{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,9,23]],"date-time":"2025-09-23T23:40:17Z","timestamp":1758670817324,"version":"3.44.0"},"reference-count":44,"publisher":"Georg Thieme Verlag KG","issue":"10","funder":[{"name":"Thailand Science Research and Innovation Fund (Fundamental Fund), Chulalongkorn University"},{"name":"Ratchadapisek Somphot Fund for Postdoctoral Fellowship, Chulalongkorn University"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Thromb Haemost"],"published-print":{"date-parts":[[2025,10]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Megakaryocytes (MK) from Bernard\u2013Soulier syndrome (BSS) induced pluripotent stem cells (iPSCs) yielded reduced numbers but increased sizes of platelets. The molecular mechanisms remain unclear. This study aims to determine roles of signaling molecules involved in this process.<\/jats:p><jats:p>Wild-type (WT) iPSCs and iPSCs from BSS patients with GP1BA (BSS-A) or GP1BB (BSS-B) mutations were differentiated into MKs and platelets with or without myosin II inhibitor (blebbistatin), ROCK inhibitor (Y27632), and procaspase-3 activator (PAC-1). Proplatelet and platelet numbers and sizes were characterized. The iPSC lines containing tubulin-green fluorescent protein (GFP) reporters were constructed to observe proplatelet formation under time-lapse microscopy.<\/jats:p><jats:p>BSS-derived MKs (BSS-MKs) yielded fewer but larger platelets compared with the WT. In the presence of blebbistatin, ROCK inhibitor, or PAC-1, WT, BSS-A, and BSS-B MKs could generate more platelets with decreased sizes, but PAC-1 caused CD42 loss on WT platelets. The proportions of proplatelet formation from MKs carrying tubulin-GFP were not different between WT and BSS-MKs, as well as among inhibitors. Notably, initially thick cytoplasmic processes were transformed into thin branching proplatelets over the observation time. The proplatelet shafts of BSS-MK became thinner in the presence of blebbistatin or ROCK inhibitor, but not of PAC-1, which displayed uneven F-actin distribution.<\/jats:p><jats:p>Inhibition of the ROCK\/myosin pathway, downstream of GpIb, could restore normal morphology of proplatelets in BSS-MKs. Procaspase-3 activation could increase platelet yields, but with abnormal proplatelet and platelet structures. Our model can be used for therapeutic drug screening and a disease model for platelet production in the future.<\/jats:p>","DOI":"10.1055\/a-2474-5644","type":"journal-article","created":{"date-parts":[[2024,12,18]],"date-time":"2024-12-18T18:53:38Z","timestamp":1734548018000},"page":"985-997","source":"Crossref","is-referenced-by-count":0,"title":["Roles of ROCK\/Myosin Pathway in Macrothrombocytopenia in Bernard\u2013Soulier Syndrome"],"prefix":"10.1055","volume":"125","author":[{"given":"Ponthip","family":"Mekchay","sequence":"additional","affiliation":[{"name":"Division of Hematology, Faculty of Medicine, Excellence Center in Translational Hematology, Chulalongkorn University, Bangkok, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Praewphan","family":"Ingrungruanglert","sequence":"additional","affiliation":[{"name":"Stem Cell and Cell Therapy Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Netchanok","family":"Leela-adisorn","sequence":"additional","affiliation":[{"name":"Stem Cell and Cell Therapy Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Noppacharn","family":"Uaprasert","sequence":"additional","affiliation":[{"name":"Division of Hematology, Faculty of Medicine, Excellence Center in Translational Hematology, Chulalongkorn University, Bangkok, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nipan","family":"Israsena","sequence":"additional","affiliation":[{"name":"Stem Cell and Cell Therapy Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ponlapat","family":"Rojnuckarin","sequence":"additional","affiliation":[{"name":"Division of Hematology, Faculty of Medicine, Excellence Center in Translational Hematology, Chulalongkorn University, Bangkok, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"194","published-online":{"date-parts":[[2024,12,18]]},"reference":[{"issue":"03","key":"ref1","first-page":"149","article-title":"Genetically engineered blood pharming: generation of HLA-universal platelets derived from CD34+ progenitor cells","volume":"9","author":"C Figueiredo","year":"2014","journal-title":"J Stem Cells"},{"issue":"01","key":"ref2","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.stemcr.2019.11.011","article-title":"iPSC-derived platelets depleted of HLA class I are inert to anti-HLA class I and natural killer cell immunity","volume":"14","author":"D Suzuki","year":"2020","journal-title":"Stem Cell Reports"},{"issue":"01","key":"ref3","doi-asserted-by":"crossref","first-page":"8472","DOI":"10.1038\/s41598-020-65577-x","article-title":"Generation and characterization of HLA-universal platelets derived from induced pluripotent stem cells","volume":"10","author":"P Norbnop","year":"2020","journal-title":"Sci Rep"},{"issue":"09","key":"ref4","doi-asserted-by":"crossref","first-page":"1461","DOI":"10.1055\/s-0039-1693409","article-title":"Study of Bernard-Soulier syndrome megakaryocytes and platelets using patient-derived induced pluripotent stem cells","volume":"119","author":"P Mekchay","year":"2019","journal-title":"Thromb Haemost"},{"issue":"08","key":"ref5","doi-asserted-by":"crossref","first-page":"1073","DOI":"10.1080\/09537104.2020.1841893","article-title":"Regulation of platelet numbers and sizes by signaling pathways","volume":"32","author":"J Pawinwongchai","year":"2021","journal-title":"Platelets"},{"issue":"22","key":"ref6","doi-asserted-by":"crossref","first-page":"2398","DOI":"10.1182\/blood.2022017296","article-title":"iPLAT1: the first-in-human clinical trial of iPSC-derived platelets as a phase 1 autologous transfusion study","volume":"140","author":"N Sugimoto","year":"2022","journal-title":"Blood"},{"key":"ref7","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1080\/21541248.2021.1885134","article-title":"Role of Rho-GTPases in megakaryopoiesis","volume":"12","author":"W Vainchenker","year":"2021","journal-title":"Small GTPases"},{"issue":"09","key":"ref8","doi-asserted-by":"crossref","first-page":"1430","DOI":"10.1111\/jth.14544","article-title":"New insights into cytoskeletal remodeling during platelet production","volume":"17","author":"D Ghalloussi","year":"2019","journal-title":"J Thromb Haemost"},{"issue":"5845","key":"ref9","doi-asserted-by":"crossref","first-page":"1767","DOI":"10.1126\/science.1146304","article-title":"Dynamic visualization of thrombopoiesis within bone marrow","volume":"317","author":"T Junt","year":"2007","journal-title":"Science"},{"issue":"06","key":"ref10","doi-asserted-by":"crossref","first-page":"785","DOI":"10.1083\/jcb.201304054","article-title":"The incredible journey: from megakaryocyte development to platelet formation","volume":"201","author":"K R Machlus","year":"2013","journal-title":"J Cell Biol"},{"issue":"04","key":"ref11","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1160\/TH14-02-0123","article-title":"Platelet demand modulates the type of intravascular protrusion of megakaryocytes in bone marrow","volume":"112","author":"S Kowata","year":"2014","journal-title":"Thromb Haemost"},{"issue":"02","key":"ref12","doi-asserted-by":"crossref","first-page":"e201800061","DOI":"10.26508\/lsa.201800061","article-title":"Multiple membrane extrusion sites drive megakaryocyte migration into bone marrow blood vessels","volume":"1","author":"E Brown","year":"2018","journal-title":"Life Sci Alliance"},{"issue":"03","key":"ref13","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1083\/jcb.201410052","article-title":"IL-1\u03b1 induces thrombopoiesis through megakaryocyte rupture in response to acute platelet needs","volume":"209","author":"S Nishimura","year":"2015","journal-title":"J Cell Biol"},{"issue":"09","key":"ref14","doi-asserted-by":"crossref","first-page":"e20191206","DOI":"10.1084\/jem.20191206","article-title":"Membrane budding is a major mechanism of in vivo platelet biogenesis","volume":"217","author":"K S Potts","year":"2020","journal-title":"J Exp Med"},{"issue":"19","key":"ref15","doi-asserted-by":"crossref","first-page":"2411","DOI":"10.3390\/cells12192411","article-title":"Multiphoton in vivo microscopy of embryonic thrombopoiesis reveals the generation of platelets through budding","volume":"12","author":"H Liu","year":"2023","journal-title":"Cells"},{"issue":"04","key":"ref16","doi-asserted-by":"crossref","first-page":"540","DOI":"10.1161\/CIRCRESAHA.124.323579","article-title":"What it takes to be a platelet: evolving concepts in platelet production","volume":"135","author":"E Carminita","year":"2024","journal-title":"Circ Res"},{"key":"ref17","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1007\/978-1-61779-307-3_15","article-title":"In vitro generation of megakaryocytes and platelets from human embryonic stem cells and induced pluripotent stem cells","volume":"788","author":"N Takayama","year":"2012","journal-title":"Methods Mol Biol"},{"issue":"34","key":"ref18","doi-asserted-by":"crossref","first-page":"35557","DOI":"10.1074\/jbc.M405319200","article-title":"Mechanism of blebbistatin inhibition of myosin II","volume":"279","author":"M Kov\u00e1cs","year":"2004","journal-title":"J Biol Chem"},{"issue":"01","key":"ref19","doi-asserted-by":"crossref","first-page":"e0270288","DOI":"10.1371\/journal.pone.0270288","article-title":"The non-selective Rho-kinase inhibitors Y-27632 and Y-33075 decrease contraction but increase migration in murine and human hepatic stellate cells","volume":"18","author":"N Bachtler","year":"2023","journal-title":"PLoS One"},{"issue":"12","key":"ref20","doi-asserted-by":"crossref","first-page":"1637","DOI":"10.1016\/j.febslet.2008.04.009","article-title":"ROCK and PRK-2 mediate the inhibitory effect of Y-27632 on polyglutamine aggregation","volume":"582","author":"J Shao","year":"2008","journal-title":"FEBS Lett"},{"issue":"10","key":"ref21","doi-asserted-by":"crossref","first-page":"1025","DOI":"10.1038\/s41419-018-1038-3","article-title":"Procaspase-3-activating compound 1 stabilizes hypoxia-inducible factor 1\u03b1 and induces DNA damage by sequestering ferrous iron","volume":"9","author":"F Li","year":"2018","journal-title":"Cell Death Dis"},{"issue":"03","key":"ref22","doi-asserted-by":"crossref","first-page":"525","DOI":"10.1182\/blood-2014-05-576462","article-title":"Myosin-II repression favors pre\/proplatelets but shear activation generates platelets and fails in macrothrombocytopenia","volume":"125","author":"K R Spinler","year":"2015","journal-title":"Blood"},{"issue":"10","key":"ref23","doi-asserted-by":"crossref","first-page":"4229","DOI":"10.1182\/blood-2006-04-020024","article-title":"Proplatelet formation is regulated by the Rho\/ROCK pathway","volume":"109","author":"Y Chang","year":"2007","journal-title":"Blood"},{"issue":"06","key":"ref24","doi-asserted-by":"crossref","first-page":"867","DOI":"10.1111\/bjh.12709","article-title":"Rho kinase inhibition drives megakaryocyte polyploidization and proplatelet formation through MYC and NFE2 downregulation","volume":"164","author":"M P Avanzi","year":"2014","journal-title":"Br J Haematol"},{"issue":"21","key":"ref25","doi-asserted-by":"crossref","first-page":"2854","DOI":"10.1091\/mbc.e17-03-0209","article-title":"Systematic gene tagging using CRISPR\/Cas9 in human stem cells to illuminate cell organization","volume":"28","author":"B Roberts","year":"2017","journal-title":"Mol Biol Cell"},{"key":"ref26","doi-asserted-by":"crossref","first-page":"15838","DOI":"10.1038\/ncomms15838","article-title":"A Cdc42\/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis","volume":"8","author":"S D\u00fctting","year":"2017","journal-title":"Nat Commun"},{"issue":"04","key":"ref27","doi-asserted-by":"crossref","first-page":"1054","DOI":"10.1182\/blood-2011-08-372193","article-title":"Megakaryocyte-specific RhoA deficiency causes macrothrombocytopenia and defective platelet activation in hemostasis and thrombosis","volume":"119","author":"I Pleines","year":"2012","journal-title":"Blood"},{"issue":"14","key":"ref28","doi-asserted-by":"crossref","first-page":"3182","DOI":"10.1182\/blood-2008-06-164061","article-title":"Abnormal megakaryocyte morphology and proplatelet formation in mice with megakaryocyte-restricted MYH9 inactivation","volume":"113","author":"A Eckly","year":"2009","journal-title":"Blood"},{"issue":"06","key":"ref29","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1182\/bloodadvances.2017010975","article-title":"Enhancing functional platelet release in vivo from in vitro-grown megakaryocytes using small molecule inhibitors","volume":"2","author":"D Jarocha","year":"2018","journal-title":"Blood Adv"},{"issue":"12","key":"ref30","doi-asserted-by":"crossref","first-page":"2163","DOI":"10.1111\/jth.12436","article-title":"The abnormal proplatelet formation in MYH9-related macrothrombocytopenia results from an increased actomyosin contractility and is rescued by myosin IIA inhibition","volume":"11","author":"Y Chen","year":"2013","journal-title":"J Thromb Haemost"},{"issue":"21","key":"ref31","doi-asserted-by":"crossref","first-page":"1887","DOI":"10.1182\/blood.2019003064","article-title":"Megakaryocyte migration defects due to nonmuscle myosin IIA mutations underlie thrombocytopenia in MYH9-related disease","volume":"135","author":"K Pal","year":"2020","journal-title":"Blood"},{"issue":"06","key":"ref32","doi-asserted-by":"crossref","first-page":"877","DOI":"10.1083\/jcb.200906012","article-title":"Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation","volume":"188","author":"A M Pasapera","year":"2010","journal-title":"J Cell Biol"},{"issue":"12","key":"ref33","doi-asserted-by":"crossref","first-page":"1459","DOI":"10.1134\/S0006297918120040","article-title":"Common and specific functions of nonmuscle myosin II paralogs in cells","volume":"83","author":"M S Shutova","year":"2018","journal-title":"Biochemistry (Mosc)"},{"issue":"7040","key":"ref34","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1038\/nature03550","article-title":"Non-equilibration of hydrostatic pressure in blebbing cells","volume":"435","author":"G T Charras","year":"2005","journal-title":"Nature"},{"issue":"01","key":"ref35","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1038\/ncb734","article-title":"A small-molecule inhibitor of skeletal muscle myosin II","volume":"4","author":"A Cheung","year":"2002","journal-title":"Nat Cell Biol"},{"issue":"04","key":"ref36","doi-asserted-by":"crossref","first-page":"1310","DOI":"10.1182\/blood-2002-03-0686","article-title":"Platelet formation is the consequence of caspase activation within megakaryocytes","volume":"100","author":"S De Botton","year":"2002","journal-title":"Blood"},{"issue":"04","key":"ref37","doi-asserted-by":"crossref","first-page":"e0125057","DOI":"10.1371\/journal.pone.0125057","article-title":"Actin inhibition increases megakaryocyte proplatelet formation through an apoptosis-dependent mechanism","volume":"10","author":"M P Avanzi","year":"2015","journal-title":"PLoS One"},{"issue":"07","key":"ref38","doi-asserted-by":"crossref","first-page":"798","DOI":"10.1111\/gtc.12384","article-title":"Proplatelet formation in megakaryocytes is associated with endoplasmic reticulum stress","volume":"21","author":"N Morishima","year":"2016","journal-title":"Genes Cells"},{"issue":"07","key":"ref39","doi-asserted-by":"crossref","first-page":"677","DOI":"10.1161\/01.RES.0000143899.73453.11","article-title":"Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates GPIbalpha shedding from platelets in vitro and in vivo","volume":"95","author":"W Bergmeier","year":"2004","journal-title":"Circ Res"},{"issue":"07","key":"ref40","doi-asserted-by":"crossref","first-page":"1530","DOI":"10.1111\/j.1538-7836.2007.02590.x","article-title":"Controlled shedding of platelet glycoprotein (GP)VI and GPIb-IX-V by ADAM family metalloproteinases","volume":"5","author":"E E Gardiner","year":"2007","journal-title":"J Thromb Haemost"},{"issue":"03","key":"ref41","doi-asserted-by":"crossref","first-page":"229","DOI":"10.3109\/09537100903443949","article-title":"Hyperthermia induces platelet apoptosis and glycoprotein Ibalpha ectodomain shedding","volume":"21","author":"Z Wang","year":"2010","journal-title":"Platelets"},{"issue":"06","key":"ref42","doi-asserted-by":"crossref","first-page":"1663","DOI":"10.1182\/blood-2011-04-347849","article-title":"Bcl-xL-inhibitory BH3 mimetics can induce a transient thrombocytopathy that undermines the hemostatic function of platelets","volume":"118","author":"S M Schoenwaelder","year":"2011","journal-title":"Blood"},{"issue":"05","key":"ref43","doi-asserted-by":"crossref","first-page":"373","DOI":"10.3109\/09537104.2013.821604","article-title":"The role of mitochondrial permeability transition pore in regulating the shedding of the platelet GPIb\u03b1 ectodomain","volume":"25","author":"Z Wang","year":"2014","journal-title":"Platelets"},{"issue":"04","key":"ref44","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1083\/jcb.200210111","article-title":"Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death","volume":"160","author":"M C Clarke","year":"2003","journal-title":"J Cell Biol"}],"container-title":["Thrombosis and Haemostasis"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/www.thieme-connect.de\/products\/ejournals\/pdf\/10.1055\/a-2474-5644.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,9,23]],"date-time":"2025-09-23T23:09:58Z","timestamp":1758668998000},"score":1,"resource":{"primary":{"URL":"http:\/\/www.thieme-connect.de\/DOI\/DOI?10.1055\/a-2474-5644"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,12,18]]},"references-count":44,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2025,9,23]]},"published-print":{"date-parts":[[2025,10]]}},"URL":"https:\/\/doi.org\/10.1055\/a-2474-5644","archive":["Portico","CLOCKSS"],"relation":{},"ISSN":["0340-6245","2567-689X"],"issn-type":[{"type":"print","value":"0340-6245"},{"type":"electronic","value":"2567-689X"}],"subject":[],"published":{"date-parts":[[2024,12,18]]}}}