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article i nfo Introduction: We assessed the safety and effectiveness of recombinant soluble thrombomodulin (thrombomodulin alfa, TM-α) in the treatment of disseminated intravascular coagulation (DIC) in a post-marketing surveillance. Methods: The cases of 3548 patients with DIC caused by infection (n = 2516, Infection-DIC) or hematological malignancy (n = 1032, Hemat-DIC) were analyzed and compared to the results of a phase III (P-III) study. Results: The DIC scores were significantly decreased in the Infection-DIC and Hemat-DIC groups with TM-α treat- ment (both P b 0.001). The incidences of critical bleeding adverse drug reactions (ADRs) in the Infection-DIC and Hemat-DIC groups were 2.6% and 2.4%, and the survival rates were 64.1% and 70.7%, respectively. Patients with DIC were subcategorized into three groups (Infection-DIC-1 or Hemat-DIC-1, P-III criteria-matched patients; Infection-DIC-2 or Hemat-DIC-2, P-III criteria-non-matched patients treated solely with TM-α ;a nd Infection-DIC-3 or Hemat-DIC-3, P-III criteria-non-matched patients treated with TM-α and other concomitant anticoagulants). Subcategory analysis revealed that the incidences of critical bleeding ADRs of Hemat-DIC-2 and Hemat-DIC-3 were significantly higher and their survival rates were significantly lower than those of Hemat-DIC-1. By multivariate analysis in Hemat-DIC, younger age (odds ratio: 2.629, P = 0.0033) and pre- existing bleeding (odds ratio: 2.044, P = 0.019) were found to affect bleeding ADRs and the severity of underlying disease was the most important factor for survival rate (odds ratio: 0.288, P b 0.001). Conclusions:This surveillance provided real-world data for the safety and effectiveness of TM-α in the treatment of Infection-DIC and Hemat-DIC in general practice settings.

Introduction: Vascular endothelial cell damage plays an important role in microvascular thrombogenesis. In vivo administration of cyclosporin A or mitomycin C sometimes results in thrombotic microangiopathy in patients. Materials and methods: The effects of cyclosporin A, mitomycin C and/or prednisolone on the cell cycle in cultured human umbilical vein endothelial cells were investigated to evaluate drug-induced endothelial cell damage and the protective effect of prednisolone on endothelial cells against the damage by cyclosporin A or mitomycin C in vitro. Results: The addition of cyclosporin A to cultures caused proliferation arrest in the G1-phase in a dose-dependent manner, while mitomycin C inhibited DNA synthesis, which resulted in cell cycle arrest and inhibition of BrdUrd incorporation in the S-phase. The administration of prednisolone also caused cell cycle arrest in the G1 by itself, and protected the cells from the damage caused by mitomycin C. The inhibitory effects of cyclosporin A and prednisolone on the cell cycle were reversible, while mitomycin C was not. The highly phosphorylated retinoblastoma protein expressed in human umbilical vein endothelial cells decreased in the presence of mitomycin C. Soluble thrombomodulin levels in the culture supernatant were elevated by the addition of cyclosporin A. Conclusion: These effects of the drugs may cause the cell cycle arrest and the prolonged repair of damaged endothelial cells in patients. D 2004 Elsevier Ltd. All rights reserved.

A patient presented with chronic large granular lymphocyte leukemia associated with chronic active Epstein-Barr virus infection (CAEBV). Cell cycle analysis revealed a minimal growth compatible with chronic lymphocytic leukemia After 5 months of treatment, the patient died from acute transformation of the leukemia. Cell harvested during chronic phase were analyzed for sensitivity to interleukin 2 (IL-2) and interferon alpha (IFN alpha) in vitro by means of surface phenotyping and cell cycle assay. IL-2 induced remarkable growth of the cells, whereas IFN alpha did not confer a growth advantage. Since IFN alpha was expected to have no growth induction effect on the leukemia cells, it was administered to the patient to treat the CAEBV.

Serum thrombomodulin (sTM) is an endothelial cell marker which would reflect the endothelial damage. In order to examine whether some antiplatelet agents decrease the endothelial damage in diabetic patients, sTM concentrations were measured by enzyme-linked immunosorbent assay before and after oral administration of a daily 100 mg of cilostazol for 4 weeks in 9 diabetics or a daily 81 mg of acetyl salicylic acids (ASA) for 4 weeks in 8 diabetics. Basal concentrations of sTM were elevated in most of these patients as compared with healthy subjects. The sTM concentrations were decreased after administration of cilostazol from 28.1 +/- 7.1 ng/ml to 23.6 +/- 5.4 ng/ml (p0.01), and after ASA from 30.7 +/- 10.9 ng/ml to 27.9 +/- 11.6 ng/ml (p0.05). These results suggest that such drugs can decrease the endothelial damage, resulting in the reduced risk of diabetic vascular complications.

Abnormal factor IX was isolated from the plasma of a patient with hemophilia B Kashihara and two patients with hemophilia BM. The F.IX was purified to homogeneity by using monoclonal anti-F.IX-Sephrose, heparin-Sepharose and DEAE-Sephadex A-50 affinity chromatography successively. The isolated proteins have the same molecular weight and the same mobility on crossed immunoelectrophoresis as normal F.IX. The limited proteolysis of purified proteins was induced by F.XIa/Ca 2+ or by RVV-X/ Ca 2+ . A time course study showed that F.IX Nagoya seemed to be cleaved by neither F.XIa nor RVV-X, F.IX Kashihara was cleaved partially by F.XIa but not by RVV-X, and that F.IX Niigata was cleaved completely at the rate similar to normal F.IX, though the resultant product of F.IX Niigata did not show any F.IXa activity. These results favored the view that hemophilia B or BM is a heterogeneous disorder.

Both anticoagulant and amidolytic activities of protein C (PC) were measured using a snake venom activator in 4 patients with hereditary PC deficiency, 37 with disseminated intravascular coagulation (DIC), and 30 under stabilized warfarin therapy. The results were compared with those obtained by an immunological assay (ELISA). PC levels measured by different functional and immunological assays were very close in patients with hereditary PC deficiency and DIC. In patients under stable oral anticoagulant therapy, there was no detectable difference between amidolytic activity and antigen levels of PC in each patient plasma, whereas a decrease in anticoagulant activity was much more pronounced. These results indicate that the present activity assays measure PC specifically, and that the snake venom activator is capable of activating both carboxylated and hypocarboxylated forms of PC, but only anticoagulant assay can evaluate the physiological PC function in vitamin K-deficient states.

Blood platelets from patients with platelet-type von Willebrand disease (vWD) aggregate upon the addition of human von Willebrand factor (vWf) in the absence of ristocetin or other stimulating factors. We measured quantitatively the ability of vWf to induce directly aggregation of platelet-type vWD platelets (platelet-type vWD platelet aggregating factor [PT-PAF]). Cryoprecipitate and factor VIII concentrates were used as a source of vWf of various multimeric composition. The PT-PAF activity was dependent on the multimer size of vWf, like in the case of ristocetin cofactor (RCof) activity. However, PT-PAF activity was not equivalent to RCof activity and the relative PT-PAF/RCof ratio ranged from 1.00 to 0.18 in the materials studied. The preparations containing the higher-molecular-weight multimers had higher PT-PAF/RCof ratio. These findings suggest that PT-PAF activity is a functional expression of more highly polymerized multimers of vWf as compared with RCof activity. Measurement of PT-PAF would serve as a novel functional assay of vWf.

We studied the multimeric composition of plasma von Willebrand factor (vWf) in 26 patients with chronic myelocytic leukaemia (CML); 13 in chronic phase, 8 in blast crisis, 5 in both chronic phase and blast crisis. The relative amount of large (multimer band greater than or equal to 11) multimers calculated by densitometer scan following SDS-agarose gel electrophoresis was 14.6 +/- 2.9% (mean +/- SD) in normal controls, 8.7 +/- 4.7% in chronic phase and 15.0 +/- 5.2% in blast crisis. CML patients in chronic phase (but not in blast crisis) had a significantly lower percentage of large multimers than normal controls (p less than 0.001). The relative amount of large multimers was positively correlated with a ratio of ristocetin cofactor/vWf antigen (p less than 0.005), and was negatively correlated with platelet count (p less than 0.001), WBC count (p less than 0.05) and granulocyte count (p less than 0.05). We conclude that some patients with CML, especially in chronic phase, lack large multimers. The negative correlation of the relative amount of large multimers with platelet and granulocyte count suggests that large multimers may be consumed in high platelet count states or degraded by protease(s) from increased blood cells.

The binding of factor VIII to platelets in the presence of ristocetin was investigated in four patients with von Willebrand's disease that showed an increased ristocetin-induced platelet aggregation (RIPA). The binding of plasma factor VIII-related antigen (VIIIR:Ag) from these patients to normal washed or formalin-fixed platelets was decreased in three patients and was normal in one patient. Washed platelets prepared from these patients, however, showed an increased binding affinity for normal VIIIR:Ag. It was suggested that the increased RIPA was brought about by an increased ristocetin-induced binding affinity of patients' platelets for factor VIII.

Plasmin generation in vivo was assessed by measuring plasma levels of plasmin-a2-plasmin inhibitor complex (PAP) with an ELISA in 42 patients with arterial or venous thromboembolic diseases. Plasma concentration of PAP was markedly elevated in patients with venous thromboembolic diseases during acute illness (3.32 +/- 3.71 ug/ml, mean +/- SD) as compared to healthy subjects (0.24 +/- 0.13 ug/ml, n = 14), while it was nearly normal (0.30 +/- 0.13 ug/ml) in patients with venous thromboembolic diseases in chronic stage. Patients with arterial thromboembolism had modestly elevated PAP; 1.05 +/- 0.77 ug/ml during acute episode, and 0.84 +/- 0.40 ug/ml in chronic stage. These results indicate that excessive activation of fibrinolytic system (plasmin generation in vivo) occurs actually in many patients with thrombotic diseases, especially in venous thromboembolic diseases during acute illness.

To evaluate the usefulness of measuring circulating endothelial cells as an in vivo indicator for vascular injury, studies were performed in vitro using cultured human endothelial cells and Factor VIII-related antigen (VIIIR:Ag) as the specific cell marker. Cultured human endothelial cells mixed with normal whole blood were processed by Ficoll-Hypaque gradient sedimentation to remove red cells and granulocytes, followed by pellet formation of the mononuclear supernatant fraction to separate the bulk of platelets. Cell films, prepared from a measured volume of the final cell suspension, were stained with fluorescent antibody against VIII R:Ag, and positively stained cells were counted under fluorescence microscopy. The final recovery of endothelial cells was 64.8 +/- 19.3% (n = 41), and the minimal detectable concentration of endothelial cells was about 0.02 cells/microliter whole blood. No endothelial cells were detected in the blood of normal individuals.