Nihon Ketsueki Gakkai zasshi : journal of Japan Haematological Society最新文献

When aequorin-loaded platelets were stimulated with thrombin, the luminescence of aequorin showed two peaks. From experiments with 1 mM external Ca2+ or 1 mM EGTA, both one-half of the first peak and the entire second peak reflected the influx of Ca2+ from the external medium, and the remaining half of the first peak reflected the mobilization of Ca2+ from its storage site. A monoclonal antibody (TM83), that recognizes the GPIIb/IIIa complex which has binding sites for fibrinogen, and synthetic peptide GRGDSP are known to inhibit fibrinogen binding and platelet aggregation. Both of them eliminated the second peak of intracellular free calcium. Similar effects were observed during activation by collagen, but not by TPA. Also dihydrocytochalasin B inhibited the second peak of Ca2+ influx by thrombin, suggesting that the signal, which was caused by fibrinogen-binding to GPIIb/IIIa (aggregation) in thrombin-activated platelets, is transferred to the inner sites of GPIIb/IIIa complex and induces the cytoskeletal reorganization such as actin polymerization. This in turn, induces the secondary increase in [Ca2+] i of platelets. It is interesting that ticlopidine inhibited the Ca2+ influx through the GPIIb/IIIa complex. This result suggests the importance of such kinds of antiplatelet drugs to prevent thrombus formation.

Anticoagulantly active heparin-like glycosaminoglycans are apparently present on the vascular surface. We have tried to modulate heparin-like substances on endothelial cells using an experimental cell culture system. Perturbation of the endothelial proteoglycan metabolism by beta-D-xyloside resulted in a reduced biosynthesis of cell surface heparan sulfate, and impaired antithrombin III binding to endothelial cells in parallel with an inhibition of endothelial cell heparin-like activity. In a separate series of experiments, treatments of endothelial cells with interleukin 1 beta and tumor necrosis factor alpha, physiological mediators of immunologic and inflammatory responses, were shown to cause an inhibition of the synthesis of endothelial cell surface heparan sulfate. The endothelial heparin-like activity was partially diminished by these cytokines, suggesting that cytokine-mediated suppression of heparin-like substance on endothelial cells is another cytokine-inducible endothelial effect affecting coagulation. The modulation of endothelial heparin-like activity by these pharmacological and physiological agents may have pathophysiological implications in thrombosis.

The organization of immunoglobulin heavy chain (IgH) gene of B precursor cell acute lymphoblastic leukemia (ALL) was examined in order to elucidate the mechanism causing simultaneous TCR gene rearrangements. Study using a 5'D probe lying 20 kb upstream of IgH D region genes was useful to distinguish diversity(D)-joining(J) recombination(DJ) from variable(V)-DJ recombination(VDJ). Indeed, IgH gene structures determined by 5'D study well correlated to those recognized by DJ or VDJ transcripts. IgH gene rearrangements of B precursor cell ALL showed developmentally restricted gene recombination; DJ/DJ genotype in the most immature stage and VDJ/VDJ genotype in the relatively mature stage. B precursor cell ALL with simultaneous rearrangements were frequently found in relatively mature cells, i.e., CD20 expressing cells on their surface. Furthermore, most of such dual genotypic ALL showed that at least one allele of IgH genes was VDJ recombination. This finding suggests that dual genotype was the incidental product by a putative common recombinase during the process of VH gene rearrangements. Moreover, since IgH gene rearrangements of acute unclassified leukemia with dual genotype were DQ52 genotype, which indicates abortive gene rearrangements, it was also thought that dual genotype occurred due to the pluripotentiality of the stem cell.

A survey of the results of marrow transplantation for severe aplastic anemia (SAA) and myelodysplastic syndrome (MDS) in Japan is reported. Of the 152 patients with SAA, 109 were alive between 2 and 132 months following transplantation and the probability of survival at 5 years was 70% (for the patients with grafts from HLA-matched siblings) and 100% (for the patients with grafts from monozygous twins). Survival rate at 3 years for the patients with grafts from family members other than HLA-matched siblings was 46%. The chance of survival was influenced by conditioning regimen and recipient's age. Recipients with sustained engraftment had a significantly higher survival rate than those with graft failure (83% vs 11%, p less than 0.001). Since 1985, the results of transplantation from HLA-matched siblings have improved and the 3-year survival is more than 90% for patients under 20 years old. For MDS, the actuarial survival at 3 years was 42%. The chance of survival was not influenced by the FAB classification, patient's age, patient's sex, interval from diagnosis to transplant, karyotype anomaly or graft versus host disease.

The clonal composition of each cell population was determined from the characteristic methylation pattern of DNA and the restriction fragment length polymorphism (RFLP) of the hypoxanthine phosphoribosyltransferase (HPRT) and phosphoglycerate kinase (PGK) genes, both located on the X chromosome. About 71% of Japanese females are heterozygous in terms of the RFLP of either HPRT or PGK genes, which was demonstrated by using 5' genomic DNA or cDNA probes for these genes. All 3 cases of chronic myeloproliferative disorders showed monoclonal patterns. AML or ALL cases demonstrated either monoclonal or polyclonal patterns depending upon the percentage of blastic cells. Monoclonal patterns were seen in 3 of 4 cases of myelodysplastic syndromes and both PNH cases.

A multicenter prospective study on the treatment of chronic idiopathic thrombocytopenic purpura (ITP), conducted by the Idiopathic Disorders of Hematopoietic Organ Research Committee, the Ministry of Health and Welfare of Japan, is currently in progress. In this study we analyzed the clinical records of 256 patients with chronic ITP in order to define the prognostic factors. As of November, 1988 after a median observation period of 34 months, 174 of the 256 patients (68%) were alive, 11 (4%) dead and 71 (28%) lost to follow-up. Bleeding was a direct cause of death in only one patient. Assessment of the status of patients based on platelet count at the final observation revealed that 48% of patients were in remission, 21% showed improvement, and 31% remained unchanged or worsened. Univariate analyses identified 4 parameters associated with favorable prognosis: presenting platelet count less than 2 x 10(4)/microliters, platelet count greater than 10 X 10(4)/microliters after one-year follow-up, maximal platelet count greater than 10 X 10(4)/microliters during administration of the initial dose of corticosteroids and splenectomy.

The incidence of gene amplification in leukemia/lymphoma is significantly lower than that in solid tumors. For understanding the biological characteristics of leukemia/lymphoma, it is necessary to clarify whether the incidence of gene amplification is actually lower or gene amplification specific to hemopoietic malignancies has not yet been found. We describe a new version of in-gel DNA renaturation technique for general screening of gene amplification that allows detection and cloning of amplified genes of unknown nature as few as 7- to 8-fold amplification.

Types II and III protein kinase C were expressed in human platelets and showed slightly different modes of activation and kinetic properties. Type III isozyme was more sensitive than type II for the activation of each isozyme with arachidonate (AA) although both isozymes were activated by diacylglycerol and phosphatidylserine in a similar manner. When human platelets were stimulated by AA, two types of platelet activation, a low level of AA (0.1-2.5 micrograms/ml)- and a high level of AA (10-50 micrograms/ml)-induced activations, were observed. These activations were associated with the phosphorylation of 40K and 20K proteins. Although a low level of AA (0.45-10.0 micrograms/ml) induced the formation of [32P] phosphatidate in intact platelets prelabeled with [32P] Pi, AA at high concentrations (20-50 micrograms/ml) did not stimulate phospholipase C. The incubation of fura 2 loaded platelets with a low level of AA evoked a rapid and transient elevation in [Ca2+] i. In contrast, a high level of AA induced an irreversible increase in [Ca2+] i but this [Ca2+] i elevation alone was not associated with platelet activation. These results suggest that the signal transduction system by a high level of AA on human platelets is different from that seen with a low level of AA. A high level of AA induces platelet activation, without phospholipase C stimulation, and therefore, the ability of AA to directly activate protein kinase C (pre-dominantly type III isozyme) may contribute toward the activation of platelets by a high level of AA.

The explosive nature of the coagulation cascade led many scientists to investigate how it is regulated. Proteinase inhibitors such as antithrombin III inhibit active proteases of the coagulation cascade. Cofactors such as factor Va and factor VIIIa are proteolytically inactivated by activated protein C. Protein C is activated by the thrombin-thrombomodulin complex on the endothelial cell surface. Thus, the independent actions of the proteinase inhibitor system and the thrombomodulin-protein C system complement each other to maintain regulation of blood coagulation. The thrombin binding site of thrombomodulin was identified to be the fifth and sixth repeats of the epidermal growth factor-like domain. The same binding template contains sufficient information to block the functions of thrombin as a procoagulant. However, additional repeats are required for the activation of protein C. Thrombomodulin is the first example which illustrates that the epidermal growth factor-like domain functions as a binding template for thrombin and as a switch to turn off the procoagulant activity of thrombin as well as to trigger the protein C anticoagulant pathway. Epidermal growth factor-like structures are found in many of the coagulation factors. Complex formation is a repeated theme not only in the blood coagulation cascade but also in the thrombomodulin-protein C anticoagulant pathway.