All-trans retinoic acid is partially effective against lipopolysaccharide-induced but not against tissue-factor-induced disseminated intravascular coagulation in rat models.

All-trans retinoic acid (ATRA) has been introduced to the management of acute promyelocytic leukemia (APL) as a differentiation treatment. This drug not only causes complete remission, but also improves disseminated intravascular coagulation (DIC) without adding anticoagulants in APL. We have attempted to determine whether ATRA is effective against DIC in rat models induced by tissue factor (TF) or lipopolysaccharide (LPS), because the anticoagulant effect of ATRA has been considered to induce thrombomodulin upregulation and TF downregulation on endothelial cells as well as on APL cells. In male Wistar rats, DIC was induced by a 4-h infusion of thromboplastin (3.75 U/kg) or lipopolysaccharide (30 mg/kg). The rats were given ATRA orally each day at a dose of 100 mg/kg per day for 1 week before the injection of TF or LPS in ATRA treatment groups, or given low molecular weight heparin (LMWH) 10 min before the injection of TF or LPS (200 U/kg, bolus intravenously) in LMWH treatment groups. No significant changes in hemostatic parameters or markers of organ dysfunction were caused by the ATRA administration, while DIC was significantly improved by LMWH in the TF-induced model. DIC was significantly improved by both ATRA and LMWH in the LPS-induced model. These findings suggested that ATRA was useful for treating DIC only in the LPS-induced model, and that drug efficacy should be carefully assessed because the agents used to induce DIC considerably influenced the outcome.

One missense mutation in the factor X gene causing factor X deficiency--factor X Kanazawa.

We investigated the molecular basis of factor X deficiency in a Japanese patient whose factor X activity and antigen level were 45% and 50% of normal control values, respectively. All exons and intron/exon junctions of the factor X gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and nonradioactive single-strand conformational polymorphism (SSCP) analysis. Exon 5, containing the DNA fragment of the proband, showed aberrant migration by SSCP analysis. All exon-containing DNA fragments amplified by PCR were sequenced, and it was revealed that the proband was a heterozygote for a G --> A substitution in exon 5 of the factor X gene of the proband. This mutation predicts an amino acid replacement of arginine (Arg) for glycine (Gly) at codon 114 in the second EGF-like domain.

Elevated levels of free tissue factor pathway inhibitor antigen in cases of disseminated intravascular coagulation caused by various underlying diseases.

Tissue factor pathway inhibitor (TFPI) is primarily synthesized by vascular endothelial cells and is found in vivo in association with endothelial cells, lipoproteins, or in free form. Free TFPI is the most potent and important type, because it is released from endothelial cells following an injection of heparin, or as a result of pathological stimuli. In order to study the role of TFPI in disease, the concentration of free form TFPI was measured in the plasma of 114 patients suffering from disseminated intravascular coagulation (DIC), as the result of several underlying diseases. Plasma antigen levels of free TFPI were significantly higher even in those patients not exhibiting DIC than in normal healthy subjects. These levels were even higher among patients exhibiting DIC, especially those with acute promyelocytic leukemia or cancer, receiving continuous heparin drip infusions. A significant correlation was observed between the plasma antigen levels of free form TFPI and those of fibrin/fibrinogen degradation products, and free form TFPI and plasmin inhibitor complex (r = 0.428, P < 0.0001 and r = 0.329, P < 0.0001, respectively) among 114 DIC patients. There were no significant differences between the plasma levels of free TFPI in DIC patients with or without multiple organ failure. It has been suggested that the plasma levels of free TFPI are closely related to the levels of fibrinolysis occurring in DIC patients, although further study is required to clarify the degree to which TFPI is expressed by endothelial cells during DIC.

Treatment of disseminated intravascular coagulation (DIC) with all-trans retinoic acid in an endotoxin-induced rat model.

Anticoagulant drugs such as heparin are often administered to patients with disseminated intravascular coagulation (DIC) who are also being treated for their underlying disease. The pathophysiology of DIC is so varied that treatment with medications other than anticoagulants may be useful. All-trans retinoic acid (ATRA), which is used for the treatment of acute promyelocytic leukemia (APL), improves DIC in APL. In vitro studies have reported that ATRA caused downregulation of tissue factor and upregulation of thrombomodulin (TM) on endothelial cells as well as APL cells. We examined the effect of ATRA in an endotoxin-induced rat DIC model. DIC was induced in male Wistar rats with a 4-h sustained infusion of endotoxin at a dose of 30 mg/kg. ATRA (20 mg/day) was given every day for 1 week before the injection of endotoxin. ATRA improved the increase in thrombin-antithrombin III (TAT) complex and D-dimer in this model. Fibrin deposition in renal glomeruli was inhibited by ATRA administration, with an increase in the intensity of immunohistochemical TM staining. These findings suggest that ATRA has beneficial effects in the endotoxin-induced rat DIC model. The mechanism may be an upregulation of TM expression on endothelial cells.

Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death.

Recently, erythropoietin has been shown to be produced by astrocytes and its production is hypoxia-inducible. In the present study, we demonstrated, using a reverse transcription-polymerase chain reaction assay and immunostaining of the cells, that the erythropoietin receptor was expressed in cultured hippocampal and cerebral cortical neurons of day 19 rat embryo. Erythropoietin protected the cultured neurons from glutamate neurotoxicity. Neurons cultured for seven to 10 days were exposed to glutamate for 15 min and after culture for a further 24 h in the absence of glutamate the neuron survival was assayed. Significant protection was observed with erythropoietin from 3 pM (c. 100 pg/ml) in a dose-dependent manner. The protection was completely reversed by co-application of a soluble erythropoietin receptor, an extracellular domain capable of binding with erythropoietin. For exhibition of the neuroprotective effect, exposure of neurons to erythropoietin approximately 8 h prior to exposure to glutamate was required. Experiments with the inhibitors indicated that RNA and protein syntheses were necessary for the protection. However, exposure to erythropoietin for a short period (5 min or less) was sufficient to elicit the protective effect. The protective effect of erythropoietin was blocked by the simultaneous addition of EGTA. These findings and the previous finding that erythropoietin induces a rapid and transient increase in intracellular Ca2+ concentration in neuronal cells suggest that erythropoietin plays a neuroprotective role in brain injury caused by hypoxia or ischemia and that erythropoietin-induced Ca2+ influx from outside of the cells is a critical initial event yielding an enhanced resistance of the neurons to glutamate toxicity.

Anti-erythropoietin receptor monoclonal antibody: epitope mapping, quantification of the soluble receptor, and detection of the solubilized transmembrane receptor and the receptor-expressing cells.

A hybridoma cell line producing the monoclonal antibody against erythropoietin receptor (EpoR) was established using the soluble ectodomain of mouse erythropoietin receptor (sEpoR) as an antigen. The monoclonal antibody termed 1G3 bound to the denatured sEpoR. Epitope mapping with peptide library revealed that 1G3 recognized the amino terminal region including the hexapeptide (positions 6 to 11; LeuProAspProLysPhe). The amino acid sequence in this hexapeptide was identical in mice, rats, and humans, and therefore 1G3 bound to EpoR from all of these sources. Using 1G3, we evaluated sEpoR by a sandwich enzyme-linked immunoassay, and EpoR in the solubilized membrane preparation was detected by Western blotting. The cells expressing EpoR were identified with immunochemical staining. We confirmed the presence of EpoR in a neuronal cell line and PC12 cells, and EpoR was expressed in primary cultured hippocampal neurons.

Role of tissue factor in disseminated intravascular coagulation.

We examined plasma antigen levels of tissue factor (TF) in 95 cases of disseminated intravascular coagulation (DIC), to investigate the role of TF in DIC. A significant elevation of plasma antigen levels of TF was observed in cases of DIC associated with cancer. However, no such significant elevation was observed in cases of DIC associated with acute promyelocytic leukemia (APL), acute leukemia except APL, blastic crisis of chronic myelogenous leukemia, non-Hodgkin lymphoma (NHL), sepsis or fulminant hepatitis. No significant elevation of TF was observed in patients without DIC, except 4 cases of cancer who developed DIC thereafter. Plasma antigen levels of TF were higher in both cases of DIC with renal failure and chronic renal failure without DIC than its levels in those without renal failure. Therefore, plasma antigen levels of TF in DIC patients with renal failure were considered to be carefully estimated. The levels of TF were decreased with the clinical improvement in some cases of DIC but were further increased or remained at high levels in patients who showed no improvement of DIC. Thus, plasma antigen levels of TF is an important marker to predict the development and/or prognosis of DIC, especially in patients with cancer.

N-glycosylation-defective receptor for erythropoietin can transduce the ligand-induced cell proliferation signal.

Erythropoietin receptor (EPOR) contains a single N-linked sugar in an extracellular domain. It has been suggested that an erythroleukemia cell line with high sensitivity to EPO expresses a high molecular mass form of EPOR, which appears to be a highly N-glycosylated form responsible for EPO-mediated signal transduction [Sawyer and Hankins (1993) Proc. Natl. Acad. Sci. USA 90, 6849-6853]. To examine the role of the N-linked sugar chain, we prepared EPO-dependent cell lines expressing the wild-type EPOR and N-glycosylation-defective EPOR. There was little difference in the expression of EPOR on the cell surface, EPO binding kinetics, and EPO-induced cell proliferation between the clones expressing the mutant EPOR and those expressing the wild-type EPOR.

Haemostatic and fibrinolytic parameters in septic patients with leukopenia or leukocytosis.

Induction of leukocytopenia by cytotoxic drugs protects against the generalized Shwartzman reaction induced by endotoxin. To elucidate the relationship between leukocyte number and in haemostatic and fibrinolytic disturbances in human sepsis, we studied 32 septic patients with abnormal leukocyte counts. Twenty patients had sepsis in the setting of leukopenia after chemotherapy for haematological malignancies. Twelve patients with leukocytosis developed sepsis associated with benign disorders. Concentrations of thrombin-antithrombin III complex (TAT), plasminogen activator inhibitor-1 (PAI-1) and plasma thrombomodulin (TM) in the leukocytosis group of (12.0 +/- 11.0, 40.2 +/- 27.0 and 5.5 +/- 2.3 ng/ml, respectively) were significantly elevated compared to the leukopenia group of (3.8 +/- 2.3, 18.0 +/- 15.0 and 3.1 +/- 1.0 ng/ml, respectively) and controls (3.3 +/- 0.4, 10.5 +/- 5.3, 3.0 +/- 0.5 ng/ml, respectively). On the other hand, there were no significant differences in these values between leukopenia group and controls. Thus leucocytes may play important roles in thrombin generation, PAI-1 release and injury to endothelial cells.

Recombinant hirudin for the treatment of disseminated intravascular coagulation in patients with haematological malignancy.

The purpose of this pilot study was to determine the effect of recombinant hirudin (r-hirudin) on coagulopathy and the relationship between concentrations of thrombin-antithrombin III (ATIII) complex (TAT) and thrombin-hirudin complex (THC) in patients with disseminated intravascular coagulation (DIC). Five patients with haematological malignancy associated with DIC were studied. r-Hirudin was administered by continuous intravenous infusion at a dose of 0.005 mg/kg/h for 4-9 days to each patient. Fibrin/fibrinogen degradation products (FDP), D-dimer, TAT and plasmin-alpha 2 antiplasmin complex (PAP) concentrations decreased after treatment with r-hirudin in four patients studied. However, in one patient, serum creatinine increased to 1.7 mg/dl and aPTT was prolonged to 74.4s. Statistical analysis disclosed significant positive correlations between plasma concentrations of hirudin and THC, and between concentrations of THC and TAT. The concentrations of THC were much higher than those of TAT. In conclusion, these findings indicate that r-hirudin more strongly inhibited thrombin than did ATIII without heparin, and that administration of r-hirudin to renal insufficiency required individual adjustment of dosage. The present findings also suggest that r-hirudin can be considered a new agent for the treatment of DIC.

Study of the balance between coagulation and fibrinolysis in disseminated intravascular coagulation using molecular markers.

Plasma levels of thrombin-antithrombin III complex (TAT), plasmin-alpha 2-plasmin inhibitor complex (PIC) and active plasminogen activator inhibitor (PAI) were assayed in 66 cases of disseminated intravascular coagulation (DIC). Significant elevation of both TAT and PIC was observed in all cases of DIC. Most elevated levels of TAT were seen in DIC with acute promyelocytic leukaemia (APL) and sepsis. The highest levels of PIC were seen in DIC with APL but were much lower in sepsis. A significant elevation in active PAI was observed in DIC due to acute leukaemia (apart from APL), chronic myeloid leukaemia and sepsis, but not in APL, non-Hodgkin lymphoma and cancer. Active PAI was higher in patients with multiple organ failure (MOF) than in those without MOF while PIC was lower in patients with this complication. Thus, the balance of coagulation and fibrinolysis varied according to the underlying cause of DIC; APL had more dominant activation of fibrinolysis, while sepsis had greater activation of coagulation. It is suggested that the inhibition of secondary fibrinolytic activation plays an important role in the progression of MOF by the disturbance of the microcirculation.

Two genetic defects in a patient with complete deficiency of the b-subunit for coagulation factor XIII.

A genomic DNA obtained from a female patient with complete b-subunit deficiency was examined by Southern blotting analysis and in vitro amplification. Nucleotide sequence analysis showed that adenosine-4161 at the acceptor splice junction of intron A/exon II was deleted in half of the amplified DNAs, resulting in a loss of the obligatory AG splicing sequence. The absence of adenosine-4161 was confirmed by cleavage with TaqI endonuclease of the amplified DNAs. Moreover, sequence analysis showed that guanosine-11499 coding for Cys 430 (TGC) in exon VIII was replaced by thymidine in half of the amplified DNAs, resulting in an amino acid change to Phe (TTC) and the destruction of a disulfide bond in the seventh Sushi domain. This mutation was also confirmed by cleavage with MboII endonuclease. Thus, the proband turned out to be a compound heterozygote of two separate defective alleles. Although half of the amplified DNAs for exon VIII of her daughter or son were cleaved by MboII, those for intron A were not cleaved by TaqI. The replacement of guanosine-11499 by thymidine in their exon VIII has also been confirmed by sequence analysis, indicating that they are heterozygous for one normal and one defective allele.

Changes in plasma levels of prothrombin fragment F 1 + 2 in cases of disseminated intravascular coagulation.

Plasma levels of prothrombin fragment F 1 + 2 (PTF) and thrombin-antithrombin III complex (TAT) were assayed in 86 cases of disseminated intravascular coagulation (DIC). A significant elevation of both parameters was observed in most cases of DIC, which suggested that the in vivo generation of thrombin is highly accelerated by the cleavage of the prothrombin molecule by factor Xa. On the contrary, no significant elevation of plasma levels of PTF was observed in cases of DIC with severe hepatic failure or fulminant hepatitis in spite of significant elevation of TAT. Plasma levels of PTF were directly proportional to those of TAT in 86 cases of DIC as a whole (r = 0.682, p < 0.001). The measurement of both parameters was considered to be useful to estimate the hemostatic activation in DIC.

Role of endothelin in disseminated intravascular coagulation.

We examined the changes in plasma levels of endothelin-1 (ET-1), a potent vasoconstrictor peptide, in 47 cases of disseminated intravascular coagulation (DIC) to investigate the role of ET-1 in DIC and its relation to multiple organ failure (MOF). A significant elevation of plasma levels of ET-1 was observed in some cases of DIC, especially in patients with sepsis, blastic crisis of chronic myelogenous leukemia, and cancer. However, no such significant elevation was observed in patients with acute promyelocytic leukemia (APL), acute leukemias except for APL, or non-Hodgkin lymphoma. Plasma levels of ET-1 were higher in patients with DIC with MOF than in those without MOF. Although the levels of ET-1 were decreased or remained low with clinical improvement in most DIC patients, the levels were further increased or remained high in patients who showed no improvement in DIC. It is suggested that ET-1 must play an important role in further progression of MOF with the vasoconstriction and microcirculatory disorders.

Increased levels of plasma thrombomodulin in chronic myelogenous leukemia.

Circulating blood plasma contains proteinase-degraded forms of thrombomodulin that are soluble. We quantitatively assayed the plasma levels of thrombomodulin in 15 patients with chronic myelogenous leukemia (CML) in chronic phase by method of an enzyme-linked immunosorbent assay using a monoclonal antibody to protease-degraded products of thrombomodulin. Plasma levels of thrombomodulin in patients with CML at diagnosis were significantly increased (19.5 +/- 6.2 ng/ml: means +/- SD) compared with the levels in normal controls (8.0 +/- 1.9 ng/ml, n = 20) (P less than 0.001). Fibrin degradation products (D-dimer), thrombin-antithrombin III complex, and plasmin alpha 2-antiplasmin complex were almost normal, suggesting that intravascular coagulation or plasmin-mediated fibrinolysis little occurred in these patients. On the other hand, the plasma levels of elastase-alpha 1-proteinase inhibitor (E-alpha 1PI) complex, which was the indicator of released leukocyte elastase, were significantly increased in CML (P less than 0.0001). The plasma levels of thrombomodulin and E-alpha 1PI complex were decreased in parallel with decline of leukocyte counts in 10 patients with CML following anti-leukemic therapy. Furthermore, a statistically significant correlation was observed between the plasma levels of thrombomodulin and E-alpha 1PI complex obtained at 39 time points in 15 patients with CML (r = 0.81, P less than 0.001). These results suggest that the increased plasma levels of thrombomodulin in CML may be partly caused by leukocyte elastase, which may split the surface thrombomodulin and release protease-degraded fragments of it into the circulation.

Plasma levels of soluble thrombomodulin increase in cases of disseminated intravascular coagulation with organ failure.

We examined the changes in plasma levels of soluble thrombomodulin in 66 cases of disseminated intravascular coagulation (DIC), to investigate the damage to vascular endothelial cells and its relationship to multiple organ failure. A significant elevation of plasma levels of soluble thrombomodulin was observed in most cases of DIC, especially in patients with sepsis. However, no such significant elevation was observed in patients with acute promyelocytic leukemia. Plasma levels of both soluble thrombomodulin and active plasminogen activator inhibitor were higher in the cases of DIC with multiple organ failure than in those without multiple organ failure. The levels of soluble thrombomodulin were decreased with the clinical improvement in most cases of DIC but were further increased or remained at high levels in patients who showed no improvement of DIC. It was suggested that an increase in soluble thrombomodulin indicates the damage to the vascular endothelial cells in cases of DIC and that the damage to vascular endothelial cells plays some role in further progression of multiple organ failure.

Changes in plasma levels of tissue-plasminogen activator/inhibitor complex and active plasminogen activator inhibitor in patients with disseminated intravascular coagulation.

Plasma levels of tissue-plasminogen activator.plasminogen activator inhibitor (t-PA.PAI) complex and active PAI were assayed in 58 cases of disseminated intravascular coagulation (DIC). A significant elevation of both parameters was observed in most cases of DIC, especially in patients with non-Hodgkin lymphoma, sepsis, or some patients with acute leukemia, but no such elevation was observed in patients with acute promyelocytic leukemia (APL). The levels of both parameters were higher in cases of DIC with multiple organ failure (MOF) than in those without MOF. Since no elevation of t-PA.PAI complex was observed in most cases of APL, t-PA did not seem to play an important role in the activation of fibrinolytic system in APL. Active PAI, which reflects the inhibitory regulation in fibrinolytic system, was considered to play a role in the progression of MOF. Plasma levels of active PAI were low in the cases of APL, which had no complication of MOF.

[A case of invasive thymoma with pulmonary tuberculosis].

A 68-year old male was referred to our hospital for the treatment of pulmonary tuberculosis. Chest X-ray film revealed left diffuse pleural thickening. Treatment of pulmonary tuberculosis was started with streptomycin, isoniazid and rifampicin. Three months later, although smear and culture of the patient's sputum became negative for M. tuberculosis, he started to complain of dyspnea on exercise and left chest pain. Biopsies of a pleural tumor and a left subclavicular lymph node were done and a diagnosis of invasive thymoma with pleural dissemination and bone and lymph node metastasis was established. After three cycles of combination chemotherapy consisting of cyclophosphamide, adriamycin and vincristine, left chest pain disappeared and pleural thickening showed shrinkage.