The central feature of hematopoiesis is life-long, stable cell renewal. This process is supported by hemopoietic stem cells which, in the steady state, appear to be dormant in cell cycling. The recruitment of the dormant stem cells into cell cycle may be promoted by such factors as interleukin (IL)-1, IL-6, granulocyte-colony stimulating factor (G-CSF), and newly discovered IL-11. The effects of IL-1 on stem cells may be indirect. Once the stem cells leave Go and begin proliferation, the subsequet process is characterized by continued proliferation and differentiation. Though several models of stem cell differentiation have been proposed, micromanipulation studies of individual progenitors suggest that the commitment of multipotential progenitors to single lineages is a stochastic process. The proliferation of early hemopoietic progenitors requires the presence of IL-3 and/or IL-4, and the intermediate process appears to be supported by granulocyte/macrophage-CSF (GM-CSF). Once the progenitors are committed to individual lineages, the subsequent maturation process appears to be supported by late-acting, lineage-specific factors such as erythropoietin (erythropoiesis), G-CSF (neutrophil production), and IL-5 (eosinophilopoiesis). Thus, hemopoietic proliferation appears to be regulated by a cascade of factors directed at different developemental stages.

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Information on the presence of specific chromosomal structural abnormalities in certain tumors has been increasing. Although the tumor specific chromosomal abnormalities were deemed important, it was not until the chromosomal location of several oncogenes was determined that the real molecular significance became apparent. It now appears that many of the genes associated with animal tumors are located near specific translocations in human cancers. The specificity of chromosomal changes have not only been used diagnostically and prognostically, but also they present key information for the molecular analysis in determining the nature of the genes of human neoplasia. In recent years, great advances have been made in our understanding of the molecular structure of the specific chromosomal translocations in certain hematologic disorders. The present report will breifly describe chromosomal reaarrangements and how oncogenes or cancer related genes involved can be affected.

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Non-Hodgkin's lymphomas was considered an incurable disease 20 years ago, but it is now possible to administered intensive combination chemotherapy thanks to the development of new anticancer agents and the sophistication of their administration, making it possible to increase both the complete response rate and the 2-year survival rate to more than 80%. The outcome is that more than 50% of cases are now curable. One significant feature of non-Hodgkin's lymphoma in Japan is the fact that the prevalence of T-cell lymphomas, which is unfavorable, is higher than in Western nations. A check of the survival time of non-Hodgkin's lymphomas between the five years ending in 1970 and those beginning in 1971 indicates that it was significantly extended. In cases to which BACOP, HOP and other agents were concurrently administered during the initial course of treatment, the complete response rate increased over 70%, and the median survival time at 45 months, making it possible to hopes on the future development of chemotherapy.

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One of the most important factor of thrombosis is intimal injury. We examined the morphology of thrombogenesis in the large artery using the rabbit as an experimental animal. When the intima was injured by a balloon catheter, we observed the adhesion of platelets onto the denuded area of the intima with various degrees of aggregation. Fibrinogen, fibronectin and vWF were observed on the surface of the aggregated platelets. In the raised thrombi induced by a polyethylene tubing fibrin thread formation was observed among the aggregated platelets. In the injured site showing interruption of internal elastic lamina by polyethylene tubing or wire tubing prominent fibrin formation was observed. The intima showing fibrous thickening which was induced by polyethylene tubing 3 weeks before the second injury also revealed prominent fibrin formation after the second injury. These thickened intima is considered to have an availability of a large amount of tissue factor for the activation of coagulation system than the normal intima.

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It has been generally accepted that platelets play etiological roles for the development of atherosclerosis and arterial thrombosis. Platelet activation may be dependent upon the cytosolic free Ca²⁺ concentration ([Ca²⁺]i), and regulated by PGI₂ and endothelium-derived relaxing factor (EDRF) released by vascular endothelium. We have studied here the effect of endothelial cells (EC) on platelet activation and intracellular Ca²⁺ mobilization. Effluent of non-stimulated EC collumn inhibited thrombin-induced platelet aggregation and intracellular Ca²⁺ mobilization. An addition of this effluent to platelet suspension leaded to increase in intraplatelet cyclic AMP (cAMP) which was inhibited by the treatment of indomethacin to EC, suggesting that this effect was involved in PGI₂ released by EC. On the other hand, effluent of thimerosal-stimulated EC collumn inhibited platelet aggregation and increase in [Ca²⁺]i stimulated with thrombin, and leaded to increase in intraplatelet cyclic GMP (cGMP). But the treatment of indomethacin to EC had no effect of this inhibition. The effect of thimerosal-stimulated EC was inhibited by the addition of 1-N_G-monomethylarginine (NMA), EDRF/NO inhibitor, suggesting that EDRF released by thimerosal-stimulated EC produced an increase in cGMP and inhibited platelet activation. Although forskolin-induced in cAMP caused a marked prevention of inositol 1, 4, 5-trisphosphate (IP₃) production stimulated with thrombin, 8-bromo cGMP and EDRF-induced increase in cGMP had no effect of IP₃ production. An increase in cAMP and cGMP was considered to inhibit intracellular Ca²⁺ mobilization by different mechanisms in platelets.

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von Willebrand factor (vWF), a macromolecular glycoprotein synthesized in endothelial cells and megakayocytes ciculates in blood as heterogenous multimers and plays a critical role in the formation of platelet plugs. vWF is composed of an identical subunit with a molecular weight (MW) of 270 kDa, which is held together by disulfide bonds. Functional domains of vWF for factor VIII, heparin, platelet glycoprotein (GP) Ib, collagen, and GP IIb/IIIa locate in this order from the N-to C-terminus of subunit. The GP Ib binding domain is cyptic in normal circulation and only becomes expressed when vWF is associated with the subendothelial matrix or with fibrin. In vitro, an antibiotic ristocetin or the snake venom botrocetin isolated from Bothrops jararaca mimics the active component of subendothelial matrix and causes the binding of vWF to GP Ib.
To elucidate the mechanism of in vitro process of vWF-GPIb binding, we describe here the isolation and characterization of two distinct forms of botrocetin. Since ristocetin is a heavily glycosylated proteoid, it is hard to characterize the structure on the basis of amino acid analysis. The apparent MW of the one-chain botrocetin was 28 kDa before and 32 kDa after reduction of disulfide bonds, while that of the two-chain botrocetin was 27 kDa before and 15/14.5 kDa after reduction. Amino acid composition of the two species revealed a similar high content of potentially acidic residues (greater that 60 Asx and Glx residues/molecules) but significant differences in the content of Cys and Phe residues. The N-terminal sequence of the one-chain was Ile-Ile/Val-Ser-Pro-Pro-Val-Cys-Gly-Asn-Glu-. Two constituent polypeptides of the two-chain botrocetin showed similar but different N-terminal sequences, distinct from that of the one-chain species: (α) Asp-Cys-Pro-Ser-Gly-Try-Ser-Ser-Tyr-Glu and (β) Asp-Cys-Pro-Pro-Asp-Trp-Ser-Ser-Tyr-Glu-. The carbohydrate content of the both species was less than 2% of the total mass. Both the one- and two-chain botrocetins bound to purified vWF and formed an activated complex, which then bound to GPIb. On a weight basis, the two-chain botrocetin was 34 times more active than the one-chain form in promoting vWF binding to platelets. Furthermore, when the concentration of botrocetin was not limiting, the parameters of vWF binding to platelets were identical with either species. These results provide an initial chemical and functional characterization of two-distinct forms of botrocetin.

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Antithrombin III (AT III) has been confirmed to play an important role as a serine protease inhibitor in the mechanism of blood coagulation, and its deficiency or abnormality is found to cause thromboembolic disorders by reducing the anticoagulant activity. In this paper AT III gene of patient with congenital AT III deficiency, which was suggested to have qualitative abnormality by isoelectric focusing, was investigated. Analysis of the genomic structure by Southern blot hybridization with a cDNA probe (pAT6) revealed no detectable changes indicating any deletions, rearragements and translocations. Therefore, we focused to analyze the sequence of exon 6 of AT III gene by polymerase chain reaction (PCR) methods followed by direct sequencing analysis. Nucleotide sequencing of exon 6 of AT III gene showed a G to T transitional mutation resulting in the conversion of arginine-406 to methionine coexisted with normal allele which encodes arginine. The mutation is located near the reactive center of AT III molecule, which region has been proved to be highly conserved during the evolution of serine pretease inhibitor (serpin) family. From these results, it is concluded tha the new type of mutation at amino acid site 407, which is similar to AT III Utah, is important for maintaining the structural and biological function of this inhibitor.

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The endothelial cells (ECs) are antithrombotic in the physiological states and maintains the integrity of blood circulation. However, ECs turn to be thrombotic upon being stimulated by various physiological mediators. These functions are mainly achieved by changing specific protein synthesis in ECs. Type 1 plasminogen activator inhibitor (PAI-1) is a serine protease inhibitor synthesized by ECs and thought to play a crucial role in the regulation of fibrinolysis. Basic research as well as clinical studies support this hypothesis. PAI-1 is a physiological inhibitor of both tissue-type plasminogen activator and urokinase-type plasminogen activator, key enzymes in the initiation of fibrinolysis. Thus PAI-1 regulates not only blood clot lysis but also a wide variety of biological reactions occurring in extracellular matrices such as tumor metastasis, neovascularization, inflammation, and cell migration. PAI-1 is a glycoprotein, of which molecular weight is approximately 50,000. Molecular biological analyses indicate that PAI-1 is synthesized as a single polypeptide composed of 402 amino acids containing a signal peptide. After post-translational modification, PAI-1 is secreted from ECs as a polypeptide composed of 379 amino acids and three N-linked carbohydrates. PAI-1 lacks Cys residues, indicating that PAI-1 may not be rigid and thus thermolabie. In fact, PAI-1 is unstable even at 37°C decaying into an inactive form with a biological half life of 2-3 hours. PAI-1 binds to a cell adhesion molecule, vitronectin. The association of PAI-1 with vitronectin appears to stabilize PAI-1. PAI-1 in complex with vitronectin is still accessible to plasminogen activators. These facts suggest that PAI-1 may play a role in cell adhesion and in wound healing. Since PAI-1 is the primary regulator of fibrinolysis, changes of the level of PAI-1 may influence the fibrinolytic activity in blood and in tissue. Inflammatory mediators (TNF, IL-1), growth factors (TGFβ, basic FGF), bacterial endotoxin are known to stimulate PAI-1 biosynthesis in ECs dramatically. Thus the level of PAI-1 may change in a variety of physiological and pathological states, and thereby modulate fibrinolysis-dependent biological phenomenons.

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Differentiation-linked expression of plasminogen activator inhibitor-2 (PAI-2) was investigated by adding cell-differentiation promoting agents [such as phorbol myristate acetate (PMA), retinoic acid, dexamethasone (Dex), and recombinant cytokines, including tumor necrosis factor-α (TNF-α), transforming growth factor-β, granulocyte-colony stimulating factor, and interleukin-6 (IL-6)] into the culture medium of a promyelocytic leukemia cell line PL-21. PAI activity both in the culture medium and in the cell lysate increased approximately 70-fold after exposure to PMA. Both PAI-1 and PAI-2 antigens increased, but the amounts of the latter in the culture medium and in the cell lysate were approximately 10 times and 2,500 times, as much, respectively, as those of the former. Dex also increased the intracellular PAI activity approximately 6-fold, parallel with PAI-2 antigen. PAI-1 antigen increased only slightly in the culture medium but not in the cell lysate after Dex-stimulation. As with the case of PMA, TNF-α and IL-6 induced PL-21 cells to macrophage-like cells, but did not affect the PAI activity. Thus, the increase of the PAI-2 production by PMA may not necessarily depend on differentiation into macrophages. Other cytokines examined did not increase the PAI activity. PAI-2 antigen was demonstrated in the cell lysates of various leukemia cells by Western blotting technique using a monoclonal antibody against the PAI-2 purified from PL-21 culture medium. PAI-2 antigen was frequently detected in the plasmas from the patients whose peripheral leukocytes were more than 10,000/μl.

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Antithrombotic effect of recombinant human thrombomodulin (TM) on experimentally induced thrombosis in mice was studied. The soluble recombinant human TM (TMD 123), which was expressed in Chinese hamster ovary cells and purified from the conditioned medium, prolonged thrombin clotting time for mouse plasma in a dose-dependent fashion. Thrombosis was induced by the injection of lipopolysaccharide (LPS) from E. coli into a lateral tail vein of mice and was identified as disseminated intravascular coagulation (DIC) syndrome by hematological and histological examinations. Simultaneous injection of TMD 123 with LPS into another lateral tail vein of mice significantly corrected the hematological abnormalities compatible with DIC, and also corrected the histological abnormalities i.e. fibrin deposition and decrease of cellular TM in the target organs including kidney, liver and lung. These results indicate that recombinant human TM is a potent antithrombotic agent and that this would be an expectative anticoagulant for DIC or thrombosis in man.

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Treatment results in the 61 adult patients with AML in first relapse were analyzed to establish a better strategy for this group of patients. These patients received reinduction chemotherapy during 1979∼1988. Complete remission (CR) was obtained in 57.4% of the cases, and the probability of survival and remaining in CR at five years was 11.9% and 17.9%, respectively. The longer duration of initial remission was favorable factor for achieving second CR. Type of reinduction regimens which were different from those used in the initial induction phase did not influence the second CR rate. The use of different consolidation regimen appeared to favorably affect the survival and probability of remaining in CR.

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The influence of bone marrow fiborblasts from healthy subjects on differentiation and clonal proliferation of HL-60 cells was studied. Clonal proliferation of HL-60 cells was examined by using agar culture method, DNA synthesis was done by counting ³H-thymidine incorporation into the cells, and differentiation of the cells was checked by non-specific esterase staining. The bone marrow fibroblasts and their conditioned-medium significantly stimulated colony formation and DNA synthesis of HL-60 cells, but they inhibited differentiation of the HL-60 cells. From these results, it was suggested that the bone marrow fibroblasts stimulated clonal proliferation of HL-60 cells and inhibited differentiation of the cells through humoral factors secreted by the fibroblasts.

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Thirty-six patients with chronic myeloproliferative disorders (CMPD) were studied as regards blood coagulation and fibrinolysis. These studies revealed various mild abnormalities: activated thromboplastin time (APTT) tended to prolong and the level of factor V decreased significantly. In several cases, the levels of D-dimer, thrombin-antithrombin III complex and plasmin-α₂-plasmin inhibitor complex were elevated compared to normal. These results suggest that abnormal coagulation system in the patients with CMPD is related to low grade disseminated intravascular coagulation. Many coagulation factors did not correlate with peripheral blood cell counts. Two patients with polycythemia vera were evaluated for several abnormalities of the coagulation system before and during treatment. Coagulation abnormalities persisited after hematologic control had been achieved. Our results suggest that patients with CMPD have a chronic state of abnormal blood coagulation system even after normalization of blood cell counts.

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We reported rare manifestations of adult T cell lymphoma/leukemia. Patient was 41 year-old woman who has had progressive alopecia from childhood and developed multiple huge tumors (largest 10×15 cm) of scalp and wide destruction of cerebral bone at age of 39. A few abnormal lymphocytes (10%) appeared in the peripheral smear. Surface markers of peripheral lymphocytes showed two populations of CD4+/CD8- and CD4+/CD8+. HTLV-1 proviral DNA was demonstrated in the peripheral lymphocytes. Serum anti-ATLA antibodies was positive (X80). Biopsy of tumor revealed diffuse and large cell type of malignant lymphoma. Magnetic resonance imaging of head showed that tumors lysed the bone and invated into cerebral regions. She was treated with CHOP regimen and irradiation, resulting of decreased tumor size and population of CD4+/CD8+.

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A 63 year-old male, who was first diagnosed as primary macroglobulinemia (IgM-κ type) developed non-Hodgin's lymphoma after 10 month clinical course. He had huge, multiple intrahepatic nodular lesions and had consciousness disturbance due to marked hypercalcemia. Since the treatment with gluco-corticoid and caltitonin was not effective for the improvement of patient's general condition, calcium-free hemodialysis was performed. After 2-hour dialysis, serum-Ca level was decreased from 15.2 mg/dl to 10.0 mg/dl. Histology of the aspiration biopsy specimen obtained from the liver showed malignant lymphoma, diffuse, large cell type (B cell origin). Combined chemotherapy (CHOP) was started and was quite effective not only for the regression of the primary lesions but also for the normalization of the serum-Ca level. The existence of PTH-like substance produced by the tumor cell was suspected and may be related to the hypercalcemia in this case. Finally, our results demonstrated that calcium-free hemodialysis is safe and highly effective for the management of hypercalcemia caused by malignancy.

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A 61-year-old male was admitted to our hospital because of progressive bilateral exophthalmos and visual disturbance. He was diagnosed as chronic lymphocytic leukemia (CLL) with stage I of Rai system. Ophthalmologic examinations suggested that CLL cells might have invaded diffusely to bilateral orbits. Radiation to orbital lesions might result in other ophthalmologic complications such as cataract, therefore we tried to treat him with chemotherapy alone. As a result, combination chemotherapy consisting of vincristine, cyclophosphamide, prednisolone and doxorubicin (VEPA) and additional daily oral administration of cyclophosphamide were effective enough for his ophthalmologic recovery.

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