA) of bone marrow blasts, the TF level of cell lysate and the transcription of TF mRNA all remarkably elevated at diagnosis, while reduced after ATRA or As2O3 therapy.. Both ATRA and As2O3 downregulated the expression of TF mRNA, decreased the PCA and TF levels in APL cells, inhibited coagulation activation and secondary hyperfibrinolysis, thus greatly relieved the bleeding symptom in the early stage of treatment.

Topics: Adolescent; Adult; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Female; Hemostatics; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Oxides; RNA, Messenger; Thromboplastin; Tretinoin

Effects of uromodulin (URO) and Tamm-Horsfall protein (THP), the most abundant proteins in the urine of pregnant and normal women, respectively, on the induction of TNF-alpha secretion and tissue factor (TF) expression of human monocytes were studied. THP, URO, and its fragments stimulated human mononuclear cells to proliferate and secrete TNF-alpha. The release of URO and THP-induced TNF-alpha in monocytes was dependent upon protein tyrosine kinase activation that results in tyrosine phosphorylation. URO and THP also induced TF expression of human monocytes and monocytic cell line U937 in a dose-dependent manner. TF expression was transient, reached its peak at 6 h and declined toward basal levels by 24 h. Reverse transcriptase-PCR and dot-blot analysis confirmed the induction of TF mRNA synthesis. URO and THP-induced TF expression were inhibited by actinomycin D and pentoxifylline further supporting the requirement of de novo TF mRNA synthesis. The possibility of LPS contamination of URO and THP was excluded because: 1) URO and THP-induced TF expression were inhibited by specific Ab; 2) URO was less capable of inducing TF in HUVEC as compared with LPS; 3) polymyxin B blocked the induction of Limulus clotting by LPS but not by URO and THP; 4) both LPS-sensitive (C3H/HeN) and -resistant (C3H/HeJ) mice produced little or no TNF-alpha after URO challenge. Therefore, our findings suggest that URO and THP play a significant role in the innate immunity of the urinary system and that the immunostimulatory activity of URO is potentially useful for immunotherapy.

Topics: Animals; Endothelium, Vascular; Female; Humans; Leukemia, Promyelocytic, Acute; Limulus Test; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Monocytes; Mucoproteins; Pregnancy; RNA, Messenger; Thromboplastin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Uromodulin

We recently found that retinoic acids (RAs) exert anticoagulant effects by upregulating thrombomodulin (TM) and downregulating tissue factor (TF) expression in acute promyelocytic leukemia (APL) cells and monoblastic leukemia cells. Two classes of nuclear RA receptors, termed retinoic acid receptors (RARs) and retinoid X receptors, have been identified. Each receptor class consists of three subtypes. In the present study, we have used several synthetic retinoids to determine which receptor subtypes are involved in the regulation of TM and TF expression in NB4 APL cells, U937 monoblastic leukemia cells, and human umbilical vein endothelial cells (HUVECs). Am80, which has no binding affinity for RAR gamma, and Ch55, which does not bind to cytoplasmic retinoic acid binding protein (CRABP), upregulated TM and downregulated TF in NB4 and U937 cells, similar to all-trans RA (ATRA). A specific RAR alpha antagonist, Ro41-5253, significantly suppressed the upregulation of TM by ATRA and Am80 in NB4 cells, U937 cells, and HUVECs. In contrast, only with preincubation with both RAR alpha and RAR beta antagonists was downregulation of TF by retinoids suppressed in NB4 cells. These findings indicate that the mechanism of transactivation and transrepression functions of RARs are distinct and also elucidate the major role of RAR alpha in TM upregulation by retinoids in leukemic cells and HUVECs and the cooperation of RAR alpha and RAR beta in TF downregulation by retinoids. They also indicate that binding to CRABP is not required for the anticoagulant effect of retinoids and that synthetic retinoids will prove very useful in controlling distinct targets, the TM and TF genes, at the level of transcription, and will permit the development of retinoids with a new type of anticoagulant effect.

Topics: Anticoagulants; Benzoates; Blotting, Western; Chromans; Dibenzazepines; Down-Regulation; Endothelium, Vascular; Humans; Leukemia, Promyelocytic, Acute; Polymerase Chain Reaction; Receptors, Retinoic Acid; Retinoids; RNA, Messenger; Thrombomodulin; Thromboplastin; Umbilical Veins; Up-Regulation

Therapy with all-trans-retinoic acid (ATRA) can rapidly improve the coagulopathy of acute promyelocytic leukemia (APL). This study was designed to evaluate whether the APL cell line NB4 induces the procoagulant activity (PCA) of human endothelial cells (ECs) in vitro, and whether this property is modified after ATRA-induced NB4 maturation. EC monolayers were incubated for 4 hours at 37 degrees C with the conditioned media (CM) of NB4 treated with 1 mumol/L ATRA (ATRA-NB4-CM) or the vehicle (control-NB4-CM). EC lysates were tested for PCA. ATRA-NB4-CM induced significantly more PCA:tissue factor (TF) than control-NB4-CM (P < .01). To identify the cause of TF induction, interleukin (IL)-1 beta antigen levels were measured in CM samples. ATRA-NB4-CM contained significantly more IL-1 beta than control-NB4-CM. EC PCA was significantly inhibited by an anti-IL-1 beta antibody. The addition to the media of 10 mumol/L ATRA counteracted the EC TF expression induced by NB4-CM. These data indicate that ATRA increases the promyelocyte-induced EC TF, partly through increased IL-1 beta production. However, ATRA can protect the endothelium from the procoagulant stimulus of leukemic cells.

Topics: Blood Coagulation Factors; Cell Differentiation; Cells, Cultured; Culture Media, Conditioned; Endothelium, Vascular; Humans; Interleukin-1; Leukemia, Promyelocytic, Acute; Thromboplastin; Tretinoin; Tumor Cells, Cultured; Umbilical Veins

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

Patients with acute leukemia are at increased risk for thrombotic and hemorrhagic complications, particularly those patients with acute promyelocytic leukemia (APL) undergoing induction chemotherapy. These serious complications have been attributed by some authors to the release of tissue factor (TF) procoagulant activity (PCA), particularly during cytotoxic chemotherapy. In previous studies of normal peripheral blood cells, only cells of the monocyte lineage have been found to express TF PCA. Therefore, several questions remain regarding the origin and characterization of the PCA in malignant leukemic cells, particularly those thought to be derived from granulocyte progenitor cells. We utilized a full-length cDNA probe, several monoclonal antibodies (MAbs) and a sensitive one-stage PCA assay to study the expression of TF in the human cell line, HL-60, in human peripheral blood monocytes/macrophages (Mo/Mø) and in highly purified populations of human polymorphonuclear leukocytes (PMN). In the HL-60 cells we detected low but significant levels of TF mRNA and TF antigen (TF:Ag). In unstimulated cells, coordinate increased levels of TF mRNA, TF:Ag and TF PCA expression were noted following phorbol-ester-induced macrophage differentiation of the cells, but a decreased level of TF mRNA with no change in the basal level of TF:Ag expression occurred following retinoic acid-induced granulocyte differentiation of this cell line. Long-term cultures of stimulated mature Mo/Mø demonstrated initial coordinate expression of TF mRNA, TF:Ag and TF PCA, but TF:Ag expression persisted even after 7 days (when TF PCA was undetectable). No TF PCA, TF:Ag or TF mRNA was demonstrated in highly purified populations of human PMN, regardless of culture conditions. Discordant expression of TF mRNA, TF:Ag and TF PCA in HL-60 cells suggests the possibility of novel, post-synthetic mechanisms for the regulation of TF PCA expression, which might be dependent on the phenotypic differentiation level of the cell. Such mechanisms (yet to be defined) might account for the ability of some leukemic cells, which frequently express characteristics of more than one cell line (e.g. monocytes and granulocytes), to express a TF gene product capable of activating blood coagulation.

Topics: Antibody Specificity; Cells, Cultured; Gene Expression Regulation; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Monocytes; Neutrophils; RNA, Messenger; Thromboplastin; Tretinoin

Topics: Blood Coagulation Disorders; Humans; Leukemia, Promyelocytic, Acute; Thromboplastin; Tretinoin

Topics: Cell Line; Colony-Stimulating Factors; Culture Media, Conditioned; Cytokines; Gene Expression; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, Promyelocytic, Acute; Thromboplastin; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The mechanisms underlying acute promyelocytic leukemia (APL) coagulopathy and its reversal by administration of all-trans retinoic acid (ATRA) have been investigated. Bone marrow promyelocytic blasts from nine patients with APL were cultured with or without ATRA 1 mumol/L. Cultured blasts (days 0, 3, 6, and 9) were washed, resuspended in phosphate buffer, lysed by freezing and thawing, and then assayed for procoagulant activity (PCA), elastase activity, tissue factor (TF) antigen, tissue-type plasminogen activator (t-PA) antigen and urokinase-type plasminogen activator (u-PA) antigen. PCA was determined by a recalcification assay. Elastase was measured by an amidolytic assay (S-2484). TF, t-PA, and u-PA antigens were measured by an enzyme-linked immunosorbent assay (ELISA). Malignant promyelocytes isolated from the patients had increased levels of PCA and TF as compared with the control polymorphonucleates, and low levels of elastase, t-PA, and u-PA; the patient blast PCA level was significantly related to the degree of hypofibrinogenemia. In this system, blast PCA depended on the tissue factor and was significantly correlated to the TF antigen values. In the cultures without ATRA, PCA, TF, and u-PA progressively increased, whereas elastase and t-PA levels remained essentially unchanged. In the presence of ATRA, all parameters (except u-PA) decreased during the culture time. Thus, a major role of the promyelocytic blast cell PCA in the pathogenesis of M3-related coagulopathy is suggested; the ATRA effect on coagulopathy seems mainly mediated by a downregulation of the PCA.

Topics: Adolescent; Adult; Aged; Aprotinin; Blood Coagulation; Cell Differentiation; Cysteine Endopeptidases; Female; Fibrinolysis; Gene Expression Regulation, Leukemic; Hemorrhagic Disorders; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Neoplasm Proteins; Neoplastic Stem Cells; Pancreatic Elastase; Thromboplastin; Tissue Plasminogen Activator; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

All-trans-retinoic acid (ATRA) induces complete remission (CR) in up to 90% of acute promyelocytic leukemia (APL) patients with rapid amelioration of the bleeding syndrome. Previous studies indicate that ATRA treatment in vitro of the APL NB4 cell line can affect their procoagulant activity (PCA). To assess whether ATRA has this effect also in vivo, we prospectively studied the PCA of bone marrow blasts from APL patients on therapy with ATRA alone or associated with chemotherapy. Samples were obtained before, during, and after ATRA. To characterize the coagulopathy, we measured a series of plasma hemostatic variables before and during the first two weeks of therapy, as follows: (1) markers of hypercoagulability; (2) natural anticoagulants; (3) fibrinolysis proteins; and (4) elastase. The results by enzymatic and immunologic methods show that both total (tissue factor-like) and factor VII-independent (cancer procoagulant-like) blast cell PCAs, present before therapy, were reduced during (69% and 65% decrement, respectively) and virtually undetectable after ATRA. The plasma hemostatic assessment of patients before treatment was elevated hypercoagulability markers, low mean protein C, normal fibrinolysis proteins, and increased elastase. After starting ATRA, hypercoagulability markers were reduced within 4 to 8 days, protein C augmented, the overall fibrinolytic balance was unmodified, and elastase remained elevated. These results were not different either with or without chemotherapy and are consistent with the clinical findings of rapid improvement of the coagulopathy.

Topics: Adolescent; Adult; Aged; Blood Coagulation; Blood Coagulation Factors; Bone Marrow Cells; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Female; Fibrinolysis; Hemostasis; Humans; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Neoplasm Proteins; Thromboplastin; Tretinoin

Tissue 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 micrograms/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.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antigens; Calcimycin; Calcium; Humans; Ionophores; Kinetics; Leukemia, Promyelocytic, Acute; Lipopolysaccharides; Thromboplastin; Tumor Cells, Cultured

The expressions of thrombomodulin (TM) and tissue factor (TF) by all-trans retinoic acid (ATRA) were studied in human leukemic cell lines including NB4 (acute promyelocytic leukemia) and U937 (monoblastic leukemia). ATRA remarkably upregulated TM antigen expression in cell lysates as well as TM cofactor activity on the cell surfaces of NB4. The level of TM mRNA in NB4 cells was increased by ATRA. Inherently procoagulant NB4 cells contained markedly higher content of TF, which was efficiently reduced by ATRA. Modest increase of TM and decrease of TF were observed when NB4 cells were treated with dibutyryl cyclic adenosine monophosphate (dbcAMP). On the other hand, both ATRA and dbcAMP showed dramatic increase of TM antigen level and modest decrease of TF antigen in U937 cells. These results suggest that ATRA regulates expressions of TM and TF antigens and activity in NB4 and U937 cell lines, and provide evidence for a potential efficiency of ATRA as a preventive and therapeutic agent for disseminated intravascular coagulation in promyelocytic and monocytic leukemia.

Topics: Bucladesine; Gene Expression; Humans; Interleukin-1; Leukemia, Promyelocytic, Acute; RNA, Messenger; Thrombomodulin; Thromboplastin; Tretinoin; Tumor Cells, Cultured

Tissue factor activity and antigen were measured in promyelocytes freshly isolated from a patient with acute promyelocytic leukemia (APL), FAB M3. Determination of functional activity revealed that physically disrupted cells expressed considerable tissue factor of which less than two percent was available prior to physical disruption of the cells. No tissue factor antigen was detectable on the cell surface by fluorescence flow cytometry. In contrast, endotoxin-stimulated peripheral blood monocytes and monoblastic cells isolated from a patient with monoblastic leukemia had notable populations of tissue factor-positive cells by flow cytometry, and expressed higher proportions of total tissue factor activity without disruption. While some cell types may express both tissue factor antigen and activity when intact, others, which can be extremely rich in tissue factor, may express neither antigen nor activity without a triggering event such as cell damage.

Topics: Antigens; Cell Membrane; Flow Cytometry; Humans; Leukemia, Promyelocytic, Acute; Thromboplastin