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Shogo Tamura
Faculty of Health Sciences　Health Sciences　Medical Laboratory Science
Associate Professor
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Last Updated :2024/05/20

Researcher Profile and Settings

Affiliation

  • Faculty of Health Sciences　Health Sciences　Medical Laboratory Science

Job Title

  • Associate Professor

URL

Research funding number

  • 60722626

J-Global ID

Research Interests

  • 巨核球   骨髄造血微小環境   オルガノイド   血栓止血学（血小板・出血性・血栓性素因）   先天性血液凝固異常症   

Research Areas

  • Life sciences / Biomedical engineering
  • Life sciences / Medical biochemistry
  • Life sciences / Hematology and oncology

Educational Organization

Academic & Professional Experience

  • 2022/04 - Today Hokkaido University Faculty of Health Sciences Associate professor
  • 2020/04 - 2022/03 Nagoya University Graduate School of Medicine Lecturer
  • 2020/03 - 2020/03 Nagoya University Graduate School of Medicine Department of Radiological and Medical Laboratory Sciences Pathophysiological Laboratory Sciences Lecturer
  • 2015/10 - 2020/02 Nagoya University Graduate School of Medicine Department of Radiological and Medical Laboratory Sciences Pathophysiological Laboratory Sciences Assistant Professor
  • 2013/04 - 2015/09 日本学術振興会特別研究員PD (山梨大学医学部臨床検査医学講座)
  • 2011/04 - 2013/03 旭川大学保健福祉学部保健看護学科 非常勤講師
  • 2010/04 - 2013/03 日本学術振興会特別研究員DC1 (北海道大学大学院保健科学院)
  • 2006/07 - 2010/03 KKR札幌医療センター 非常勤臨床検査技師

Education

  •        - 2013/03  Hokkaido University  Graduate School of Health Sciences
  •        - 2010/03  Hokkaido University  Graduate School of Health Sciences
  •        - 2008/03  Hokkaido University  Faculty of Medicine
  •        - 2006/03  Hokkaido University

Association Memberships

  • 日本検査血液学会   日本血栓止血学会   日本血液学会   International Society of Thrombosis and Hemostasis   

Research Activities

Published Papers

  • Atsuo Suzuki, Nobuaki Suzuki, Takeshi Kanematsu, Shuichi Okamoto, Naruko Suzuki, Shogo Tamura, Ryosuke Kikuchi, Akira Katsumi, Tetsuhito Kojima, Tadashi Matsushita
    International journal of laboratory hematology 2024/03/01 
    INTRODUCTION: An investigation of the suitability of reagents for measuring FVIII products in a one-stage clotting assay (OSA) showed variations in their FVIII activity (FVIII:C). Most studies have focused on the activated partial thromboplastin time (APTT) reagent rather than FVIII-deficient plasma (F8DP), even though the APTT-based OSA is comprised of APTT reagents and factor-deficient plasma. AIM: A single-centre study was conducted to clarify variations in measurements of FVIII products in an OSA using a total of 12 reagent combinations, including four APTT reagents and three types of F8DP. METHODS: FVIII:C in nine types of FVIII product-spiked plasma was measured using an OSA with four different APTT reagents and three types of F8DP. RESULTS: F8DP-dependent variations were found in addition to differences derived from APTT reagents. Variations in target recovery (TR) were observed for NovoEight®, Eloctate®, and Jivi®. Reduced TR for Jivi was found only for Pathromtin SL in combination with congenital F8DP (F8DP-3). This lower TR was not observed with alternative manufacturing lots of F8DP-3. The reduced TR for Jivi might be related to impaired contact activation due to lower factor XI activity in F8DP-3. CONCLUSION: In addition to APTT reagents, variations in F8DPs used for OSAs can also affect FVIII:C results. F8DPs as well as the APTT reagent used for OSA should be chosen with caution, and laboratories should evaluate reagents for F8DPs as they currently do for APTT reagents, especially when lot changes occur.
  • Hinako Notoh, Satoshi Yamasaki, Nobuaki Suzuki, Atsuo Suzuki, Shuichi Okamoto, Takeshi Kanematsu, Naruko Suzuki, Akira Katsumi, Tetsuhito Kojima, Tadashi Matsushita, Shogo Tamura
    Biochemical and biophysical research communications 701 149583 - 149583 2024/01/30 [Refereed][Not invited]
     
    Endochondral ossification is a developmental process in the skeletal system and bone marrow of vertebrates. During endochondral ossification, primitive cartilaginous anlages derived from mesenchymal stem cells (MSCs) undergo vascular invasion and ossification. In vitro regeneration of endochondral ossification is beneficial for research on the skeletal system and bone marrow development as well as their clinical aspects. However, to achieve the regeneration of endochondral ossification, a stem cell-based artificial cartilage (cartilage organoid, Cart-Org) that possesses an endochondral ossification phenotype is required. Here, we modified a conventional 3D culture method to create stem cell-based Cart-Org by mixing it with a basement membrane extract (BME) and further characterized its chondrogenic and ossification properties. BME enlarged and matured the bone marrow MSC-based Cart-Orgs without any shape abnormalities. Histological analysis using Alcian blue staining showed that the production of cartilaginous extracellular matrices was enhanced in Cart-Org treated with BME. Transcriptome analysis using RNA sequencing revealed that BME altered the gene expression pattern of Cart-Org to a dominant chondrogenic state. BME triggered the activation of the SMAD pathway and inhibition of the NK-κB pathway, which resulted in the upregulation of SOX9, COL2A1, and ACAN in Cart-Org. BME also facilitated the upregulation of genes associated with hypertrophic chondrocytes (IHH, PTH1R, and COL10A1) and ossification (SP7, ALPL, and MMP13). Our findings indicate that BME promotes cartilaginous maturation and further ossification of bone marrow MSC-based Cart-Org, suggesting that Cart-Org treated with BME possesses the phenotype of endochondral ossification.
  • Yukino Kojima, Fumika Kawashima, Takahiko Yasuda, Koya Odaira, Yuichiro Inagaki, Chiharu Yamada, Ami Muraki, Mina Noura, Shuichi Okamoto, Shogo Tamura, Eisuke Iwamoto, Masashi Sanada, Itaru Matsumura, Yasushi Miyazaki, Tetsuhito Kojima, Hitoshi Kiyoi, Shinobu Tsuzuki, Fumihiko Hayakawa
    International journal of hematology 2023/05/07 
    Gene aberrations of B-cell regulators and growth signal components such as the JAK-STAT pathway are frequently found in B-cell acute lymphoblastic leukemia (B-ALL). EBF1 is a B-cell regulator that regulates the expression of PAX5 and co-operates with PAX5 to regulate B-cell differentiation. Here, we analyzed the function of the fusion protein of EBF1 and JAK2, EBF1-JAK2 (E-J). E-J caused constitutive activation of JAK-STAT and MAPK pathways and induced autonomous cell growth in a cytokine-dependent cell line. E-J did not affect the transcriptional activity of EBF1 but inhibited that of PAX5. Both the physical interaction of E-J with PAX5 and kinase activity of E-J were required for E-J to inhibit PAX5 function, although the detailed mechanism of inhibition remains unclear. Importantly, gene set enrichment analysis using the results of our previous RNA-seq data of 323 primary BCR-ABL1-negative ALL samples demonstrated repression of the transcriptional target genes of PAX5 in E-J-positive ALL cells, which suggests that E-J also inhibited PAX5 function in ALL cells. Our results shed new light on the mechanisms of differentiation block by kinase fusion proteins.
  • Naruko Suzuki, Nobuaki Suzuki, Yuka Kawaguchi, Shuichi Okamoto, Takeshi Kanematsu, Akira Katsumi, Atsuo Suzuki, Shogo Tamura, Tetsuhito Kojima, Hitoshi Kiyoi, Tadashi Matsushita
    Thrombosis journal 21 (1) 10 - 10 2023/01/25 [Refereed]
     
    BACKGROUND: Tranexamic acid (TXA) is an antifibrinolytic drug that blocks lysine-binding sites on the profibrinolytic enzyme plasminogen. Aortic diseases with chronic consumption coagulopathy may lead to disseminated intravascular coagulation (DIC) and cause fatal bleeding. Although the use of antifibrinolytic agents in DIC is generally not recommended due to enhanced fibrin deposition risking thrombotic symptoms, the efficacy of TXA has been reported in several cases of DIC with aortic diseases. However, the efficacy and safety of TXA for bleeding symptoms of chronic consumption coagulopathy with aortic diseases have not been studied in detail. METHODS: We evaluated the efficacy of TXA in 14 patients with chronic consumptive coagulopathy due to aortic disease complicated by bleeding symptoms. Changes in coagulation and fibrinolysis parameters from baseline were analyzed with Wilcoxon matched-pairs signed-rank tests, excluding missing values. Kaplan-Meier curves were used to analyze overall survival. RESULTS: Median age was 78.5 years (range, 66-89 years) and median observation period was 448 days (range, 0-2282 days). Twelve patients had chronic renal failure and 1 patient had chronic liver failure. Before starting treatment, median Japanese Ministry of Health and Welfare DIC diagnostic criteria score was 8 (range, 4-11) and median platelet count was 64 × 109/L (range, 25-97 × 109/L). Twelve patients underwent evaluation of bleeding symptoms after introduction of TXA, and 10 of those 12 patients showed improved bleeding tendencies within 30 days (median, 5.0 days). One patient with chronic liver failure showed worsening of bleeding symptoms. Although only one patient was initiated TXA in combination with anticoagulants, no significant worsening of thrombotic events was observed within 30 days. CONCLUSIONS: TXA therapy appears effective against chronic consumptive coagulopathy with bleeding due to aortic disease, with few side effects.
  • Koya Odaira, Takahiko Yasuda, Kentaro Okada, Takuya Shimooka, Yukino Kojima, Mina Noura, Shogo Tamura, Shingo Kurahashi, Eisuke Iwamoto, Masashi Sanada, Itaru Matsumura, Yasushi Miyazaki, Tetsuhito Kojima, Hitoshi Kiyoi, Shinobu Tsuzuki, Fumihiko Hayakawa
    Cancer science 114 (3) 781 - 792 2022/11/07 [Refereed]
     
    CEBPA-IGH, a fusion gene of the immunoglobulin heavy-chain locus (IGH) and the CCAAT enhancer-binding protein α (C/EBPα) gene, is recurrently found in B-ALL cases and causes aberrant expression of C/EBPα, a master regulator of granulocyte differentiation, in B cells. Forced expression of C/EBPα in B cells was reported to cause loss of B-cell identity due to the inhibition of Pax5, a master regulator of B-cell differentiation; however, it is not known whether the same mechanism is applicable for B-ALL development by CEBPA-IGH. It is known that a full-length isoform of C/EBPα, p42, promotes myeloid differentiation, whereas its N-terminal truncated isoform, p30, inhibits myeloid differentiation through the inhibition of p42; however, the differential role between p42 and p30 in ALL development has not been clarified. In the present study, we examined the effect of the expression of p42 and p30 in B cells by performing RNA-seq of mRNA from LCL stably transfected with p42 or p30. Unexpectedly, suppression of PAX5 target genes was barely observed. Instead, both isoforms suppressed the target genes of MEF2 family members (MEF2s), other regulators of B-cell differentiation. Similarly, MEF2s target genes rather than PAX5 target genes were suppressed in CEBP-IGH-positive ALL (n = 8) compared with other B-ALL (n = 315). Furthermore, binding of both isoforms to MEF2s target genes and the reduction of surrounding histone acetylation were observed in ChIP-qPCR. Our data suggest that the inhibition of MEF2s by C/EBPα plays a role in the development of CEBPA-IGH-positive ALL and that both isoforms work co-operatively to achieve it.
  • Keiji Nogami, Masashi Taki, Tadashi Matsushita, Tetsuhito Kojima, Toshiaki Oka, Shouichi Ohga, Kiyoshi Kawakami, Michio Sakai, Takashi Suzuki, Satoshi Higasa, Yasuo Horikoshi, Keiko Shinozawa, Shogo Tamura, Koji Yada, Masue Imaizumi, Yoshitoshi Ohtsuka, Fuminori Iwasaki, Masao Kobayashi, Junki Takamatsu, Hideyuki Takedani, Hisaya Nakadate, Yoko Matsuo, Takeshi Matsumoto, Teruhisa Fujii, Katsuyuki Fukutake, Akira Shirahata, Akira Yoshioka, Midori Shima
    Haemophilia : the official journal of the World Federation of Hemophilia 28 (5) 745 - 759 2022/09 [Refereed]
     
    BACKGROUND: Inhibitor-development is a serious complication in patients with haemophilia (PwH). Previous studies reported that therapeutic and genetic factors could be associated with these alloantibodies. Relevant clinical features such as genetic-background and different treatment regimens in Japan remain unclear, however. AIMS: To analyse a nation-wide Japanese registry for PwH, and to examine risk factors for inhibitor-development. METHODS AND RESULTS: Newly diagnosed patients with haemophilia A (PwHA) or haemophilia B (PwHB) without inhibitors after 2007, and with treatment records traceable from 0 to 75 exposure days (ED), were enrolled in the Japan Hemophilia Inhibitor Study 2 (J-HIS2) initiated in 2008. Of 417 patients (340 PwHA, 77 PwHB) from 46 facilities, 83 (76 PwHA, 7 PwHB) were recorded with inhibitors by July 2020. Inhibitors were observed in 31.0% of severe PwHA, 8.0% moderate and 1.6% mild and in 17.1% of severe PwHB. The majority of inhibitors (89.7% in severe PwHA and 71.4% in severe PwHB) were detected on or before 25ED (median 12ED in PwHA and 19ED in PwHB). Genotyping in these severe patients identified an association between inhibitor-development and null variants of F8 (P < .01) or F9 (P < .05). A lower incidence of inhibitors was recorded in severe PwHA treated with prophylaxis than in those treated on-demand (P < .01). A past-history of intracranial-haemorrhage appeared to be associated with inhibitor-development, while FVIII-concentrates infusion and routine vaccination on the same day was not related to inhibitor-development. CONCLUSION: The J-HIS2 study has identified significant clinical variables associated with inhibitor-development in Japanese PwH, consistent with other global studies.
  • Shuichi Okamoto, Shogo Tamura, Naomi Sanda, Koya Odaira, Yuri Hayakawa, Masato Mukaide, Atsuo Suzuki, Takeshi Kanematsu, Fumihiko Hayakawa, Akira Katsumi, Hitoshi Kiyoi, Tetsuhito Kojima, Tadashi Matsushita, Nobuaki Suzuki
    Journal of thrombosis and haemostasis : JTH 20 (8) 1784 - 1796 2022/08 [Refereed]
     
    BACKGROUND: Von Willebrand factor (VWF) is a multimeric glycoprotein that plays important roles in hemostasis and thrombosis. C-terminal interchain-disulfide bonds in the cystine knot (CK) domain are essential for VWF dimerization. Previous studies have reported that missense variants of cysteine in the CK domain disrupt the intrachain-disulfide bond and cause type 3 von Willebrand disease (VWD). However, type 3 VWD-associated noncysteine substitution variants in the CK domain have not been reported. OBJECTIVE: To investigate the molecular mechanism of a novel non-cysteine variant in the CK domain, VWF c.8254 G>A (p.Gly2752Ser), which was identified in a patient with type 3 VWD as homozygous. METHODS: Genetic analysis was performed by whole exome sequencing and Sanger sequencing. VWF multimer analysis was performed using SDS-agarose electrophoresis. VWF production and subcellular localization were analyzed using ex vivo endothelial colony forming cells (ECFCs) and an in vitro recombinant VWF (rVWF) expression system. RESULTS: The patient was homozygous for VWF-Gly2752Ser. Plasma VWF enzyme-linked immunosorbent assay showed that the VWF antigen level of the patient was 1.2% compared with healthy subjects. A tiny amount of VWF was identified in the patient's ECFC. Multimer analysis revealed that the circulating VWF-Gly2752Ser presented only low molecular weight multimers. Subcellular localization analysis of VWF-Gly2752Ser-transfected cell lines showed that rVWF-Gly2752Ser was severely impaired in its ER-to-Golgi trafficking. CONCLUSION: VWF-Gly2752Ser causes severe secretory impairment because of its dimerization failure. This is the first report of a VWF variant with a noncysteine substitution in the CK domain that causes type 3 VWD.
  • Masahiro Nishihori, Yoshio Araki, Nobuaki Suzuki, Shogo Tamura, Mayo Hattori, Takashi Izumi, Shunsaku Goto, Kinya Yokoyama, Kenji Uda, Tadashi Matsushita, Ryuta Saito
    Journal of stroke and cerebrovascular diseases : the official journal of National Stroke Association 31 (7) 106526 - 106526 2022/07 [Refereed]
     
    OBJECTIVES: Congenital afibrinogenemia is an autosomal recessive inherited disorder that can cause thrombotic as well as hemorrhagic events. We describe a case of repeated mild ischemic strokes due to a mural thrombus in the carotid artery and our medical treatment. CASE DESCRIPTION: A 49-year-old woman with congenital afibrinogenemia developed two minor ischemic strokes in two months. Clinical images revealed scattered fresh infarcts in the right middle cerebral artery region and mild cervical carotid artery stenosis. The risk for surgical treatment was considered to be extraordinarily high. The patient was treated with 100 mg/day of aspirin and 3 g fibrinogen infusion every two weeks. After the one-year course of medication, the mural thrombus gradually decreased, and there were no bleeding or ischemic stroke events. CONCLUSION: This case report highlights the successful treatment of an ischemic stroke in a patient with a congenital afibrinogenemia with an antiplatelet agent and fibrinogen replacement. There are no guidelines for managing ischemic stroke in patients with congenital afibrinogenemia, and further studies are needed.
  • Nobuaki Suzuki, Takeshi Kanematsu, Mayuko Kishimoto, Naruko Suzuki, Shuichi Okamoto, Shogo Tamura, Hitoshi Kiyoi, Tadashi Matsushita
    Japanese Journal of Transfusion and Cell Therapy 68 (3) 422 - 427 1881-3011 2022/06/24 [Refereed][Not invited]
  • Shogo Tamura, Masato Mukaide, Yumi Katsuragi, Wataru Fujii, Koya Odaira, Nobuaki Suzuki, Nagaharu Tsukiji, Shuichi Okamoto, Atsuo Suzuki, Takeshi Kanematsu, Akira Katsumi, Akira Takagi, Katsuhide Ikeda, Jun Ueyama, Masaaki Hirayama, Katsue Suzuki-Inoue, Tadashi Matsushita, Tetsuhito Kojima, Fumihiko Hayakawa
    Journal of Biological Chemistry 298 (5) 101833 - 101833 0021-9258 2022/05 [Refereed]
  • Koya Odaira, Fumika Kawashima, Shogo Tamura, Nobuaki Suzuki, Mahiru Tokoro, Yuri Hayakawa, Atsuo Suzuki, Takeshi Kanematsu, Shuichi Okamoto, Akira Takagi, Akira Katsumi, Tadashi Matsushita, Midori Shima, Keiji Nogami, Tetsuhito Kojima, Fumihiko Hayakawa
    Thrombosis Research 213 91 - 96 0049-3848 2022/05 [Refereed]
  • Kentaro Okada, Shogo Tamura, Nobuaki Suzuki, Koya Odaira, Masato Mukaide, Wataru Fujii, Yumi Katsuragi, Atsuo Suzuki, Takeshi Kanematsu, Shuichi Okamoto, Naruko Suzuki, Akira Katsumi, Tadashi Matsushita, Tetsuhito Kojima, Fumihiko Hayakawa
    Thrombosis Research 210 26 - 32 0049-3848 2022/02 [Refereed][Not invited]
  • Atsuo Suzuki, Nobuaki Suzuki, Takeshi Kanematsu, Sho Shinohara, Hiroshi Kurono, Nobuo Arai, Shuichi Okamoto, Naruko Suzuki, Shogo Tamura, Ryosuke Kikuchi, Akira Katsumi, Tetsuhito Kojima, Tadashi Matsushita
    Scientific Reports 12 (1) 2022/01/21 
    Abstract Plasma fibrinogen is commonly examined by Clauss fibrinogen assay, which cannot distinguish between quantitative and qualitative fibrinogen anomalies. However, our previously reported Clauss fibrinogen assay utilizing clot waveform analysis (Clauss-CWA) provides additional information that contributes to the classification of fibrinogen anomalies. In this study, we adopted the Clauss-CWA method for an autoanalyzer to automatically measure the antigenic estimate (eAg) of fibrinogen in addition to the functional amount (Ac), and to thus provide the Ac/eAg ratio as a qualitative indicator. Performance was validated by receiver operating characteristics (ROC) and precision recall (PR) curve analyses using a patient cohort, consisting of a training cohort (n = 519) and a validation cohort (n = 523), both of which contained cases of congenital (hypo)dysfibrinogenemia as qualitative defects. We obtained an optimal cutoff of 0.65 for Ac/eAg by ROC curve analysis of the training cohort, offering superior sensitivity (> 0.9661) and specificity (1.000). This cutoff was validated in the validation cohort, providing positive predictive value > 0.933 and negative predictive value > 0.998. PR curve analysis also showed that Clauss-CWA provided excellent performance for detecting qualitative fibrinogen anomalies. The Clauss-CWA method may represent a useful approach for detecting qualitative fibrinogen abnormalities in routine laboratory testing.
  • Akira Katsumi, Akihiro Abe, Shogo Tamura, Tadashi Matsushita
    Geriatrics & gerontology international 21 (7) 549 - 554 2021/07 
    Anemia, a frequently occurring condition in older patients, has no standard definition; however, in most studies, it is defined as hemoglobin level <12 and <13 g/dL in women and men, respectively. Approximately 10% of older adults living in the community have anemia. The prevalence of anemia is significantly correlated with advanced age and male sex. Anemia is associated with falls, frailty and other negative outcomes, including early mortality. However, there remains little consensus regarding whether anemia treatment favorably affects these adverse outcomes. Therefore, this article reviews the prevalence of anemia, and provides updates on its common causes and treatments in older adults. While excluding well-established hematopoietic diseases, the etiology of anemia in older adults has been grouped into four categories: (i) nutritional deficiency; (ii) inflammation; (iii) clonal hematopoiesis; and (iv) "unexplained anemia," when there is no clear mechanism to account for the anemia. Recently, clonal leukocytes were detected in a considerable number of older individuals. The number of somatic mutations in blood leukocytes increases with age; however, single mutations of DNMT3A, TET2 and ASXL1 are not correlated with the presence of unexplained anemia in older adults. With an increased understanding of anemia etiology and the availability of innovative anti-anemic drugs, future studies that evaluate the causes and benefits of treatment are required. Geriatr Gerontol Int 2021; 21: 549-554.
  • Shimon Otake, Tomoyuki Sasaki, Toshiaki Shirai, Nagaharu Tsukiji, Shogo Tamura, Katsuhiro Takano, Yukio Ozaki, Katsue Suzuki-Inoue
    Journal of thrombosis and haemostasis : JTH 19 (6) 1572 - 1584 2021/06 
    BACKGROUND: Erythropoiesis is a complex multistep process by which erythrocytes are produced. C-type lectin-like receptor 2 (CLEC-2) is a podoplanin (PDPN) receptor almost exclusively expressed on the surface of platelets and megakaryocytes. Deletion of megakaryocyte/platelet CLEC-2 was reported to cause anemia along with thrombocytopenia in mice. PDPN-expressing stromal cells in the bone marrow (BM) were also reported to facilitate megakaryocyte expansion and maturation depending on the CLEC-2/PDPN interaction. OBJECTIVES: We investigated how specific deletion of CLEC-2 in megakaryocytes/platelets leads to anemia. METHODS: We used flow cytometry to analyze maturation of erythroblasts, apoptotic cell death, and cell cycle distribution. CLEC-2 stimulated PDPN-expressing stromal cell-conditioned medium was analyzed by cytokine array and ELISA, and co-cultured with immature erythroblasts. Cytokine levels in serum and BM extracellular fluid were quantified by ELISA. RESULTS: We observed increased apoptosis of BM erythroblasts in megakaryocyte/platelet-specific CLEC-2 conditional knockout (Clec1bΔPLT ) mice. Moreover, PDPN-expressing stromal cells in the BM secreted insulin-like growth factor 1 (IGF-1) depending on the CLEC-2/PDPN interaction. Pretreatment with IGF-1 receptor inhibitor increased apoptosis rate and decreased the proliferation of erythroblasts in vitro. Furthermore, in Clec1bΔPLT mice, IGF-1 concentrations in serum and BM extracellular fluid were decreased, and IGF-1 replacement in Clec1bΔPLT mice attenuated anemia. CONCLUSIONS: Our findings suggest that IGF-1 secretion from PDPN-expressing stromal cells by CLEC-2 stimulation positively regulates erythroblasts. This novel mechanism of erythropoiesis regulation indicates that a microenvironment consisting of megakaryocytes and PDPN-expressing stromal cells supports erythropoiesis.
  • 第VIII因子製剤の活性測定における試薬バリエーションを把握するための単施設研究
    鈴木 敦夫, 鈴木 伸明, 兼松 毅, 岡本 修一, 田村 彰吾, 小嶋 哲人, 松下 正
    日本血栓止血学会誌 (一社)日本血栓止血学会 32 (2) 198 - 198 0915-7441 2021/05
  • Yuri Hayakawa, Shogo Tamura, Nobuaki Suzuki, Koya Odaira, Mahiru Tokoro, Fumika Kawashima, Fumihiko Hayakawa, Akira Takagi, Akira Katsumi, Atsuo Suzuki, Shuichi Okamoto, Takeshi Kanematsu, Tadashi Matsushita, Tetsuhito Kojima
    Journal of thrombosis and haemostasis : JTH 19 (4) 920 - 930 2021/04 [Refereed][Not invited]
     
    BACKGROUND: Coagulation factor XI (FXI) is a plasma serine protease zymogen that contributes to hemostasis. However, the mechanism of its secretion remains unclear. OBJECTIVE: To determine the molecular mechanism of FXI secretion by characterizing a novel FXI mutant identified in a FXI-deficient Japanese patient. PATIENT/METHODS: The FXI gene (F11) was analyzed by direct sequencing. Mutant recombinant FXI (rFXI) was overexpressed in HEK293 or COS-7 cells. Western blotting and enzyme-linked immunosorbent assay were performed to examine the FXI extracellular secretion profile. Immunofluorescence microscopy was used to investigate the subcellular localization of the rFXI mutant. RESULTS: We identified a novel homozygous frameshift mutation in F11 [c.1788dupC (p.E597Rfs*65)], resulting in a unique and extended carboxyl-terminal (C-terminal) structure in FXI. Although rFXI-E597Rfs*65 was intracellularly synthesized, its extracellular secretion was markedly reduced. Subcellular localization analysis revealed that rFXI-E597Rfs*65 was abnormally retained in the endoplasmic reticulum (ER). We generated a series of C-terminal-truncated rFXI mutants to further investigate the role of the C-terminal region in FXI secretion. Serial rFXI experiments revealed that a threonine at position 622, the fourth residue from the C-terminus, was essential for secretion. Notably, Thr622 engages in the formation of an α-helix motif, indicating the importance of the C-terminal α-helix in FXI intracellular behavior and secretion. CONCLUSION: FXI E597Rfs*65 results in the pathogenesis of a severe secretory defect resulting from aberrant ER-to-Golgi trafficking caused by the lack of a C-terminal α-helix motif. This study demonstrates the impact of the C-terminal structure, especially the α-helix motif, on FXI secretion.
  • Takeshi Kanematsu, Nobuaki Suzuki, Shogo Tamura, Atsuo Suzuki, Yuichi Ishikawa, Akira Katsumi, Hitoshi Kiyoi, Hidehiko Saito, Shinji Kunishima, Tetsuhito Kojima, Tadashi Matsushita
    Nagoya journal of medical science 83 (1) 75 - 86 2021/02 
    MYH9 disorders are characterized by giant platelets, thrombocytopenia, and Döhle body-like cytoplasmic inclusion bodies in granulocytes. However, whether these disorders cause any changes in erythroid cells has yet to be determined. This study analyzed the influence of Myh9 R702C, as one of the most commonly detected MYH9 disorders, on erythroid cells in a mouse model. Knock-in mice expressing Myh9 R702C mutation either systemically or specific to hematological cells (R702C and R702C vav1 mice, respectively) were used in this study. Both displayed lower hemoglobin and higher erythropoietin levels than wild-type (WT) mice, along with significant splenomegaly. Flow cytometric analysis revealed erythroblasts present at a higher rate than WT mice in the spleen. However, no obvious abnormalities were seen in erythroid differentiation from megakaryocyte/erythroid progenitor to erythrocyte. Cell culture assay by fetal liver and colony assay also showed normal progression of erythroid differentiation from erythroid burst-forming unit to red blood cell. In conclusion, R702C and R702C vav1 mice displayed erythroid abnormality with splenomegaly. However, erythroid differentiation showed no obvious abnormality. Further research is required to elucidate the underlying mechanisms.
  • Suzuki Atsuo, Suzuki Nobuaki, Kanematsu Takeshi, Okamoto Shuichi, Tamura Shogo, Kikuchi Ryosuke, Katsumi Akira, Kiyoi Hitoshi, Kojima Tetsuhito, Matsushita Tadashi
    INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY 43 (1) 131 - 138 1751-5521 2020/09/11 [Refereed][Not invited]
     
    INTRODUCTION: Factor VIII activity (FVIII:C) is measured by one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). Significant differences in FVIII:C between OSA (FVIII:C1st ) and CSA (FVIII:CChr ) are described as assay discrepancy in nonsevere haemophilia A (HA). A large number of reagent combinations (APTT reagent and FVIII-deficient plasma) are used for OSA, but the impact of variations in reagent combinations on assay discrepancy has not been fully characterized. AIM: To clarify the variations in FVIII:C1st /FVIII:CChr ratios according to OSA reagent combination in HA subjects with/without assay discrepancy. METHODS: Thirty-nine patients previously diagnosed with nonsevere HA were enrolled, and their FVIII genes were investigated and FVIII:C levels were assessed by a single CSA reagent and 11 OSA reagent combinations. Receiver operating characteristic (ROC) curve analysis was used to predict possible cut-off values of the FVIII:C1st /FVIII:CChr ratio to define FVIII assay discrepancy for each reagent combination. RESULTS: Patients were categorized into nondiscrepant (n = 25), discrepant (n = 5) and unclassified (n = 9) groups according to their genotypes and information in the database. The FVIII:C1st /FVIII:CChr ratio in nondiscrepant HA varied widely, depending on the APTT reagents and FVIII-deficient plasma used. The ratio in discrepant HA patients differed with respect to their genotype and the reagent combination used. ROC curve analyses revealed that cut-off values to distinguish the assay discrepancy differed depending on the reagents used, but revealed two novel genotype variants, p.Cys573Gly and p.Gly582Arg, associated with FVIII assay discrepancy. CONCLUSION: Our findings showed that the FVIII:C1st /FVIII:CChr ratio is dependent on the reagent combination used for OSA.
  • 鈴木 敦夫, 鈴木 伸明, 兼松 毅, 岡本 修一, 田村 彰吾, 篠原 翔, 新井 信夫, 菊地 良介, 安藤 善孝, 小嶋 哲人, 松下 正
    日本血栓止血学会誌 (一社)日本血栓止血学会 31 (4) 409 - 419 0915-7441 2020/08 
    INNOVANCE VWF Acは血小板膜糖蛋白質GPIbの変異体を用いるフォンヴィレブランド因子(von Willebrand factor:VWF)活性(VWF:GPIbM)測定試薬である。本研究では本邦で初めてその基本性能評価を行った。併行精度はCVが3%未満、室内再現性はCVが2%未満と良好な成績であった。希釈直線性も良好であり、最小検出感度は1.3IU/dLであった。また、干渉物質や未分画ヘパリン混入による明らかな影響は見られなかった。正常検体100例を用いた相関性試験ではVWF抗原量およびVWFリストセチンコファクター活性(VWF:RCo)と良好な相関性を示した。フォンヴィレブランド病患者検体においてもVWF:RCoとの測定値に大きな差はなく、VWF:GPIbMでは特に低濃度域を評価することが可能であった。INNOVANCE VWF AcはVWF:RCo測定試薬と比べても高い性能を有することが明らかとなった。(著者抄録)
  • Tokoro Mahiru, Tamura Shogo, Suzuki Nobuaki, Kakihara Misaki, Hattori Yuna, Odaira Koya, Suzuki Sachiko, Takagi Akira, Katsumi Akira, Hayakawa Fumihiko, Okamoto Shuichi, Suzuki Atsuo, Kanematsu Takeshi, Matsushita Tadashi, Kojima Tetsuhito
    MOLECULAR GENETICS & GENOMIC MEDICINE 8 (9) e1390  2324-9269 2020/07/05 [Refereed][Not invited]
     
    BACKGROUND: Hemophilia A (HA) is an X-linked recessive bleeding disorder caused by pathogenic variants of the coagulation factor VIII gene (F8). Half of the patients with severe HA have a recurrent inversion in the X chromosome, that is, F8 intron 22 or intron 1 inversion. Here, we characterized an abnormal F8 due to atypical complex X chromosome rearrangements in a Japanese patient with severe HA. METHODS: Recurrent F8 inversions were tested with inverse shifting-PCR. The genomic structure was investigated using PCR-based direct sequencing or quantitative PCR. RESULTS: The proband's X chromosome had a 119.5 kb insertion, a reverse duplex of an extragenic sequence on the F8 telomere region into the F8 intron 1 with two breakpoints. The telomeric breakpoint was a joining from the F8 intron 1 to the inverted FUNDC2 via a two-base microhomology, and the centromeric breakpoint was a recombination between F8 intron 1 homologous sequences. The rearrangement mechanism was suggested as a multi-step rearrangement with template switching such as fork stalling and template switching (FoSTeS)/microhomology-mediated break-induced replication (MMBIR) and/or homologous sequence-associated recombination during a sister chromatid formation. CONCLUSION: We identified the aberrant X chromosome with a split F8 due to a multi-step rearrangement in a patient with severe HA.
  • 凝固一段法による第VIII因子活性測定における試薬の組み合わせに関する検討
    鈴木 敦夫, 鈴木 伸明, 兼松 毅, 岡本 修一, 田村 彰吾, 安藤 善孝, 清井 仁, 松下 正
    日本血栓止血学会誌 (一社)日本血栓止血学会 31 (2) 231 - 231 0915-7441 2020/05
  • 高齢者における静脈血栓塞栓症発症の症例対照研究
    勝見 章, 室谷 健太, 鴨下 園子, 伊藤 美由紀, 田村 彰吾, 鈴木 伸明, 松下 正, 小嶋 哲人
    日本血栓止血学会誌 (一社)日本血栓止血学会 31 (2) 277 - 277 0915-7441 2020/05 [Refereed][Not invited]
  • Ogawa M, Suzuki N, Takahashi N, Tamura S, Suzuki A, Suzuki S, Hattori Y, Kakihara M, Kanematsu T, Kojima T, Katsumi A, Hayakawa F, Kojima T, Ishiguro N, Kiyoi H, Matsushita T
    Thrombosis research 188 103-105 - 105 0049-3848 2020/02/22 [Refereed][Not invited]
  • 先天性凝固異常症の遺伝子解析 「解析のStrategyとPitfall」
    田村彰吾, 高木明, 早川文彦, 小嶋哲人
    日本検査血液学会雑誌 21 71 - 80 2020/02 [Refereed][Invited]
  • Okamoto S, Suzuki N, Suzuki A, Suzuki S, Tamura S, Suzuki M, Takahashi N, Kojima T, Kanematsu T, Kojima T, Kiyoi H, Ishiguro N, Matsushita T
    TH open : companion journal to thrombosis and haemostasis 3 (4) e364-e366  2567-3459 2019/10 [Refereed][Not invited]
     
    We managed perioperative hemostasis for a 72-year-old man with hemophilia A and low inhibitor titers (3 BU/mL), who underwent osteosynthesis for supracondylar fracture of the left humerus. He was treated perioperatively using the combination of high doses of factor VIII (FVIII) with recombinant human Factor VIII Fc fusion protein (rFVIIIFc), followed by emicizumab. On the day of surgery (day 0), he was administered bolus infusion of 150 IU/kg rFVIIIFc, followed by continuous infusion at a dose of 4 IU/kg/h. Emicizumab, 3 mg/kg, was injected subcutaneously once a week, on days 5, 12, 19, and 26. Inhibitors were detected on day 6 at a titer of 4 BU/mL and FVIII:C decreased to below assay sensitivity limits on day 10. The rate of increase in inhibitor titers was high, with inhibitors increasing to 343.4 BU/mL on day 14. The transition of thrombin production by thrombin generation assay (TGA) showed temporary decrease in thrombin production on day 7, although it was restored by day 10, i.e., five days after commencement of emicizumab therapy. Rotational thromboelastometry displayed consistent results with TGA, showing that clotting time was prolonged and the alpha angle decreased to less than measurable levels on day 6, although they were improved by day 10. There were no bleeding-related events or other adverse events throughout the perioperative period. In conclusion, emicizumab was effective for the management of perioperative hemostasis after development of an anamnestic response in a patient with hemophilia A with inhibitors. Combination therapy with high doses of FVIII followed by emicizumab could be a workable alternative for patients with hemophilia A with inhibitors.
  • 田村 彰吾, 井上 克枝, 尾崎 由基男, 早川 文彦, 小嶋 哲人
    臨床血液 60 (7) 834 - 842 2019/08/06 [Refereed][Not invited]
  • Koya Odaira, Shogo Tamura, Nobuaki Suzuki, Misaki Kakihara, Yuna Hattori, Mahiru Tokoro, Sachiko Suzuki, Akira Takagi, Akira Katsumi, Fumihiko Hayakawa, Shuichi Okamoto, Atsuo Suzuki, Takeshi Kanematsu, Tadashi Matsushita, Tetsuhito Kojima
    Thrombosis research 179 95 - 103 2019/07 [Refereed][Not invited]
     
    INTRODUCTION: Hemophilia B is an X-linked recessive bleeding disorder caused by coagulation factor IX (FIX) gene (F9) mutations. Several F9 synonymous mutations have been known to cause hemophilia B; however, the deleterious mechanisms underlying the development of hemophilia B have not been completely understood. To elucidate the molecular pathogenesis causing hemophilia B, we investigated the synonymous F9 mutation: c.87A>G, p.(Thr29=). MATERIALS AND METHODS: The influence of F9 c.87A>G on mRNA splicing was analyzed by exon-trap assay and in silico prediction. We prepared FIX expression vectors using mutant F9 cDNA and transfected HepG2 cells to investigate intracellular transport and extracellular secretion of FIX. Intracellular kinetics of the expressed FIX was examined by treatment with the proteasome inhibitor MG132. RESULTS: Exon-trap analysis revealed that F9 c.87A>G resulted in almost (99.1%) aberrant splicing (r.83_88del). In silico analysis predicted that F9 c.87A>G influenced the splicing pattern by generating an available aberrant 5' splice site. The aberrant F9 mRNA (r.83_88del) was translated to a mutant FIX p.Cys28_Val30delinsPhe with an in-frame mutation at the signal peptide cleavage site. Simultaneously, a small amount (0.9%) of mutant F9 r.87A>G translating into WT FIX p.Thr29 = was also observed. The mutant FIX was abnormally retained in the endoplasmic reticulum (ER) and was not extracellularly secreted. It appeared to be intracellularly degraded via proteasome-dependent degradation machinery. CONCLUSION: F9 c.87A>G was found to cause abnormal mRNA splicing, r.83_88del, and produce the mutant FIX p.Cys28_Val30delinsPhe. The mutant FIX is an abnormal protein with extracellular secretory defects and is intracellularly eliminated via proteasome-dependent ER-associated degradation.
  • Osamu Inoue, Makoto Osada, Junya Nakamura, Fuminori Kazama, Toshiaki Shirai, Nagaharu Tsukiji, Tomoyuki Sasaki, Hiroshi Yokomichi, Tomotaka Dohi, Makoto Kaneko, Makoto Kurano, Mitsuru Oosawa, Shogo Tamura, Kaneo Satoh, Katsuhiro Takano, Katsumi Miyauchi, Hiroyuki Daida, Yutaka Yatomi, Yukio Ozaki, Katsue Suzuki-Inoue
    INTERNATIONAL JOURNAL OF HEMATOLOGY The Japanese Society of Hematology 110 (3) 285 - 294 0925-5710 2019/06/05 [Refereed][Not invited]
  • Tamura S, Hashimoto E, Suzuki N, Kakihara M, Odaira K, Hattori Y, Tokoro M, Suzuki S, Takagi A, Katsumi A, Hayakawa F, Suzuki A, Okamoto S, Kanematsu T, Matsushita T, Kojima T
    Thrombosis research 178 159 - 170 0049-3848 2019/04 [Refereed][Not invited]
  • 特発性血栓症 アンチトロンビン抵抗性の診断 検査法を中心に
    高木 明, 田村 彰吾, 小嶋 哲人
    日本検査血液学会雑誌 (一社)日本検査血液学会 19 (3) 393 - 400 1347-2836 2018/11 
    アンチトロンビン抵抗性とは、家族性血栓症家系より発見したプロトロンビンの1アミノ酸置換(p.Arg596Leu)に由来するトロンビンが凝固活性を保持したまま、変異によりアンチトロンビンとの親和性が低下しトロンビンの不活化が遅れてしまう病態である。我々は、アンチトロンビン抵抗性を検出する臨床検査室で実施可能なトロンビン不活化動態検査法を考案したが、プロトロンビンアクチベータに使用するOxyuranus scutellatus蛇毒が入手困難となったためプロトロンビンアクチベータをウシFXa・FVaに変更した検査法を考案した。さまざまな抗血栓薬の影響、回避法を検討した。ヘパリン類の影響はポリブレンの添加で回避できたが、ダビガトランの共存はトロンビン・アンチトロンビン複合体形成に阻害的に作用し、アンチトロンビン抵抗性を示した。(著者抄録)
  • Nagaharu Tsukiji, Osamu Inoue, Mitsuru Morimoto, Norifumi Tatsumi, Hiroaki Nagatomo, Koji Ueta, Toshiaki Shirai, Tomoyuki Sasaki, Shimon Otake, Shogo Tamura, Toshiaki Tachibana, Masataka Okabe, Masanori Hirashima, Yukio Ozaki, Katsue Suzuki-Inoue
    Blood 132 (11) 1167 - 1179 0006-4971 2018/09/13 [Refereed][Not invited]
     
    Platelets participate in not only thrombosis and hemostasis but also other pathophysiological processes, including tumor metastasis and inflammation. However, the putative role of platelets in the development of solid organs has not yet been described. Here, we report that platelets regulate lung development through the interaction between the platelet-activation receptor, C-type lectin-like receptor-2 (Clec-2; encoded by Clec1b), and its ligand, podoplanin, a membrane protein. Clec-2 deletion in mouse platelets led to lung malformation, which caused respiratory failure and neonatal lethality. In these embryos, α-smooth muscle actin-positive alveolar duct myofibroblasts (adMYFs) were almost absent in the primary alveolar septa, which resulted in loss of alveolar elastic fibers and lung malformation. Our data suggest that the lack of adMYFs is caused by abnormal differentiation of lung mesothelial cells (luMCs), the major progenitor of adMYFs. In the developing lung, podoplanin expression is detected in alveolar epithelial cells (AECs), luMCs, and lymphatic endothelial cells (LECs). LEC-specific podoplanin knockout mice showed neonatal lethality and Clec1b-/--like lung developmental abnormalities. Notably, these Clec1b-/--like lung abnormalities were also observed after thrombocytopenia or transforming growth factor-β depletion in fetuses. We propose that the interaction between Clec-2 on platelets and podoplanin on LECs stimulates adMYF differentiation of luMCs through transforming growth factor-β signaling, thus regulating normal lung development.
  • S. Tamura, Y. Suga, M. Tanamura, M. Murata-Kawakami, Y. Takagi, Y. Hottori, M. Kakihara, S. Suzuki, A. Takagi, T. Kojima
    International Journal of Laboratory Hematology 40 (3) 312 - 319 1751-5521 2018/06 [Not refereed][Not invited]
  • T. Sasaki, T. Shirai, N. Tsukiji, S. Otake, S. Tamura, J. Ichikawa, M. Osada, K. Satoh, Y. Ozaki, K. Suzuki-Inoue
    Journal of Thrombosis and Haemostasis 16 (5) 960 - 972 1538-7836 2018/05/01 [Not refereed][Not invited]
     
    Essentials We generated recombinant rhodocytin that could aggregate platelets via CLEC-2. Recombinant wild-type rhodocytin formed heterooctamer with four α- and β-subunits. Asp 4 in α-subunit of rhodocytin was required for binding to CLEC-2. Inhibitory mutant of rhodocytin blocked podoplanin-dependent hematogenous metastasis. Summary: Background Rhodocytin, a disulfide-linked heterodimeric C-type lectin from Calloselasma rhodostoma consisting of α-subunits and β-subunits, induces platelet aggregation through C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a physiological binding partner of podoplanin (PDPN), which is expressed on some tumor cell types, and is involved in tumor cell-induced platelet aggregation and tumor metastasis. Thus, modified rhodocytin may be a possible source of anti-CLEC-2 drugs for both antiplatelet and antimetastasis therapy. However, its molecular function has not been well characterized, because of the lack of recombinant rhodocytin that induces platelet aggregation. Objective To produce recombinant rhodocytin, in order to verify its function with mutagenesis, and to develop an anti-CLEC-2 drug based on the findings. Methods We used Chinese hamster ovary cells to express recombinant rhodocytin (wild-type [WT] and mutant), which was analyzed for induction/inhibition of platelet aggregation with light transmission aggregometry, the formation of multimers with blue native PAGE, and binding to CLEC-2 with flow cytometry. Finally, we investigated whether mutant rhodocytin could suppress PDPN-induced metastasis in an experimental lung metastasis mouse model. Results Functional WT] rhodocytin (αWTβWT) was obtained by coexpression of both subunits. Asp4 in α-subunits of rhodocytin was required for CLEC-2 binding. αWTβWT formed a heterooctamer similarly to native rhodocytin. Moreover, an inhibitory mutant of rhodocytin (αWTβK53A/R56A), forming a heterotetramer, bound to CLEC-2 without inducing platelet aggregation, and blocked CLEC-2–PDPN interaction-dependent platelet aggregation and experimental lung metastasis. Conclusion These findings provide molecular characterization information on rhodocytin, and suggest that mutant rhodocytin could be used as a therapeutic agent to target CLEC-2.
  • 凝固一段法と合成基質法による第VIII因子活性測定において乖離を示した血友病A症例
    鈴木 敦夫, 鈴木 伸明, 兼松 毅, 岸本 磨由子, 田村 彰吾, 高木 明, 川上 萌, 梶浦 容子, 小嶋 哲人, 松下 正
    日本血栓止血学会誌 (一社)日本血栓止血学会 29 (2) 210 - 210 0915-7441 2018/05
  • Shogo TAMURA
    日本血栓止血学会誌 29 (4) 389-397  2018 [Refereed][Invited]
  • S. Suzuki, Y. Nakamura, N. Suzuki, T. Yamazaki, Y. Takagi, S. Tamura, A. Takagi, T. Kanematsu, T. Matsushita, T. Kojima
    Haemophilia 24 (1) e13 - e16 1365-2516 2018/01/01 [Not refereed][Not invited]
  • Miki Kamiyama, Toshiaki Shirai, Shogo Tamura, Katsue Suzuki-Inoue, Shogo Ehata, Kei Takahashi, Kohei Miyazono, Yoshihiro Hayakawa, Takehiro Sato, Kohsuke Takeda, Isao Naguro, Hidenori Ichijo
    CELL DEATH AND DIFFERENTIATION 24 (12) 2066 - 2076 1350-9047 2017/12 [Refereed][Not invited]
     
    Tumor metastasis is the major cause of deaths in cancer patients and is modulated by intertwined stress-responsive signaling cascades. Here we demonstrate that deletion of stress-responsive apoptosis signal-regulating kinase 1 (Ask1) in platelets results in unstable hemostasis and drastic attenuation of tumor lung metastasis, both of which are attributable to platelet dysfunction. Platelet-specific deletion of Ask1 in mice leads to defects in ADP-dependent platelet aggregation, unstable hemostasis and subsequent attenuation of tumor metastasis. We also revealed that activating phosphorylation of Akt is attenuated in Ask1-deficient platelets, contrary to the previous reports suggesting that Akt is negatively regulated by ASK1. Mechanistically, ASK1-JNK/p38 axis phosphorylates an ADP receptor P2Y(12) at Thr345, which is required for the ADP-dependent sustained Akt activity that is vital to normal platelet functions. Our findings offer insight into positive regulation of Akt signaling through P2Y(12) phosphorylation as well as MAPK signaling in platelets by ASK1 and suggest that ASK1-JNK/p38 axis provides a new therapeutic opportunity for tumor metastasis.
  • Shogo Tamura, Moe Murata-Kawakami, Yuki Takagi, Sachiko Suzuki, Akira Katsumi, Akira Takagi, Tetsuhito Kojima
    THROMBOSIS RESEARCH 159 (159) 33 - 38 0049-3848 2017/11 [Refereed][Not invited]
     
    Introduction: Antithrombin resistance (ATR) prothrombinemia is an inherited thrombophilic disorder caused by missense mutations in prothrombin gene (F2) at Arg596 of the sodium-binding region. Previously, prothrombin mutants Yukuhashi (Arg596Leu), Belgrade (Arg596Gln), and Padua 2 (Arg596Trp) were reported as ATR-prothrombins possessing a risk of familial venous thrombosis. To identify additional F2 mutations causing the ATR-phenotype, we investigated the coagulant properties of recombinant prothrombins mutated at amino acid residues within the sodium-binding region by single nucleotide substitutions (Thr540, Arg541, Glu592, and Lys599). Materials and methods: We constructed expression vectors of prothrombin mutants, established stably transfected HEK293 cells, and isolated the recombinant prothrombin proteins. We evaluated procoagulant activity and ATR-phenotypes of those mutants in reconstituted plasma by mixing with prothrombin deficient plasma. Results: The secreted quantity of all prothrombin mutants was the same as that of the wild-type prothrombin. Procoagulant activity of each mutant varied from 1.7% to 79.5% in a one-stage clotting assay and from 2.0% to 104.5% in a two-stage chromogenic assay. Most prothrombin mutants tested presented with a severe ATR-phenotype. To estimate the thrombosis risk of these mutations, we determined the residual clotting activity (RCA) after 30 min inactivation with antithrombin. RCA scores, normalized to the wild-type, revealed that prothrombin mutants Lys599Arg (5.35) and Glu592Gln (4.71) had high scores, which were comparable with prothrombins Yukuhashi (4.36) and Belgrade (5.19). Conclusions: Mutation of prothrombin at the sodium-binding site caused ATR-phenotypes. Of those tested, Lys599Arg and Glu592Gln may possess a thrombosis risk as large as the known pathogenic prothrombins Yukuhashi and Belgrade.
  • T. Shirai, O. Inoue, S. Tamura, N. Tsukiji, T. Sasaki, H. Endo, K. Satoh, M. Osada, H. Sato-Uchida, H. Fujii, Y. Ozaki, K. Suzuki-Inoue
    JOURNAL OF THROMBOSIS AND HAEMOSTASIS 15 (3) 513 - 525 1538-7933 2017/03 [Refereed][Not invited]
     
    Background C-type lectin-like receptor 2 (CLEC-2) is a platelet activation receptor of sialoglycoprotein podoplanin, which is expressed on the surface of certain types of tumor cells. CLEC-2-podoplanin interactions facilitate hematogenous tumor metastasis. However, direct evidence of the role of CLEC-2 in hematogenous metastasis and cancer progression is lacking. Objective and methods We generated immunological CLEC-2-depleted mice by using anti-mouse CLEC-2 monoclonal antibody 2A2B10 and investigated whether CLEC-2 promoted hematogenous tumor metastasis and tumor growth and exacerbated the prognosis of mice bearing podoplanin-expressing B16F10 melanoma cells. Results Our results showed that hematogenous metastasis was significantly inhibited in CLEC-2-depleted mice. B16F10 cells co-cultured with wild-type platelets, but not with CLEC-2-deficient platelets, showed increased proliferation. However, B16F10 cell proliferation was not inhibited in CLEC-2-depleted mice. Histological analysis showed that thrombus formation in tumor vessels was significantly inhibited and functional vessel density was significantly increased in CLEC-2-depleted mice. These data suggest that CLEC-2 deficiency may inhibit thrombus formation in tumor vessels and increase the density of functional vessels, thus improving oxygen and nutrient supply to tumors, indirectly promoting tumor proliferation. Furthermore, the overall survival of CLEC-2-depleted mice was significantly prolonged, which may be due to the suppression of thrombus formation in the lungs and subsequent inhibition of systemic inflammation and cachexia. Conclusions These data provide a rationale for the targeted inhibition of CLEC-2 as a new strategy for preventing hematogenous tumor metastasis and for inhibiting cancer-related thromboembolism.
  • Fumihiko Furuya, Toshihisa Ishii, Shogo Tamura, Kazuya Takahashi, Hidetoshi Kobayashi, Masashi Ichijo, Soichi Takizawa, Masahiro Kaneshige, Katsue Suzuki-Inoue, Kenichiro Kitamura
    SCIENTIFIC REPORTS 7 43960  2045-2322 2017/03 [Refereed][Not invited]
     
    In chronic kidney disease (CKD) patients, inflammation plays a pivotal role in the progression of renal fibrosis. Hypothyroidism is associated with an increased occurrence of atherosclerosis and inflammation, suggesting protective roles of thyroid hormones and their receptors against inflammatory processes. The contribution of thyroid hormone receptors to macrophage differentiation has not been well documented. Here, we focused on the endogenous thyroid hormone receptor a (TR alpha) in macrophages and examined the role of ligand-bound TRa in macrophage polarization-mediated anti-inflammatory effects. TR alpha-deficient irradiated chimeric mice showed exacerbated tubulointerstitial injury in a unilateral ureteral obstruction model. Compared with wild-type macrophages, macrophages isolated from the obstructed kidneys of mice lacking TRa displayed increased expression of proinflammatory cytokines that was accompanied by enhanced nuclear translocation of p65. Comparison of TRa-deficient bone marrow-derived macrophages with wild-type macrophages confirmed the propensity of the former cells to produce excessive IL-1 beta levels. Co-culture of these macrophages with renal epithelial cells induced more severe damage to the epithelial cells via the IL-1 receptor. Our findings indicate that ligand-bound TRa on macrophages plays a protective role in kidney inflammation through the inhibition of NF-kappa B pathways, possibly by affecting the pro-and anti-inflammatory balance that controls the development of CKD.
  • 田村彰吾, 井上克枝, 小嶋哲人
    血液フロンティア 医薬ジャーナル社 27 (6) 37-46 - 820,785 1344-6940 2017 [Not refereed][Invited]
  • 血小板・巨核球造血微小環境を構成する骨髄間質細胞
    田村彰吾, 尾崎由基男, 小嶋哲人, 井上克枝
    日本血栓止血学会誌 28 (1) 59-63  2017 [Not refereed][Not invited]
  • Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance.
    Takagi Yuki, Murata Moe, Kozuka Toshihiro, Nakata Yukiko, Hasebe Ryo, Tamura Shogo, Takagi Akira, Matsushita Tadashi, Saito Hidehiko, Kojima Tetsuhito
    Thrombosis and Haemostasis 116 (6) 1-10  2016/09/08 [Refereed][Not invited]
  • New horizon in platelet function: with special reference to a recently-found molecule, CLEC-2.
    Ozaki Yukio, Tamura Shogo, Suzuki-Inoue Katsue
    Thrombosis journal 14 27-36  2016/09/06 [Refereed][Not invited]
  • Progestin isoforms provide different levels of protein S expression in HepG2 cells.
    Kozuka Toshihiro, Tamura Shogo, Kawamura Nami, Nakata Yukiko, Hasebe Ryo, Makiyama, Ayumi, Takagi Yuki, Murata Moe, Mizutani Naoki, Takagi Akira, Kojima Tetsuhito
    Thrombosis Research 145 40-45  2016/07/16 [Refereed][Not invited]
  • Shogo Tamura, Katsue Suzuki-Inoue, Nagaharu Tsukiji, Toshiaki Shirai, Tomoyuki Sasaki, Makoto Osada, Kaneo Satoh, Yukio Ozaki
    BLOOD 127 (13) 1701 - 1710 0006-4971 2016/03 [Refereed][Not invited]
     
    Megakaryopoiesis is the hierarchical differentiation of hematopoietic stem cells into megakaryocytes. Differentiating megakaryocytes undergo maturation characterized by endomitosis and produce numerous platelets through proplatelet formation. C-type lectin-like receptor 2 (CLEC-2) is a podoplanin (PDPN) receptor mainly expressed on platelets and megakaryocytes. Deletion of platelet/megakaryocyte CLEC-2 causes thrombocytopenia in mice; however, its contribution to megakaryopoiesis remains unknown. Here, we show that megakaryopoiesis is promoted through the CLEC-2/PDPN interaction in the vicinity of arterioles in the bone marrow (BM). We have also identified PDPN-expressing BM arteriolar stromal cells, tentatively termed as BM fibroblastic reticular cell (FRC)-like cells. Platelet/megakaryocyte-specific CLEC-2 conditional knockout (cKO) mice showed a decrease in the number of immature megakaryocytes. CLEC-2 wild-type megakaryocyte expansion was augmented in vitro by the addition of recombinant PDPN, but not cKO megakaryocytes. Moreover, megakaryocyte colonies were colocalized with periarteriolar BM FRC-like cells in the BM. Coculture of megakaryocytes with BM FRC-like cells augmented megakaryocyte expansion, which was dependent upon the CLEC-2/PDPN interaction. Furthermore, we found that the CLEC-2/PDPN interaction induces BM FRC-like cells to secrete chemokine (C-C motif) ligand 5 (CCL5) to facilitate proplatelet formation. These observations indicate that a reciprocal interaction between CLEC-2 on megakaryocytes and PDPN on BM FRC-like cells contributes to the periarteriolar megakaryopoietic microenvironment in mouse BM.
  • Takanori Moriyama, Shogo Tamura, Keiichi Nakano, Kohei Otsuka, Masahiko Shigemura, Naoyuki Honma
    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1854 (6) 658 - 667 1570-9639 2015/06 [Refereed][Not invited]
     
    We report the analysis of unusual macroenzymes, performed in our laboratory, and review the relevant literature. In particular, we focused on macro AST, macroamylase, macro LD and macro CK. Macroenzymes are seen in healthy subjects, but can also be related to disease; thus, accurate detection is useful in day-to-day clinical practice. The macroenzyme is thought to be a specific antigen-antibody complex from the following findings: (1) the complex could be dissociated under acidic pH levels; (2) binding specificity of immunoglobulin in the complex was observed; (3) the binding site of immunoglobulin in the complex was Fab portion; and (4) the maternal IgG involved with macroenzyme was transferred to her children. This article is part of a Special Issue entitled: Medical Proteomics. (C) 2014 Elsevier B.V. All rights reserved.
  • Yuya Masuda, Shogo Tamura, Kazuhiko Matsuno, Ayumi Nagasawa, Koji Hayasaka, Chikara Shimizu, Takanori Moriyama
    THROMBOSIS RESEARCH 135 (5) 951 - 957 0049-3848 2015/05 [Refereed][Not invited]
     
    Introduction: CD36 is a multifunctional glycoprotein expressed on various human cells, including platelets and monocytes. Five CD36 gene mutations (C268T, 949insA, 329-339del, 1228-1239del and 629-631del/insAAAAC) are mainly responsible for CD36-deficient phenotypes in Japan. It has also been reported that platelet CD36 expression varies widely among normal phenotype individuals. Here, in order to obtain further insight into CD36 expression, we investigated the association between platelet and monocyte CD36 expression levels and defective mutations in the Japanese population. Materials and Methods: Blood samples were collected from 135 healthy Japanese volunteers. CD36 expression levels on platelets and monocytes were quantitatively analyzed by flow cytometry. Real-time PCR, PCR-RFLP and allele-specific PCR were performed to detect mutant genotypes. Results: In this population, we found 2 (1.5%) and 9 (6.7%) CD36-deficient subjects as type I and type II, respectively. Among normal phenotype subjects, CD36 expression levels ranged from 1,259 to 11,002 (4,487 +/- 2,017) molecules/platelet and from 211 to 5,150 (1,628 +/- 986) molecules/monocyte. Genotyping assay showed that heterozygotes with the defective mutations were present in normal (12.9%) and type II-deficient (66.7%) subjects, and that these heterozygous mutations led to decreases in CD36 surface expression on platelets and monocytes. Conclusions: Heterozygous CD36 mutations, previously known to lead to deficiency in this molecule, are one of the factors responsible for the diversity of CD36 surface expression levels on platelets and monocytes in normal phenotype subjects. (C) 2015 Elsevier Ltd. All rights reserved.
  • Fuminori Kazama, Junya Nakamura, Makoto Osada, Osamu Inoue, Mitsuru Oosawa, Shogo Tamura, Nagaharu Tsukiji, Kaoru Aida, Akio Kawaguchi, Soichi Takizawa, Masahiro Kaneshige, Shoichiro Tanaka, Katsue Suzuki-inoue, Yukio Ozaki
    PLATELETS 26 (8) 711 - 719 0953-7104 2015 [Refereed][Not invited]
     
    Detection of platelet activation in vivo is useful to identify patients at risk of thrombotic diseases. Platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) are used for this purpose; however, they are easily released upon the minimal platelet activation that occurs during sampling. Soluble forms of several platelet membrane proteins are released upon platelet activation; however, the soluble form of C-type lectin-like receptor 2 (sCLEC-2) has not yet been fully investigated. Western blotting with an anti-CLEC-2 antibody showed that sCLEC-2 was released from washed human platelets stimulated with collagen mimetics. To detect sCLEC-2 in plasma, we established a sandwich enzyme-linked immunosorbent assay (ELISA) using F(ab')2 anti-CLEC-2 monoclonal antibodies. Although plasma mixed with citrate, adenosine, theophylline and adenosine (CTAD) is needed for the PF4 and beta-TG assays, effects of anti-coagulants (EDTA, citrate and CTAD) on the sCLEC-2 ELISA were negligible. Moreover, while special techniques are required for blood sampling and sample preparation for PF4 and beta-TG assay, the standard blood collections procedures used in daily clinical laboratory tests have shown to suffice for sCLEC-2 analysis. In this study, we found that two forms of sCLEC-2 are released after platelet activation: a shed fragment and a microparticle-bound full-length protein, both of which are detected by the sCLEC-2 ELISA. The average concentration of sCLEC-2 in the plasma of 10 healthy individuals was 97 +/- 55 pg/ml, whereas that in the plasma of 25 patients with diabetes mellitus (DM) was 149 +/- 260 pg/ml. A trend towards an increase in sCLEC-2 concentration in the DM patients may reflect in vivo platelet activation in the patients, suggesting that sCLEC-2 may have clinical significance as a biomarker of in vivo platelet activation.
  • A. Nagasawa, K. Matsuno, S. Tamura, K. Hayasaka, C. Shimizu, T. Moriyama
    INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY 35 (5) 534 - 541 1751-5521 2013/10 [Refereed][Not invited]
     
    IntroductionPlatelet activation in circulation is considered to be associated with thrombosis and inflammation; thus, sensitive and easy-to-use markers are necessary. In this study, we established a simple and rapid protocol to clinically examine leukocyte-platelet aggregate formation associated with activated platelets in circulation. MethodsWhole blood was stained with PC5-conjugated anti-CD45 monoclonal antibody and fluorescent isothiocyanate-conjugated anti-CD41 monoclonal antibody for leukocyte-platelet aggregate analysis. For platelet activation, 5m thrombin receptor-activated peptide (TRAP) or 2g/mL collagen was added. Samples were analyzed by EPICS XL (Beckman Coulter, Miami, FL, USA). Monocytes, neutrophils, and lymphocytes were gated based on differences in CD45 fluorescence intensity and side scatter. For each gate, the percentage (%) of platelets expressing CD41 was analyzed. Same drawing sample was stained with anti-CD62P monoclonal antibody. Platelet CD62P expression was then analyzed with gating for platelet cell population. ResultsWe analyzed leukocyte-platelet aggregates and platelet CD62P expression in 18 healthy individuals. Leukocyte-platelet aggregates, mainly monocyte-platelet aggregates, increased when platelets were activated by platelet agonists. Monocyte-platelet aggregates and neutrophil-platelet aggregates also increased over time with mild platelet activation. ConclusionLeukocyte-platelet aggregates, mainly monocyte-platelet aggregates, appear to be a sensitive marker of platelet activation in circulation.
  • Keiichi Nakano, Shogo Tamura, Kohei Otuka, Noriyasu Niizeki, Masahiko Shigemura, Chikara Shimizu, Kazuhiko Matsuno, Seiichi Kobayashi, Takanori Moriyama
    Analytical Biochemistry 438 (2) 117 - 123 0003-2697 2013/07/15 [Refereed][Not invited]
     
    Three-dimensional gel electrophoresis (3-DE), which combines agarose gel electrophoresis and isoelectric focusing/SDS-PAGE, was developed to characterize monoclonal proteins (M-proteins). However, the original 3-DE method has not been optimized and its specificity has not been demonstrated. The main goal of this study was to optimize the 3-DE procedure and then compare it with 2-DE. We developed a highly sensitive 3-DE method in which M-proteins are extracted from a first-dimension agarose gel, by diffusing into 150 mM NaCl, and the recovery of M-proteins was 90.6%. To validate the utility of the highly sensitive 3-DE, we compared it with the original 3-DE method. We found that highly sensitive 3-DE provided for greater M-protein recovery and was more effective in terms of detecting spots on SDS-PAGE gels than the original 3-DE. Moreover, highly sensitive 3-DE separates residual normal IgG from M-proteins, which could not be done by 2-DE. Applying the highly sensitive 3-DE to clinical samples, we found that the characteristics of M-proteins vary tremendously between individuals. We believe that our highly sensitive 3-DE method described here will prove useful in further studies of the heterogeneity of M-proteins.© 2013 Elsevier Inc. All rights reserved.
  • Reo Nozaki, Shogo Tamura, Aimi Ito, Takanori Moriyama, Kikuji Yamaguchi, Toru Kono
    FOOD CHEMISTRY 134 (4) 2332 - 2337 0308-8146 2012/10 [Refereed][Not invited]
     
    Soluble royal jelly (RJ) proteins (SRJPs) include the major RJ protein (MRJP) family, which contribute to the physiological actions of RJ. Although SRJPs are prepared using conventional methods involving dialysis and centrifugation, dialysis is a time-consuming process. We have therefore developed a simple method to isolate SRJPs from RJ. This new method produces 20-fold higher levels of SRJPs than that of the conventional procedure; hence, the levels obtained by the new and existing methods were compared. A 1-h ultracentrifugation separated SRJPs in the supernatant into upper, middle and lower layers. Each layer was analyzed by size-exclusion HPLC, SDS-PAGE and 2-DE. The upper and middle layers contained MRJP2 (52 kDa) and MRJP3 (60-70 kDa), while the lower layer contained MRJP1 (290 kDa). In nature, MRJP1 is a monomer and/or oligomer. When the lower layer was analyzed by Superose 12 HPLC, MRJP1 was predominantly an oligomer. Our MRJP isolation method reduces the procedure time by using ultracentrifugation without dialysis to obtain SRIPs and produces layers containing MRJP1 oligomers, MRJP2 and MRJP3. (C) 2012 Elsevier Ltd. All rights reserved.
  • Shogo Tamura, Ayumi Nagasawa, Yuya Masuda, Tetsuya Tsunematsu, Koji Hayasaka, Kazuhiko Matsuno, Chikara Shimizu, Yukio Ozaki, Takanori Moriyama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 427 (3) 542 - 546 0006-291X 2012/10 [Refereed][Not invited]
     
    While human platelets release endogenous brain-derived neurotrophic factor (BDNF) upon activation, a previous report on MEG-01, a megakaryocytic cell line, found no trace of BDNF production, and the pathophysiological function of platelet BDNF has remained elusive. In the present study, we demonstrate that MEG-01 produces BDNF in the presence of TPO and that this serves to potentiate cell proliferation. Our in vitro findings suggest that BDNF regulates MEG-01 proliferation in an autocrine manner, and we suggest that BDNF may be a physiological autocrine regulator of megakaryocyte progenitors. (C) 2012 Elsevier Inc. All rights reserved.
  • Shogo Tamura, Hidenori Suzuki, Yuji Hirowatari, Masanao Hatase, Ayumi Nagasawa, Kazuhiko Matsuno, Seiichi Kobayashi, Takanori Moriyama
    THROMBOSIS RESEARCH 128 (5) E55 - E61 0049-3848 2011/11 [Refereed][Not invited]
     
    Brain-derived neurotrophic factor (BDNF) is a cytokine that plays important roles in the survival, development, and plasticity of neurons. BDNF is also expressed in peripheral tissues and cells. In this article, we report the BDNF release reaction through thrombin stimulation and its localization in human platelets. Platelets from healthy volunteers were subjected to PAR1-AP or PAR4-AP stimulation. Release of BDNF was measured by ELISA. Localization of BDNF in resting and thrombin-activated platelets was examined by immunoelectron microscopy and sucrose gradient ultracentrifugation following western blotting. BDNF was released dose-dependently with PAR1-AP concentrations with drastic release at low PAR1-AP concentrations and gently release at high PAR1-AP concentrations. Maximum BDNF release was approximately 37% at 132 mu M PAR1-AP. In contrast, 3.8% BDNF was released with 1.13 mM PAR4-AP stimulation. In immunoelectron microscopy and sucrose gradient ultracentrifugation analyses, BDNF was detected not only in alpha-granules but also cytoplasm in of the resting platelets, and it was distributed in the swollen open canalicular system fused to alpha-granules at 1 min and disappeared at 5 min after stimulation by thrombin. However, BDNF in cytoplasm remained throughout platelet activation. In conclusions, we demonstrate that BDNF is released from platelets through predominately PAR1 regulation. Furthermore, we identified two pools of BDNF in the alpha-granules and cytoplasm of human platelets, and only BDNF in alpha-granules is released through platelet activation. (C) 2011 Elsevier Ltd. All rights reserved.
  • Shougo Tamura, Shizuka Amano, Toru Kono, Jun Kondoh, Kikuji Yamaguchi, Seiichi Kobayashi, Tokiyoshi Ayabe, Takanori Moriyama
    PROTEOMICS 9 (24) 5534 - 5543 1615-9853 2009/12 [Refereed][Not invited]
     
    Royal jelly contains numerous components, including proteins. Major royal jelly protein (MRJP) 1 is the most abundant protein among the soluble royal jelly proteins. In its physiological state, MRJP 1 exists as a monomer and/or oligomer. This study focuses the molecular characteristics and functions of MRJP 1 oligomer. MRJP 1 oligomer purified using HPLC techniques was subjected to the following analyses. The molecular weight of MRJP 1 oligomer was found to be 290 kDa using blue native-PAGE. MRJP 1 oligomer was separated into 55 and 5 kDa spots on 2-D blue native/SDS-PAGE. The 55 kDa protein was identified as MRJP 1 monomer by proteome analysis, whereas the 5 kDa protein was identified as Apisimin by N-terminal amino acid sequencing, and this protein may function as a subunit-joining protein within MRJP 1 oligomer. We also found that the oligomeric form included noncovalent bonds and was stable under heat treatment at 56 C. Furthermore, MRJP 1 oligomer dose dependently enhanced and sustained cell proliferation in the human lymphoid cell line Jurkat. In conclusion, MRJP 1 oligomer is a heat-resistant protein comprising MRJP 1 monomer and Apisimin, and has cell proliferation activity. These findings will contribute to further studies analyzing the effects of MRJP 1 in humans.
  • Shougo Tamura, Toru Kono, Chika Harada, Kikuji Yamaguchi, Takanori Moriyama
    FOOD CHEMISTRY 114 (4) 1491 - 1497 0308-8146 2009/06 [Refereed][Not invited]
     
    Royal jelly (RJ) contains many components, including proteins. We focused on major royal jelly proteins (MRJPs) under natural conditions, and attempted to determine the content ratios and molecular forms of MRJPs by size-exclusion HPLC, SDS-PAGE, 2-DE and MALDI TOF/TOF MS. Soluble RJ proteins were extracted by dialysis followed by several centrifugation techniques. Soluble RJ proteins were universally separated into five peaks (640 kDa, 280 kDa, 100 kDa, 72 kDa and 4.5 kDa) by size-exclusion HPLC on a Superose 12 column. Among these peaks, both the 280 kDa and 72 kDa peaks were major, but the intensity of the 280 kDa peak differed markedly among original RJ samples (n = 70). The main 280 kDa protein was separated into a 55 kDa band by reducing and non-reducing SDS-PAGE. This protein was also separated into multiple spots ranging from pH 4.2 to 6.5 by 2-DE. These spots were identified as MRJP 1 by MALDI TOF/TOF MS. From these results, MRJP I was thought to comprise an oligomer complex linked by non-covalent bonds under natural conditions. Another major protein, the 72 kDa peak on Superose 12 HPLC, was identified as MRJP 2. (c) 2008 Elsevier Ltd. All rights reserved.

Books etc

  • みんなに役立つ血友病の基礎と臨床
    田村彰吾, 小島哲人 (Joint work)
    医薬ジャーナル社 2016/08 55-61
  • 新染色法のすべて、化学検査:アイソザイム染色
    森山隆則, 中野恵一, 畑瀬正尚, 田村彰吾 (Joint work)
    医歯薬出版株式会社 2011/03 396-403

MISC

  • 最近の血栓止血異常のとらえかた 検査室や研究室から 先天性凝固異常症の遺伝子解析 解析のStrategyとPitfall
    田村 彰吾, 高木 明, 早川 文彦, 小嶋 哲人  日本検査血液学会雑誌  21-  (1)  71  -81  2020/02  [Not refereed][Not invited]
     
    先天性血液凝固異常症はその病態により「出血性疾患」と「血栓性疾患」の2つにわけられ、それぞれ遺伝性の出血性素因と血栓性素因を保有することに起因する。先天性出血性疾患の代表例は血友病であり、血友病Aは血液凝固第VIII遺伝子(F8)、血友病Bは血液凝固第IX遺伝子(F9)に生じた変異によって引き起こされる。血友病の病因遺伝子変異は多様であり、病因変異の同定は血友病の確定診断のみならず、病型・病態の把握やインヒビター発生リスクの予測につながるなど、個々の患者の特徴を把握する上で重要な情報である。先天性血栓性疾患は主に血液凝固制御因子であるアンチトロンビン、プロテインC、プロテインSの欠乏症・異常症であり、それぞれの責任遺伝子であるSERPINC1、PROC、PROS1の変異を原因とする。これらの先天性血栓性疾患は2017年4月に「特発性血栓症(遺伝性血栓性素因によるものに限る)」として指定難病に認定された。特発性血栓症の診断には血液凝固制御因子遺伝子の変異同定が不可欠であり、正確な診断のためにはそれぞれの遺伝子の特徴を理解した正しい遺伝子解析技術が必須となる。本稿では、当研究室で実施している先天性血液凝固異常症の遺伝子解析のスキームを紹介しつつ、変異同定に苦慮した遺伝子構造異常の症例を提示する。(著者抄録)
  • Nagaharu Tsukiji, Osamu Inoue, Mitsuru Morimoto, Norifumi Tatsumi, Hiroaki Nagatomo, Koji Ueta, Toshiaki Shirai, Tomoyuki Sasaki, Shimon Otake, Shogo Tamura, Toshiaki Tachibana, Masataka Okabe, Masanori Hirashima, Yukio Ozaki, Katsue Suzuki-Inoue  Blood  132-  (11)  1167  -1179  2018/09/13  [Not refereed][Not invited]
     
    Platelets participate in not only thrombosis and hemostasis but also other pathophysiological processes, including tumor metastasis and inflammation. However, the putative role of platelets in the development of solid organs has not yet been described. Here, we report that platelets regulate lung development through the interaction between the platelet-activation receptor, C-type lectin-like receptor-2 (Clec-2; encoded by Clec1b), and its ligand, podoplanin, a membrane protein. Clec-2 deletion in mouse platelets led to lung malformation, which caused respiratory failure and neonatal lethality. In these embryos, α-smooth muscle actin-positive alveolar duct myofibroblasts (adMYFs) were almost absent in the primary alveolar septa, which resulted in loss of alveolar elastic fibers and lung malformation. Our data suggest that the lack of adMYFs is caused by abnormal differentiation of lung mesothelial cells (luMCs), the major progenitor of adMYFs. In the developing lung, podoplanin expression is detected in alveolar epithelial cells (AECs), luMCs, and lymphatic endothelial cells (LECs). LEC-specific podoplanin knockout mice showed neonatal lethality and Clec1b-/--like lung developmental abnormalities. Notably, these Clec1b-/--like lung abnormalities were also observed after thrombocytopenia or transforming growth factor-β depletion in fetuses. We propose that the interaction between Clec-2 on platelets and podoplanin on LECs stimulates adMYF differentiation of luMCs through transforming growth factor-β signaling, thus regulating normal lung development.
  • S. Tamura, Y. Suga, M. Tanamura, M. Murata-Kawakami, Y. Takagi, Y. Hottori, M. Kakihara, S. Suzuki, A. Takagi, T. Kojima  International Journal of Laboratory Hematology  40-  (3)  312  -319  2018/06  [Not refereed][Not invited]
  • T. Sasaki, T. Shirai, N. Tsukiji, S. Otake, S. Tamura, J. Ichikawa, M. Osada, K. Satoh, Y. Ozaki, K. Suzuki-Inoue  Journal of Thrombosis and Haemostasis  16-  (5)  960  -972  2018/05/01  [Not refereed][Not invited]
     
    Essentials We generated recombinant rhodocytin that could aggregate platelets via CLEC-2. Recombinant wild-type rhodocytin formed heterooctamer with four α- and β-subunits. Asp 4 in α-subunit of rhodocytin was required for binding to CLEC-2. Inhibitory mutant of rhodocytin blocked podoplanin-dependent hematogenous metastasis. Summary: Background Rhodocytin, a disulfide-linked heterodimeric C-type lectin from Calloselasma rhodostoma consisting of α-subunits and β-subunits, induces platelet aggregation through C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a physiological binding partner of podoplanin (PDPN), which is expressed on some tumor cell types, and is involved in tumor cell-induced platelet aggregation and tumor metastasis. Thus, modified rhodocytin may be a possible source of anti-CLEC-2 drugs for both antiplatelet and antimetastasis therapy. However, its molecular function has not been well characterized, because of the lack of recombinant rhodocytin that induces platelet aggregation. Objective To produce recombinant rhodocytin, in order to verify its function with mutagenesis, and to develop an anti-CLEC-2 drug based on the findings. Methods We used Chinese hamster ovary cells to express recombinant rhodocytin (wild-type [WT] and mutant), which was analyzed for induction/inhibition of platelet aggregation with light transmission aggregometry, the formation of multimers with blue native PAGE, and binding to CLEC-2 with flow cytometry. Finally, we investigated whether mutant rhodocytin could suppress PDPN-induced metastasis in an experimental lung metastasis mouse model. Results Functional WT] rhodocytin (αWTβWT) was obtained by coexpression of both subunits. Asp4 in α-subunits of rhodocytin was required for CLEC-2 binding. αWTβWT formed a heterooctamer similarly to native rhodocytin. Moreover, an inhibitory mutant of rhodocytin (αWTβK53A/R56A), forming a heterotetramer, bound to CLEC-2 without inducing platelet aggregation, and blocked CLEC-2–PDPN interaction-dependent platelet aggregation and experimental lung metastasis. Conclusion These findings provide molecular characterization information on rhodocytin, and suggest that mutant rhodocytin could be used as a therapeutic agent to target CLEC-2.
  • Akitoshi Nakano, Kento Sugawara, Shinya Tamura, Naoyuki Katayama, Kazuyuki Matsubayashi, Taku Okada, Yoshiya Uwatoko, Kouji Munakata, Akiko Nakao, Hajime Sagayama, Reiji Kumai, Kunihisa Sugimoto, Naoyuki Maejima, Akihiko Machida, Tetsu Watanuki, Hiroshi Sawa  IUCrJ  5-  158  -165  2018/03/01  [Not refereed][Not invited]
     
    The crystal structure of the excitonic insulator Ta2NiSe5 has been investigated under a range of pressures, as determined by the complementary analysis of both single-crystal and powder synchrotron X-ray diffraction measurements. The monoclinic ambient-pressure excitonic insulator phase II transforms upon warming or under a modest pressure to give the semiconducting C-centred orthorhombic phase I. At higher pressures (i.e. > 3GPa), transformation to the primitive orthorhombic semimetal phase III occurs. This transformation from phase I to phase III is a pressure-induced first-order phase transition, which takes place through coherent sliding between weakly coupled layers. This structural phase transition is significantly influenced by Coulombic interactions in the geometric arrangement between interlayer Se ions. Furthermore, upon cooling, phase III transforms into the monoclinic phase IV, which is analogous to the excitonic insulator phase II. Finally, the excitonic interactions appear to be retained despite the observed layer sliding transition.
  • James Slack, on behalf of the, Michael H. Albert, Dmitry Balashov, Bernd H. Belohradsky, Alice Bertaina, Jack Bleesing, Claire Booth, Jochen Buechner, Rebecca H. Buckley, Marie Ouachée-Chardin, Elena Deripapa, Katarzyna Drabko, Mary Eapen, Tobias Feuchtinger, Andrea Finocchi, H. Bobby Gaspar, Sujal Ghosh, Alfred Gillio, Luis I. Gonzalez-Granado, Eyal Grunebaum, Tayfun Güngör, Carsten Heilmann, Merja Helminen, Kohei Higuchi, Kohsuke Imai, Krzysztof Kalwak, Nubuo Kanazawa, Gülsün Karasu, Zeynep Y. Kucuk, Alexandra Laberko, Andrzej Lange, Nizar Mahlaoui, Roland Meisel, D. Moshous, Hideki Muramatsu, Suhag Parikh, Srdjan Pasic, Irene Schmid, Catharina Schuetz, Ansgar Schulz, Kirk R. Schultz, Peter J. Shaw, Mary A. Slatter, Karl-Walter Sykora, Shinobu Tamura, Mervi Taskinen, Angela Wawer, Beata Wolska-Kuśnierz, Morton J. Cowan, Alain Fischer, Andrew R. Gennery  Journal of Allergy and Clinical Immunology  141-  (1)  322  -328.e10  2018/01/01  [Not refereed][Not invited]
     
    Background Rare DNA breakage repair disorders predispose to infection and lymphoreticular malignancies. Hematopoietic cell transplantation (HCT) is curative, but coadministered chemotherapy or radiotherapy is damaging because of systemic radiosensitivity. We collected HCT outcome data for Nijmegen breakage syndrome, DNA ligase IV deficiency, Cernunnos–XRCC4-like factor (Cernunnos-XLF) deficiency, and ataxia-telangiectasia (AT). Methods Data from 38 centers worldwide, including indication, donor, conditioning regimen, graft-versus-host disease, and outcome, were analyzed. Conditioning was classified as myeloablative conditioning (MAC) if it contained radiotherapy or alkylators and reduced-intensity conditioning (RIC) if no alkylators and/or 150 mg/m2 fludarabine or less and 40 mg/kg cyclophosphamide or less were used. Results Fifty-five new, 14 updated, and 18 previously published patients were analyzed. Median age at HCT was 48 months (range, 1.5-552 months). Twenty-nine patients underwent transplantation for infection, 21 had malignancy, 13 had bone marrow failure, 13 received pre-emptive transplantation, 5 had multiple indications, and 6 had no information. Twenty-two received MAC, 59 received RIC, and 4 were infused information was unavailable for 2 patients. Seventy-three of 77 patients with DNA ligase IV deficiency, Cernunnos-XLF deficiency, or Nijmegen breakage syndrome received conditioning. Survival was 53 (69%) of 77 and was worse for those receiving MAC than for those receiving RIC (P =.006). Most deaths occurred early after transplantation, suggesting poor tolerance of conditioning. Survival in patients with AT was 25%. Forty-one (49%) of 83 patients experienced acute GvHD, which was less frequent in those receiving RIC compared with those receiving MAC (26/56 [46%] vs 12/21 [57%], P =.45). Median follow-up was 35 months (range, 2-168 months). No secondary malignancies were reported during 15 years of follow-up. Growth and developmental delay remained after HCT immune-mediated complications resolved. Conclusion RIC HCT resolves DNA repair disorder–associated immunodeficiency. Long-term follow-up is required for secondary malignancy surveillance. Routine HCT for AT is not recommended.
  • S. Suzuki, Y. Nakamura, N. Suzuki, T. Yamazaki, Y. Takagi, S. Tamura, A. Takagi, T. Kanematsu, T. Matsushita, T. Kojima  Haemophilia  24-  (1)  e13  -e16  2018/01/01  [Not refereed][Not invited]
  • Miki Kamiyama, Toshiaki Shirai, Shogo Tamura, Katsue Suzuki-Inoue, Shogo Ehata, Kei Takahashi, Kohei Miyazono, Yoshihiro Hayakawa, Takehiro Sato, Kohsuke Takeda, Isao Naguro, Hidenori Ichijo  CELL DEATH AND DIFFERENTIATION  24-  (12)  2066  -2076  2017/12  [Refereed][Not invited]
     
    Tumor metastasis is the major cause of deaths in cancer patients and is modulated by intertwined stress-responsive signaling cascades. Here we demonstrate that deletion of stress-responsive apoptosis signal-regulating kinase 1 (Ask1) in platelets results in unstable hemostasis and drastic attenuation of tumor lung metastasis, both of which are attributable to platelet dysfunction. Platelet-specific deletion of Ask1 in mice leads to defects in ADP-dependent platelet aggregation, unstable hemostasis and subsequent attenuation of tumor metastasis. We also revealed that activating phosphorylation of Akt is attenuated in Ask1-deficient platelets, contrary to the previous reports suggesting that Akt is negatively regulated by ASK1. Mechanistically, ASK1-JNK/p38 axis phosphorylates an ADP receptor P2Y(12) at Thr345, which is required for the ADP-dependent sustained Akt activity that is vital to normal platelet functions. Our findings offer insight into positive regulation of Akt signaling through P2Y(12) phosphorylation as well as MAPK signaling in platelets by ASK1 and suggest that ASK1-JNK/p38 axis provides a new therapeutic opportunity for tumor metastasis.
  • Miki Kamiyama, Toshiaki Shirai, Shogo Tamura, Katsue Suzuki-Inoue, Shogo Ehata, Kei Takahashi, Kohei Miyazono, Yoshihiro Hayakawa, Takehiro Sato, Kohsuke Takeda, Isao Naguro, Hidenori Ichijo  CELL DEATH AND DIFFERENTIATION  24-  (12)  2066  -2076  2017/12  [Not refereed][Not invited]
     
    Tumor metastasis is the major cause of deaths in cancer patients and is modulated by intertwined stress-responsive signaling cascades. Here we demonstrate that deletion of stress-responsive apoptosis signal-regulating kinase 1 (Ask1) in platelets results in unstable hemostasis and drastic attenuation of tumor lung metastasis, both of which are attributable to platelet dysfunction. Platelet-specific deletion of Ask1 in mice leads to defects in ADP-dependent platelet aggregation, unstable hemostasis and subsequent attenuation of tumor metastasis. We also revealed that activating phosphorylation of Akt is attenuated in Ask1-deficient platelets, contrary to the previous reports suggesting that Akt is negatively regulated by ASK1. Mechanistically, ASK1-JNK/p38 axis phosphorylates an ADP receptor P2Y(12) at Thr345, which is required for the ADP-dependent sustained Akt activity that is vital to normal platelet functions. Our findings offer insight into positive regulation of Akt signaling through P2Y(12) phosphorylation as well as MAPK signaling in platelets by ASK1 and suggest that ASK1-JNK/p38 axis provides a new therapeutic opportunity for tumor metastasis.
  • Shogo Tamura, Moe Murata-Kawakami, Yuki Takagi, Sachiko Suzuki, Akira Katsumi, Akira Takagi, Tetsuhito Kojima  THROMBOSIS RESEARCH  159-  (159)  33  -38  2017/11  [Refereed][Not invited]
     
    Introduction: Antithrombin resistance (ATR) prothrombinemia is an inherited thrombophilic disorder caused by missense mutations in prothrombin gene (F2) at Arg596 of the sodium-binding region. Previously, prothrombin mutants Yukuhashi (Arg596Leu), Belgrade (Arg596Gln), and Padua 2 (Arg596Trp) were reported as ATR-prothrombins possessing a risk of familial venous thrombosis. To identify additional F2 mutations causing the ATR-phenotype, we investigated the coagulant properties of recombinant prothrombins mutated at amino acid residues within the sodium-binding region by single nucleotide substitutions (Thr540, Arg541, Glu592, and Lys599). Materials and methods: We constructed expression vectors of prothrombin mutants, established stably transfected HEK293 cells, and isolated the recombinant prothrombin proteins. We evaluated procoagulant activity and ATR-phenotypes of those mutants in reconstituted plasma by mixing with prothrombin deficient plasma. Results: The secreted quantity of all prothrombin mutants was the same as that of the wild-type prothrombin. Procoagulant activity of each mutant varied from 1.7% to 79.5% in a one-stage clotting assay and from 2.0% to 104.5% in a two-stage chromogenic assay. Most prothrombin mutants tested presented with a severe ATR-phenotype. To estimate the thrombosis risk of these mutations, we determined the residual clotting activity (RCA) after 30 min inactivation with antithrombin. RCA scores, normalized to the wild-type, revealed that prothrombin mutants Lys599Arg (5.35) and Glu592Gln (4.71) had high scores, which were comparable with prothrombins Yukuhashi (4.36) and Belgrade (5.19). Conclusions: Mutation of prothrombin at the sodium-binding site caused ATR-phenotypes. Of those tested, Lys599Arg and Glu592Gln may possess a thrombosis risk as large as the known pathogenic prothrombins Yukuhashi and Belgrade.
  • Shogo Tamura, Moe Murata-Kawakami, Yuki Takagi, Sachiko Suzuki, Akira Katsumi, Akira Takagi, Tetsuhito Kojima  THROMBOSIS RESEARCH  159-  33  -38  2017/11  [Not refereed][Not invited]
     
    Introduction: Antithrombin resistance (ATR) prothrombinemia is an inherited thrombophilic disorder caused by missense mutations in prothrombin gene (F2) at Arg596 of the sodium-binding region. Previously, prothrombin mutants Yukuhashi (Arg596Leu), Belgrade (Arg596Gln), and Padua 2 (Arg596Trp) were reported as ATR-prothrombins possessing a risk of familial venous thrombosis. To identify additional F2 mutations causing the ATR-phenotype, we investigated the coagulant properties of recombinant prothrombins mutated at amino acid residues within the sodium-binding region by single nucleotide substitutions (Thr540, Arg541, Glu592, and Lys599). Materials and methods: We constructed expression vectors of prothrombin mutants, established stably transfected HEK293 cells, and isolated the recombinant prothrombin proteins. We evaluated procoagulant activity and ATR-phenotypes of those mutants in reconstituted plasma by mixing with prothrombin deficient plasma. Results: The secreted quantity of all prothrombin mutants was the same as that of the wild-type prothrombin. Procoagulant activity of each mutant varied from 1.7% to 79.5% in a one-stage clotting assay and from 2.0% to 104.5% in a two-stage chromogenic assay. Most prothrombin mutants tested presented with a severe ATR-phenotype. To estimate the thrombosis risk of these mutations, we determined the residual clotting activity (RCA) after 30 min inactivation with antithrombin. RCA scores, normalized to the wild-type, revealed that prothrombin mutants Lys599Arg (5.35) and Glu592Gln (4.71) had high scores, which were comparable with prothrombins Yukuhashi (4.36) and Belgrade (5.19). Conclusions: Mutation of prothrombin at the sodium-binding site caused ATR-phenotypes. Of those tested, Lys599Arg and Glu592Gln may possess a thrombosis risk as large as the known pathogenic prothrombins Yukuhashi and Belgrade.
  • Changfa Shi, Yuanzhi Cheng, Jinke Wang, Yadong Wang, Kensaku Mori, Shinichi Tamura  MEDICAL IMAGE ANALYSIS  38-  30  -49  2017/05  [Not refereed][Not invited]
     
    One major limiting factor that prevents the accurate delineation of human organs has been the presence of severe pathology and pathology affecting organ borders. Overcoming these limitations is exactly what we are concerned in this study. We propose an automatic method for accurate and robust pathological organ segmentation from CT images. The method is grounded in the active shape model (ASM) framework. It leverages techniques from low-rank and sparse decomposition (LRSD) theory to robustly recover a subspace from grossly corrupted data. We first present a population-specific LRSD-based shape prior model, called LRSD-SM, to handle non-Gaussian gross errors caused by weak and misleading appearance cues of large lesions, complex shape variations, and poor adaptation to the finer local details in a unified framework. For the shape model initialization, we introduce a method based on patient-specific LRSD-based probabilistic atlas (PA), called LRSD-PA, to deal with large errors in atlas-to-target registration and low likelihood of the target organ. Furthermore, to make our segmentation framework more efficient and robust against local minima, we develop a hierarchical ASM search strategy. Our method is tested on the SLIVER07 database for liver segmentation competition, and ranks 3rd in all the published state-of-the-art automatic methods. Our method is also evaluated on some pathological organs (pathological liver and right lung) from 95 clinical CT scans and its results are compared with the three closely related methods. The applicability of the proposed method to segmentation of the various pathological organs (including some highly severe cases) is demonstrated with good results on both quantitative and qualitative experimentation; our segmentation algorithm can delineate organ boundaries that reach a level of accuracy comparable with those of human raters. (C) 2017 Elsevier B.V. All rights reserved.
  • T. Shirai, O. Inoue, S. Tamura, N. Tsukiji, T. Sasaki, H. Endo, K. Satoh, M. Osada, H. Sato-Uchida, H. Fujii, Y. Ozaki, K. Suzuki-Inoue  JOURNAL OF THROMBOSIS AND HAEMOSTASIS  15-  (3)  513  -525  2017/03  [Refereed][Not invited]
     
    Background C-type lectin-like receptor 2 (CLEC-2) is a platelet activation receptor of sialoglycoprotein podoplanin, which is expressed on the surface of certain types of tumor cells. CLEC-2-podoplanin interactions facilitate hematogenous tumor metastasis. However, direct evidence of the role of CLEC-2 in hematogenous metastasis and cancer progression is lacking. Objective and methods We generated immunological CLEC-2-depleted mice by using anti-mouse CLEC-2 monoclonal antibody 2A2B10 and investigated whether CLEC-2 promoted hematogenous tumor metastasis and tumor growth and exacerbated the prognosis of mice bearing podoplanin-expressing B16F10 melanoma cells. Results Our results showed that hematogenous metastasis was significantly inhibited in CLEC-2-depleted mice. B16F10 cells co-cultured with wild-type platelets, but not with CLEC-2-deficient platelets, showed increased proliferation. However, B16F10 cell proliferation was not inhibited in CLEC-2-depleted mice. Histological analysis showed that thrombus formation in tumor vessels was significantly inhibited and functional vessel density was significantly increased in CLEC-2-depleted mice. These data suggest that CLEC-2 deficiency may inhibit thrombus formation in tumor vessels and increase the density of functional vessels, thus improving oxygen and nutrient supply to tumors, indirectly promoting tumor proliferation. Furthermore, the overall survival of CLEC-2-depleted mice was significantly prolonged, which may be due to the suppression of thrombus formation in the lungs and subsequent inhibition of systemic inflammation and cachexia. Conclusions These data provide a rationale for the targeted inhibition of CLEC-2 as a new strategy for preventing hematogenous tumor metastasis and for inhibiting cancer-related thromboembolism.
  • Fumihiko Furuya, Toshihisa Ishii, Shogo Tamura, Kazuya Takahashi, Hidetoshi Kobayashi, Masashi Ichijo, Soichi Takizawa, Masahiro Kaneshige, Katsue Suzuki-Inoue, Kenichiro Kitamura  SCIENTIFIC REPORTS  7-  43960  2017/03  [Refereed][Not invited]
     
    In chronic kidney disease (CKD) patients, inflammation plays a pivotal role in the progression of renal fibrosis. Hypothyroidism is associated with an increased occurrence of atherosclerosis and inflammation, suggesting protective roles of thyroid hormones and their receptors against inflammatory processes. The contribution of thyroid hormone receptors to macrophage differentiation has not been well documented. Here, we focused on the endogenous thyroid hormone receptor a (TR alpha) in macrophages and examined the role of ligand-bound TRa in macrophage polarization-mediated anti-inflammatory effects. TR alpha-deficient irradiated chimeric mice showed exacerbated tubulointerstitial injury in a unilateral ureteral obstruction model. Compared with wild-type macrophages, macrophages isolated from the obstructed kidneys of mice lacking TRa displayed increased expression of proinflammatory cytokines that was accompanied by enhanced nuclear translocation of p65. Comparison of TRa-deficient bone marrow-derived macrophages with wild-type macrophages confirmed the propensity of the former cells to produce excessive IL-1 beta levels. Co-culture of these macrophages with renal epithelial cells induced more severe damage to the epithelial cells via the IL-1 receptor. Our findings indicate that ligand-bound TRa on macrophages plays a protective role in kidney inflammation through the inhibition of NF-kappa B pathways, possibly by affecting the pro-and anti-inflammatory balance that controls the development of CKD.
  • Combined deficiency of factors V and VIII by chance coinheritance of parahaemophilia and haemophilia A, but not by mutations of either LMAN1 or MCFD2, in a Japanese family
    Suzuki, S. Nakamura, Y. Suzuki, N. Yamazaki, T. Takagi, Y. Tamura, S. Takagi, A. Kanematsu, T. Matsushita, T. Kojima, T  Haemophilia  1-3  2017  [Refereed][Not invited]
  • 巨核球造血の微小環境
    田村彰吾, 井上克枝, 小嶋哲人  血液フロンティア  27-  (6)  37-46  2017  [Not refereed][Invited]
  • 血小板・巨核球造血微小環境を構成する骨髄間質細胞
    田村彰吾, 尾崎由基男, 小嶋哲人, 井上克枝  日本血栓止血学会誌  28-  (1)  59-63  2017  [Not refereed][Not invited]
  • Yuki Takagi, Moe Murata, Toshihiro Kozuka, Yukiko Nakata, Ryo Hasebe, Shogo Tamura, Akira Takagi, Tadashi Matsushita, Hidehiko Saito, Tetsuhito Kojima  Thrombosis and haemostasis  116-  (6)  1022  -1031  2016/11/30  [Refereed][Not invited]
     
    Antithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0 %, in contrast to values of all variants were maintained at above 80 %. The thrombin-AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12 %, whereas that of tested variants was 37 %-56 %. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin-TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin-TM affinity-dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM-mediated anticoagulation.
  • New horizon in platelet function: with special reference to a recently-found molecule, CLEC-2.
    Ozaki Yukio, Tamura Shogo, Suzuki-Inoue Katsue  Thrombosis journal  14-  27-36  2016/09/06  [Refereed][Not invited]
  • Progestin isoforms provide different levels of protein S expression in HepG2 cells.
    Kozuka Toshihiro, Tamura Shogo, Kawamura Nami, Nakata Yukiko, Hasebe Ryo, Makiyama, Ayumi, Takagi Yuki, Murata Moe, Mizutani Naoki, Takagi Akira, Kojima Tetsuhito  Thrombosis Research  145-  40-45  2016/07/16  [Refereed][Not invited]
  • Shogo Tamura, Katsue Suzuki-Inoue, Nagaharu Tsukiji, Toshiaki Shirai, Tomoyuki Sasaki, Makoto Osada, Kaneo Satoh, Yukio Ozaki  BLOOD  127-  (13)  1701  -1710  2016/03  [Refereed][Not invited]
     
    Megakaryopoiesis is the hierarchical differentiation of hematopoietic stem cells into megakaryocytes. Differentiating megakaryocytes undergo maturation characterized by endomitosis and produce numerous platelets through proplatelet formation. C-type lectin-like receptor 2 (CLEC-2) is a podoplanin (PDPN) receptor mainly expressed on platelets and megakaryocytes. Deletion of platelet/megakaryocyte CLEC-2 causes thrombocytopenia in mice; however, its contribution to megakaryopoiesis remains unknown. Here, we show that megakaryopoiesis is promoted through the CLEC-2/PDPN interaction in the vicinity of arterioles in the bone marrow (BM). We have also identified PDPN-expressing BM arteriolar stromal cells, tentatively termed as BM fibroblastic reticular cell (FRC)-like cells. Platelet/megakaryocyte-specific CLEC-2 conditional knockout (cKO) mice showed a decrease in the number of immature megakaryocytes. CLEC-2 wild-type megakaryocyte expansion was augmented in vitro by the addition of recombinant PDPN, but not cKO megakaryocytes. Moreover, megakaryocyte colonies were colocalized with periarteriolar BM FRC-like cells in the BM. Coculture of megakaryocytes with BM FRC-like cells augmented megakaryocyte expansion, which was dependent upon the CLEC-2/PDPN interaction. Furthermore, we found that the CLEC-2/PDPN interaction induces BM FRC-like cells to secrete chemokine (C-C motif) ligand 5 (CCL5) to facilitate proplatelet formation. These observations indicate that a reciprocal interaction between CLEC-2 on megakaryocytes and PDPN on BM FRC-like cells contributes to the periarteriolar megakaryopoietic microenvironment in mouse BM.
  • Takanori Moriyama, Shogo Tamura, Keiichi Nakano, Kohei Otsuka, Masahiko Shigemura, Naoyuki Honma  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS  1854-  (6)  658  -667  2015/06  [Refereed][Not invited]
     
    We report the analysis of unusual macroenzymes, performed in our laboratory, and review the relevant literature. In particular, we focused on macro AST, macroamylase, macro LD and macro CK. Macroenzymes are seen in healthy subjects, but can also be related to disease; thus, accurate detection is useful in day-to-day clinical practice. The macroenzyme is thought to be a specific antigen-antibody complex from the following findings: (1) the complex could be dissociated under acidic pH levels; (2) binding specificity of immunoglobulin in the complex was observed; (3) the binding site of immunoglobulin in the complex was Fab portion; and (4) the maternal IgG involved with macroenzyme was transferred to her children. This article is part of a Special Issue entitled: Medical Proteomics. (C) 2014 Elsevier B.V. All rights reserved.
  • Yuya Masuda, Shogo Tamura, Kazuhiko Matsuno, Ayumi Nagasawa, Koji Hayasaka, Chikara Shimizu, Takanori Moriyama  THROMBOSIS RESEARCH  135-  (5)  951  -957  2015/05  [Refereed][Not invited]
     
    Introduction: CD36 is a multifunctional glycoprotein expressed on various human cells, including platelets and monocytes. Five CD36 gene mutations (C268T, 949insA, 329-339del, 1228-1239del and 629-631del/insAAAAC) are mainly responsible for CD36-deficient phenotypes in Japan. It has also been reported that platelet CD36 expression varies widely among normal phenotype individuals. Here, in order to obtain further insight into CD36 expression, we investigated the association between platelet and monocyte CD36 expression levels and defective mutations in the Japanese population. Materials and Methods: Blood samples were collected from 135 healthy Japanese volunteers. CD36 expression levels on platelets and monocytes were quantitatively analyzed by flow cytometry. Real-time PCR, PCR-RFLP and allele-specific PCR were performed to detect mutant genotypes. Results: In this population, we found 2 (1.5%) and 9 (6.7%) CD36-deficient subjects as type I and type II, respectively. Among normal phenotype subjects, CD36 expression levels ranged from 1,259 to 11,002 (4,487 +/- 2,017) molecules/platelet and from 211 to 5,150 (1,628 +/- 986) molecules/monocyte. Genotyping assay showed that heterozygotes with the defective mutations were present in normal (12.9%) and type II-deficient (66.7%) subjects, and that these heterozygous mutations led to decreases in CD36 surface expression on platelets and monocytes. Conclusions: Heterozygous CD36 mutations, previously known to lead to deficiency in this molecule, are one of the factors responsible for the diversity of CD36 surface expression levels on platelets and monocytes in normal phenotype subjects. (C) 2015 Elsevier Ltd. All rights reserved.
  • Fuminori Kazama, Junya Nakamura, Makoto Osada, Osamu Inoue, Mitsuru Oosawa, Shogo Tamura, Nagaharu Tsukiji, Kaoru Aida, Akio Kawaguchi, Soichi Takizawa, Masahiro Kaneshige, Shoichiro Tanaka, Katsue Suzuki-inoue, Yukio Ozaki  PLATELETS  26-  (8)  711  -719  2015  [Refereed][Not invited]
     
    Detection of platelet activation in vivo is useful to identify patients at risk of thrombotic diseases. Platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) are used for this purpose; however, they are easily released upon the minimal platelet activation that occurs during sampling. Soluble forms of several platelet membrane proteins are released upon platelet activation; however, the soluble form of C-type lectin-like receptor 2 (sCLEC-2) has not yet been fully investigated. Western blotting with an anti-CLEC-2 antibody showed that sCLEC-2 was released from washed human platelets stimulated with collagen mimetics. To detect sCLEC-2 in plasma, we established a sandwich enzyme-linked immunosorbent assay (ELISA) using F(ab')2 anti-CLEC-2 monoclonal antibodies. Although plasma mixed with citrate, adenosine, theophylline and adenosine (CTAD) is needed for the PF4 and beta-TG assays, effects of anti-coagulants (EDTA, citrate and CTAD) on the sCLEC-2 ELISA were negligible. Moreover, while special techniques are required for blood sampling and sample preparation for PF4 and beta-TG assay, the standard blood collections procedures used in daily clinical laboratory tests have shown to suffice for sCLEC-2 analysis. In this study, we found that two forms of sCLEC-2 are released after platelet activation: a shed fragment and a microparticle-bound full-length protein, both of which are detected by the sCLEC-2 ELISA. The average concentration of sCLEC-2 in the plasma of 10 healthy individuals was 97 +/- 55 pg/ml, whereas that in the plasma of 25 patients with diabetes mellitus (DM) was 149 +/- 260 pg/ml. A trend towards an increase in sCLEC-2 concentration in the DM patients may reflect in vivo platelet activation in the patients, suggesting that sCLEC-2 may have clinical significance as a biomarker of in vivo platelet activation.
  • A. Nagasawa, K. Matsuno, S. Tamura, K. Hayasaka, C. Shimizu, T. Moriyama  INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY  35-  (5)  534  -541  2013/10  [Refereed][Not invited]
     
    IntroductionPlatelet activation in circulation is considered to be associated with thrombosis and inflammation; thus, sensitive and easy-to-use markers are necessary. In this study, we established a simple and rapid protocol to clinically examine leukocyte-platelet aggregate formation associated with activated platelets in circulation. MethodsWhole blood was stained with PC5-conjugated anti-CD45 monoclonal antibody and fluorescent isothiocyanate-conjugated anti-CD41 monoclonal antibody for leukocyte-platelet aggregate analysis. For platelet activation, 5m thrombin receptor-activated peptide (TRAP) or 2g/mL collagen was added. Samples were analyzed by EPICS XL (Beckman Coulter, Miami, FL, USA). Monocytes, neutrophils, and lymphocytes were gated based on differences in CD45 fluorescence intensity and side scatter. For each gate, the percentage (%) of platelets expressing CD41 was analyzed. Same drawing sample was stained with anti-CD62P monoclonal antibody. Platelet CD62P expression was then analyzed with gating for platelet cell population. ResultsWe analyzed leukocyte-platelet aggregates and platelet CD62P expression in 18 healthy individuals. Leukocyte-platelet aggregates, mainly monocyte-platelet aggregates, increased when platelets were activated by platelet agonists. Monocyte-platelet aggregates and neutrophil-platelet aggregates also increased over time with mild platelet activation. ConclusionLeukocyte-platelet aggregates, mainly monocyte-platelet aggregates, appear to be a sensitive marker of platelet activation in circulation.
  • Keiichi Nakano, Shogo Tamura, Kohei Otuka, Noriyasu Niizeki, Masahiko Shigemura, Chikara Shimizu, Kazuhiko Matsuno, Seiichi Kobayashi, Takanori Moriyama  Analytical Biochemistry  438-  (2)  117  -123  2013/07/15  [Refereed][Not invited]
     
    Three-dimensional gel electrophoresis (3-DE), which combines agarose gel electrophoresis and isoelectric focusing/SDS-PAGE, was developed to characterize monoclonal proteins (M-proteins). However, the original 3-DE method has not been optimized and its specificity has not been demonstrated. The main goal of this study was to optimize the 3-DE procedure and then compare it with 2-DE. We developed a highly sensitive 3-DE method in which M-proteins are extracted from a first-dimension agarose gel, by diffusing into 150 mM NaCl, and the recovery of M-proteins was 90.6%. To validate the utility of the highly sensitive 3-DE, we compared it with the original 3-DE method. We found that highly sensitive 3-DE provided for greater M-protein recovery and was more effective in terms of detecting spots on SDS-PAGE gels than the original 3-DE. Moreover, highly sensitive 3-DE separates residual normal IgG from M-proteins, which could not be done by 2-DE. Applying the highly sensitive 3-DE to clinical samples, we found that the characteristics of M-proteins vary tremendously between individuals. We believe that our highly sensitive 3-DE method described here will prove useful in further studies of the heterogeneity of M-proteins.© 2013 Elsevier Inc. All rights reserved.
  • 【事例で学ぶ 免疫検査異常値への対応】 総論 免疫検査における非特異反応
    森山 隆則, 中野 恵一, 田村 彰吾  Medical Technology  41-  (7)  724  -729  2013/07  [Not refereed][Not invited]
     
    免疫検査における非特異反応の基本的な考え方、患者血清中に想定されるさまざまな非特異(妨害)因子による偽反応について解説した。まれなヒト抗マウス抗体(HAMA)によるCA125・TSH偽陽性反応および抗TSH抗体により異常高値を示した代表的な自験例について解説し、臨床検査医学的な教訓について述べた。(著者抄録)
  • M蛋白多様性解析に向けた高感度3次元電気泳動法の確立
    中野 恵一, 田村 彰吾, 大塚 浩平, 新関 紀康, 重村 雅彦, 澁谷 斉, 松野 一彦, 清水 力, 小林 清一, 森山 隆則  臨床化学  42-  (Suppl.1)  197  -197  2013/07  [Not refereed][Not invited]
  • Reo Nozaki, Shogo Tamura, Aimi Ito, Takanori Moriyama, Kikuji Yamaguchi, Toru Kono  FOOD CHEMISTRY  134-  (4)  2332  -2337  2012/10  [Refereed][Not invited]
     
    Soluble royal jelly (RJ) proteins (SRJPs) include the major RJ protein (MRJP) family, which contribute to the physiological actions of RJ. Although SRJPs are prepared using conventional methods involving dialysis and centrifugation, dialysis is a time-consuming process. We have therefore developed a simple method to isolate SRJPs from RJ. This new method produces 20-fold higher levels of SRJPs than that of the conventional procedure; hence, the levels obtained by the new and existing methods were compared. A 1-h ultracentrifugation separated SRJPs in the supernatant into upper, middle and lower layers. Each layer was analyzed by size-exclusion HPLC, SDS-PAGE and 2-DE. The upper and middle layers contained MRJP2 (52 kDa) and MRJP3 (60-70 kDa), while the lower layer contained MRJP1 (290 kDa). In nature, MRJP1 is a monomer and/or oligomer. When the lower layer was analyzed by Superose 12 HPLC, MRJP1 was predominantly an oligomer. Our MRJP isolation method reduces the procedure time by using ultracentrifugation without dialysis to obtain SRIPs and produces layers containing MRJP1 oligomers, MRJP2 and MRJP3. (C) 2012 Elsevier Ltd. All rights reserved.
  • Shogo Tamura, Ayumi Nagasawa, Yuya Masuda, Tetsuya Tsunematsu, Koji Hayasaka, Kazuhiko Matsuno, Chikara Shimizu, Yukio Ozaki, Takanori Moriyama  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS  427-  (3)  542  -546  2012/10  [Refereed][Not invited]
     
    While human platelets release endogenous brain-derived neurotrophic factor (BDNF) upon activation, a previous report on MEG-01, a megakaryocytic cell line, found no trace of BDNF production, and the pathophysiological function of platelet BDNF has remained elusive. In the present study, we demonstrate that MEG-01 produces BDNF in the presence of TPO and that this serves to potentiate cell proliferation. Our in vitro findings suggest that BDNF regulates MEG-01 proliferation in an autocrine manner, and we suggest that BDNF may be a physiological autocrine regulator of megakaryocyte progenitors. (C) 2012 Elsevier Inc. All rights reserved.
  • Shogo Tamura, Hidenori Suzuki, Yuji Hirowatari, Masanao Hatase, Ayumi Nagasawa, Kazuhiko Matsuno, Seiichi Kobayashi, Takanori Moriyama  THROMBOSIS RESEARCH  128-  (5)  E55  -E61  2011/11  [Refereed][Not invited]
     
    Brain-derived neurotrophic factor (BDNF) is a cytokine that plays important roles in the survival, development, and plasticity of neurons. BDNF is also expressed in peripheral tissues and cells. In this article, we report the BDNF release reaction through thrombin stimulation and its localization in human platelets. Platelets from healthy volunteers were subjected to PAR1-AP or PAR4-AP stimulation. Release of BDNF was measured by ELISA. Localization of BDNF in resting and thrombin-activated platelets was examined by immunoelectron microscopy and sucrose gradient ultracentrifugation following western blotting. BDNF was released dose-dependently with PAR1-AP concentrations with drastic release at low PAR1-AP concentrations and gently release at high PAR1-AP concentrations. Maximum BDNF release was approximately 37% at 132 mu M PAR1-AP. In contrast, 3.8% BDNF was released with 1.13 mM PAR4-AP stimulation. In immunoelectron microscopy and sucrose gradient ultracentrifugation analyses, BDNF was detected not only in alpha-granules but also cytoplasm in of the resting platelets, and it was distributed in the swollen open canalicular system fused to alpha-granules at 1 min and disappeared at 5 min after stimulation by thrombin. However, BDNF in cytoplasm remained throughout platelet activation. In conclusions, we demonstrate that BDNF is released from platelets through predominately PAR1 regulation. Furthermore, we identified two pools of BDNF in the alpha-granules and cytoplasm of human platelets, and only BDNF in alpha-granules is released through platelet activation. (C) 2011 Elsevier Ltd. All rights reserved.
  • Shougo Tamura, Shizuka Amano, Toru Kono, Jun Kondoh, Kikuji Yamaguchi, Seiichi Kobayashi, Tokiyoshi Ayabe, Takanori Moriyama  PROTEOMICS  9-  (24)  5534  -5543  2009/12  [Refereed][Not invited]
     
    Royal jelly contains numerous components, including proteins. Major royal jelly protein (MRJP) 1 is the most abundant protein among the soluble royal jelly proteins. In its physiological state, MRJP 1 exists as a monomer and/or oligomer. This study focuses the molecular characteristics and functions of MRJP 1 oligomer. MRJP 1 oligomer purified using HPLC techniques was subjected to the following analyses. The molecular weight of MRJP 1 oligomer was found to be 290 kDa using blue native-PAGE. MRJP 1 oligomer was separated into 55 and 5 kDa spots on 2-D blue native/SDS-PAGE. The 55 kDa protein was identified as MRJP 1 monomer by proteome analysis, whereas the 5 kDa protein was identified as Apisimin by N-terminal amino acid sequencing, and this protein may function as a subunit-joining protein within MRJP 1 oligomer. We also found that the oligomeric form included noncovalent bonds and was stable under heat treatment at 56 C. Furthermore, MRJP 1 oligomer dose dependently enhanced and sustained cell proliferation in the human lymphoid cell line Jurkat. In conclusion, MRJP 1 oligomer is a heat-resistant protein comprising MRJP 1 monomer and Apisimin, and has cell proliferation activity. These findings will contribute to further studies analyzing the effects of MRJP 1 in humans.
  • Shougo Tamura, Toru Kono, Chika Harada, Kikuji Yamaguchi, Takanori Moriyama  FOOD CHEMISTRY  114-  (4)  1491  -1497  2009/06  [Refereed][Not invited]
     
    Royal jelly (RJ) contains many components, including proteins. We focused on major royal jelly proteins (MRJPs) under natural conditions, and attempted to determine the content ratios and molecular forms of MRJPs by size-exclusion HPLC, SDS-PAGE, 2-DE and MALDI TOF/TOF MS. Soluble RJ proteins were extracted by dialysis followed by several centrifugation techniques. Soluble RJ proteins were universally separated into five peaks (640 kDa, 280 kDa, 100 kDa, 72 kDa and 4.5 kDa) by size-exclusion HPLC on a Superose 12 column. Among these peaks, both the 280 kDa and 72 kDa peaks were major, but the intensity of the 280 kDa peak differed markedly among original RJ samples (n = 70). The main 280 kDa protein was separated into a 55 kDa band by reducing and non-reducing SDS-PAGE. This protein was also separated into multiple spots ranging from pH 4.2 to 6.5 by 2-DE. These spots were identified as MRJP 1 by MALDI TOF/TOF MS. From these results, MRJP I was thought to comprise an oligomer complex linked by non-covalent bonds under natural conditions. Another major protein, the 72 kDa peak on Superose 12 HPLC, was identified as MRJP 2. (c) 2008 Elsevier Ltd. All rights reserved.

Awards & Honors

  • 2016/07 第十二回麒麟塾 麒麟児賞

Research Grants & Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2022/04 -2025/03 
    Author : 勝見 章, 田村 彰吾
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2022/04 -2025/03 
    Author : 田村 彰吾, 築地 長治, 鈴木 伸明
  • 骨髄発生の再現により達成する骨髄オルガノイド開発
    国立研究開発法人科学技術振興機構：創発的研究支援事業
    Date (from‐to) : 2022/04 -2025/03 
    Author : 田村彰吾
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2021/04 -2024/03 
    Author : 鈴木 伸明, 高橋 伸典, 田村 彰吾, 鈴木 敦夫
     
    新たに9症例の血友病A患者の遺伝子解析を実施。研究対象症例は合計57症例の患者群となった。新たに判明したバリアントの中には1例の新規バリアントが含まれた。これらの症例に対し、合成基質法と凝固一段法による血液凝固第VIII因子(FVIII)活性測定を実施した。その結果、13症例が活性測定法の違いにより、活性値が解離する研究対象症例であった。次のステップとして、これらの活性乖離症例がどのようなメカニズムで活性乖離を呈するのかを検討することにしたが、基礎検討として、活性乖離を示すことが判明しているFVIII製剤(アルブトレペノナコグ アルファ)を用いて、活性乖離を示さないFVIII製剤であるルリオクトコグアルファを対照として、FVIII製剤を添加したスパイク検体を作製し、トロンビン生成試験やFVIII活性測定、FVIII抗原量測定、凝固波形解析、モディファイド合成基質法の実験系セットアップを行った。しかし、この検討の過程で、発売されている様々なFVIII製剤は抗原量と活性値の比である比活性(活性値/抗原量)のばらつきが非常に大きく、評価が難しいことが判明した。そのため、患者検体での実験系セットアップに方針転換したが、FVIII以外の検体条件など比較条件を揃えるのが難しく、さらには含まれているFVIII量が絶対的に少ないため、差を評価するのが難しいという課題に直面している。 今回の研究では、研究対象バリアントを保有する患者さんでは本人が関節内出血と認識していない出血、いわゆる微小出血を繰り返し、サイレントに血友病性関節症を発症するという仮説の検証も行う計画であるが、こちらの方は次年度以降に進めていく予定である。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A)
    Date (from‐to) : 2017/04 -2021/03 
    Author : Tamura Shogo
     
    This study suggested that a cellular origin of the bone marrow PDPN-expressing stromal cells discovered by the principal investigator is skeletal stem cells (SSCs). We analyzed the distribution of bone marrow PDPN-expressing stromal cells during bone and bone marrow development, especially in the secondary ossification center marrow formed at the epiphysis. We found that, during the formation of the secondary ossification center at the ends of bones, PDPN-positive cells invaded into the secondary ossification center along with periosteal vasculatures and populated the primary marrow. Furthermore, using an in vitro model, we found that PDPN-positive stromal cells maintained vascular homeostasis as a physiological function.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)
    Date (from‐to) : 2018/06 -2020/03 
    Author : Tamura Shogo
     
    The aim of this study is to establish a high efficient in vitro culture system of megakaryocyte/platelet using a novel bone marrow podoplanin (PDPN) positive stromal cells which we previously identified. PDPN positive stromal cells obviously promoted an expansion of megakaryo-progenitor in vitro. However, in the co-culture system with PDPN positive stromal cells and megakaryocytes, we found that platelet collection rate was significantly reduced because of unexpected platelet adhesion onto stromal cells.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
    Date (from‐to) : 2014/04 -2017/03 
    Author : Tamura Shogo, Inoue Katsue, Ozaki Yukio, Tsukiji Nagaharu, Shirai Toshiaki
     
    C-type lectin-like receptor 2 (CLEC-2) is a platelet activating receptor which binds to podoplanin (PDPN). In this study, we investigated a physiological function of CLEC-2 and/or PDPN for meagkaryo/thrombopoiesis. The interaction of CLEC-2 with PDPN directly accelerated expansion of megakaryocyte progenitors. We also newly identified PDPN expressing bone marrow stromal cells adjacent to arterioles in the bone marrow, and termed as BM FRC-like cells. Now, we propose that BM FRC-like cells provide a novel CLEC-2/PDPN dependent megakaryopoietic microenvironment in the vicinity of arterioles in the bone marrow.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2014/04 -2017/03 
    Author : OZAKI Yukio
     
    Previously, we reported that brain-derived neurotrophic factor (BDNF) had a novel function which involved in a megakaryocyte differentiation using human megakaryocyte cell-line model, MEG-01. In this study, we investigated whether BDNF affected normal primary megakaryocyte development in mice. BDNF accelerated clonal expansion of primary megakaryocyte progenitor in vitro. On the other hand, megakaryocyte maturation was not promoted by BDNF. These results suggested that BDNF had a potential as a Meg-CSF for primary megakaryocyte. However, BDNF did not show the physiological function to promote a platelet recovery in transient thrombocytopenic mouse model with anti-platelet antibody. Last year, it was reported that mouse megakaryocytes did not produce BDNF, whereas human megakaryocytes produce BDNF and platelets stored abundant BDNF in alpha-granules. To further investigate a BDFN pathophysiological function for in vivo megakaryopoiesis, an alternative in vivo model should be demanded.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for JSPS Fellows
    Date (from‐to) : 2013/04 -2016/03 
    Author : 田村 彰吾
     
    これまでのin vitroの検討から、BDNFはマウス正常巨核球の自己増殖（clonal expansion）を促進することが明らかになった。そこで平成27年度はin vivoにおけるBDNFの巨核球・血小板造血への関与を検討した。血小板減少症モデルマウスにおいてBDNFは血小板数の回復を促進すると考え、本検討では抗マウスGPIb抗体カクテル投与による一過性血小板減少症モデルマウスを用いたBDNF投与実験を実施した。しかしながら、今回のモデルではBDNF投与マウスの有意な血小板数回復は認められなかった。本課題の今後の方針は、骨髄抑制モデルなど他の血小板減少症モデルにおけるBDNFの血小板・巨核球増殖促進作用の検討が挙げられる。 一方、申請者は本課題以外に血小板活性化受容体CLEC-2の血小板造血における役割に関する研究も手掛けた。CLEC-2の生体内リガンドである膜蛋白、ポドプラニンを発現した新規の間質細胞を同定し、同細胞のポドプラニンと巨核球のCLEC-2が結合して全く新しいニッシェが形成されることを見出した。このニッシェで、ポドプラニン刺激で巨核球の増殖が促進され、CLEC-2の刺激で間質細胞が産生するサイトカインが巨核球のproplatelet形成を促進するという、画期的な機序を解明した。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for JSPS Fellows
    Date (from‐to) : 2010/04 -2013/03 
    Author : 田村 彰吾
     
    脳由来神経栄養因子(Brain-derived neurotrophic factor, BDNF)は循環血液中では大部分が血小板に貯蔵されていることが知られているが、血小板母細胞である巨核球でのBDNFの産生はcell lineを用いたin vitroの系では認められず、巨核球はBDNF産生能力が欠如していると考えられてきた。しかしながら我々のこれまでの検討によって血小板母細胞である巨核球によるBDNFの産生の可能性がが提示された。そこで我々は巨核球系細胞株のMEG-01を用いたBDNF産生能の解析を実施した。その結果、MEG-01はTPO存在下でBDNFを産生する事を明らかにした。また、産生されたBDNFはautocrine様式によって自己利用され、MEG-01の細胞増殖を促進する事が明らかになった。一方、MEG-01の多核化に対してBDNFは促進的作用を示さず、むしろ多核化抑制傾向が認められた。造血幹細胞から巨核球産生に至る分化成熟過程はmegakaryopoiesisと呼ばれ、早期の自己増殖能に特徴づけられる分化過程と、それに続発する多核化・細胞質膨張が起こる成熟過程に分けられる。本検討により得られた知見から、BDNFはMEG-01に対して自己細胞増殖を促進するmegakaryocyte-colony stimulating factor(MEG-CSF)様作用を呈する事が明らかになり、BDNFがmegakaryopoiesisに関与する新しいサイトカインである可能性が示唆された。

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