Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese   Publishing type：(MISC) Introduction and explanation (scientific journal)  

DOI： 10.2491/jjsth.32.504

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.7883/yoken.JJID.2021.091

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1111/jth.15317

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society of Hematology  

<title>Abstract</title>
There is increasing evidence that platelets participate in multiple pathophysiological processes other than thrombosis and hemostasis, such as immunity, inflammation, embryonic development, and cancer progression. A recent study revealed that heme (hemin)-activated platelets induce macrophage extracellular traps (METs) and exacerbate rhabdomyolysis-induced acute kidney injury (RAKI); however, how hemin activates platelets remains unclear. Here, we report that both C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI (GPVI) are platelet hemin receptors and are involved in the exacerbation of RAKI. We investigated hemin-induced platelet aggregation in humans and mice, binding of hemin to CLEC-2 and GPVI, the RAKI-associated phenotype in a mouse model, and in vitro MET formation. Using western blotting and surface plasmon resonance, we showed that hemin activates human platelets by stimulating the phosphorylation of SYK and PLCγ2 and directly binding to both CLEC-2 and GPVI. Furthermore, hemin-induced murine platelet aggregation was partially reduced in CLEC-2–depleted and FcRγ-deficient (equivalent to GPVI-deficient) platelets and almost completely inhibited in CLEC-2–depleted FcRγ-deficient (double-knockout) platelets. In addition, hemin-induced murine platelet aggregation was inhibited by the CLEC-2 inhibitor cobalt hematoporphyrin or GPVI antibody (JAQ-1). Renal dysfunction, tubular injury, and MET formation were attenuated in double-knockout RAKI mice. Furthermore, in vitro MET formation assay showed that the downstream signaling pathway of CLEC-2 and GPVI is involved in MET formation. We propose that both CLEC-2 and GPVI in platelets play an important role in RAKI development.

DOI： 10.1182/bloodadvances.2020001698

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Advance Publication by J-STAGE  

The 2019 novel coronavirus (severe acute respiratory syndrome-coronavirus: SARS-CoV-2) has currently caused a global outbreak of infection. In general, children with the coronavirus disease-2019 have been reported to show milder respiratory symptoms as a respiratory infection than adult patients. Here, we describe SARS-CoV-2 infection in an infant who presented with a severe episode of apparent life-threatening event (ALTE). An 8-month-old otherwise healthy infant who was transported to our hospital because of a sudden cardiopulmonary arrest. Approximately one hour before this episode, she was almost fine but in a slightly worse humor than usual. On arrival at our hospital, sever acidosis but no clear sign of inflammatory response was denoted. A chest computed tomography scan showed weak consolidations in the upper right lung as well as atelectasis in the lower left lung. No sign of congenital heart disease or cardiomyopathy was observed in echocardiography, and no significant arrhythmia was observed in the later clinical course. Of note, the specific SARS-CoV-2 RNA was detected in both of her tracheal aspirate and urine sample by real-time RT-PCR. Although further accumulation of the cases is indispensable, our case suggests that SARS-CoV-2 infection may be one of the underlying factors in the pathophysiology of ALTE.

DOI： 10.7883/yoken.JJID.2020.572

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/jbmr.4045

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.ijid.2020.03.062

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Hematology  

Soluble forms of platelet membrane proteins are released upon platelet activation. We previously reported that soluble C-type lectin-like receptor 2 (sCLEC-2) is released as a shed fragment (Shed CLEC-2) or as a whole molecule associated with platelet microparticles (MP-CLEC-2). In contrast, soluble glycoprotein VI (sGPVI) is released as a shed fragment (Shed GPVI), but not as a microparticle-associated form (MP-GPVI). However, mechanism of sCLEC-2 generation or plasma sCLEC-2 has not been fully elucidated. Experiments using metalloproteinase inhibitors/stimulators revealed that ADAM10/17 induce GPVI shedding, but not CLEC-2 shedding, and that shed CLEC-2 was partially generated by MMP-2. Although MP-GPVI was not generated, it was generated in the presence of the ADAM10 inhibitor. Moreover, antibodies against the cytoplasmic or extracellular domain of GPVI revealed the presence of the GPVI cytoplasmic domain, but not the extracellular domain, in the microparticles. These findings suggest that most of the GPVI on microparticles are induced to shed by ADAM10; MP-GPVI is thus undetected. Plasma sCLEC-2 level was 1/32 of plasma sGPVI level in normal subjects, but both soluble proteins significantly increased in plasma of patients with acute coronary syndrome. Thus, sCLEC-2 and sGPVI are released by different mechanisms and released in vivo upon platelet activation.

DOI： 10.1007/s12185-019-02680-4

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society of Hematology  

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.

DOI： 10.1182/blood-2017-12-823369

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1182/bloodadvances.2018016261.

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing Ltd  

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.

DOI： 10.1111/jth.13987

Scopus

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Sasaki T, Lian S, Khan A, Llop JR, Samuelson AV, Chen W, Klionsky DJ, Kishi S.  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Thrombosis and Haemostasis  

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.

DOI： 10.1111/jth.13604

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1073/pnas.

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC HEMATOLOGY  

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.

DOI： 10.1182/blood-2015-08-663708

Web of Science

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pgen.1004409.

Authorship：Lead author   Language：English   Publishing type：(MISC) Introduction and explanation (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese   Publishing type：Doctoral Thesis  

Language：English   Publishing type：Research paper (scientific journal)  

Event date： 2023.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：北九州市   Country：Japan  

Event date： 2022.7

Language：English   Presentation type：Poster presentation  

Venue：Toshiaki Shirai, Tomoyuki Sasaki, Nagaharu Tsukiji, Saori Ohishi, Ryohei Yokomori, Katsuhiro Takano, Katsue Suzuki-Inoue  

Language：English   Presentation type：Poster presentation  

Venue：Toshiaki Shirai, Tomoyuki Sasaki, Nagaharu Tsukiji, Saori Ohishi, Ryohei Yokomori, Katsuhiro Takano, Katsue Suzuki-Inoue  

Event date： 2022.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：仙台国際センター　+Web  

Event date： 2022.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：仙台国際センター　+Web  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：仙台国際センター +Web  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：仙台国際センター +Web  

Event date： 2021.9

Language：Japanese   Presentation type：Oral presentation(general)  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：完全Web開催  

Event date： 2021.5

Language：English   Presentation type：Oral presentation(invited, special)  

Event date： 2021.5

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：オンライン開催  

Language：English   Presentation type：Oral presentation(invited, special)  

Venue：オンライン開催  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：オンライン開催  

Event date： 2020.7

Language：English   Presentation type：Oral presentation(general)  

Venue：Virtual  

Language：English   Presentation type：Oral presentation(general)  

Venue：Virtual  

Event date： 2020.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：大阪（web開催）  

Event date： 2020.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：大阪（web開催）  

Event date： 2020.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：大阪（web開催）  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：大阪（web開催）  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：大阪（web開催）  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：大阪（web開催）  

Event date： 2019.7

Language：English   Presentation type：Oral presentation(general)  

OC 79.2

Language：English   Presentation type：Oral presentation(general)  

Venue：オーストラリア・メルボルン  

OC 79.2

Event date： 2019.7

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：オーストラリア・メルボルン  

Event date： 2019.6

Language：Japanese   Presentation type：Poster presentation  

Venue：三重県津市  

P-053

Event date： 2019.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：三重県津市  

O-017

Language：Japanese   Presentation type：Poster presentation  

Venue：三重県津市  

P-053

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：三重県津市  

O-017

Event date： 2018.10

Language：English   Presentation type：Oral presentation(general)  

Language：English   Presentation type：Oral presentation(general)  

Venue：大阪・大阪国際会議場  

Event date： 2018.9

Language：Japanese   Presentation type：Poster presentation  

Venue：京都・国立京都国際会館  

Language：Japanese   Presentation type：Poster presentation  

Venue：京都・国立京都国際会館  

Event date： 2018.6

Language：English   Presentation type：Symposium workshop panel(nominated)  

Event date： 2018.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：北海道・札幌  

Event date： 2018.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：北海道・札幌  

Event date： 2018.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：北海道・札幌  

Language：English   Presentation type：Symposium workshop panel(nominated)  

Venue：北海道・札幌  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：北海道・札幌  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：北海道・札幌  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：北海道・札幌  

Event date： 2017.10

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：東京国際フォーラム  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：東京国際フォーラム  

Event date： 2017.7

Language：English   Presentation type：Poster presentation  

Venue：Berlin, Germany  

Event date： 2017.7

Language：English   Presentation type：Oral presentation(general)  

Venue：Berlin, Germany  

Event date： 2017.7

Language：English   Presentation type：Poster presentation  

Venue：Berlin, Germany  

Event date： 2017.7

Language：English   Presentation type：Oral presentation(general)  

Venue：Berlin, Germany  

Language：English   Presentation type：Oral presentation(general)  

Venue：Berlin, Germany  

Language：English   Presentation type：Oral presentation(general)  

Venue：Berlin, Germany  

Language：English   Presentation type：Poster presentation  

Venue：Berlin, Germany  

Language：English   Presentation type：Poster presentation  

Venue：Berlin, Germany  

Event date： 2017.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：名古屋国際会議場  

Event date： 2017.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：名古屋国際会議場  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：名古屋国際会議場  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：名古屋国際会議場  

Event date： 2016.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：奈良  

Event date： 2016.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：奈良  

Event date： 2016.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：奈良  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：奈良  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：奈良  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：奈良  

Event date： 2015.6

Language：English   Presentation type：Oral presentation(general)  

Language：English   Presentation type：Oral presentation(general)  

Venue：トロント、カナダ  

Event date： 2015.6

Language：English   Presentation type：Oral presentation(general)  

Language：English   Presentation type：Oral presentation(general)  

Venue：トロント、カナダ  

Event date： 2015.6

Language：English   Presentation type：Oral presentation(general)  

Language：English   Presentation type：Oral presentation(general)  

Venue：トロント、カナダ  

Event date： 2015.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：名古屋  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：名古屋  

Event date： 2015.5

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Event date： 2015.5

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Event date： 2015.5

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Event date： 2015.5

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨・甲府  

Application no：2015-053625  Date applied：2015.3

Announcement no：2016-171776 

Application no：2011-53842  Date applied：2011.3

Announcement no：2012-188396 

Application no：2011-53842  Date applied：2011.3

Announcement no：2012-188396