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AOHE-D-17-00597 R2-3.Pdf A novel heterozygous ITGB3 p.T720del inducing spontaneous activation of integrin αIIbβ3 in autosomal dominant Title macrothrombocytopenia with aggregation dysfunction Miyashita, Naohiro; Onozawa, Masahiro; Hayasaka, Koji; Yamada, Takahiro; Migita, Ohsuke; Hata, Kenichiro; Okada, Author(s) Kohei; Goto, Hideki; Nakagawa, Masao; Hashimoto, Daigo; Kahata, Kaoru; Kondo, Takeshi; Kunishima, Shinji; Teshima, Takanori Annals of hematology, 97(4), 629-640 Citation https://doi.org/10.1007/s00277-017-3214-4 Issue Date 2018-04 Doc URL http://hdl.handle.net/2115/73417 Rights The final publication is available at link.springer.com Type article (author version) File Information AOHE-D-17-00597_R2-3.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP Manuscript 1 2 3 Title 4 5 6 A novel heterozygous ITGB3 p.T720del inducing spontaneous activation of 7 8 9 10 integrin αIIbβ3 in autosomal dominant macrothrombocytopenia with aggregation 11 12 13 dysfunction 14 15 16 17 18 19 Authors 20 21 22 Naohiro Miyashita1, Masahiro Onozawa1, Koji Hayasaka2, Takahiro Yamada3, Ohsuke Migita4, 23 24 25 Kenichiro Hata4, Kohei Okada1, Hideki Goto1, Masao Nakagawa1, Daigo Hashimoto1, Kaoru Kahata1, 26 27 28 1 5 1 29 Takeshi Kondo , Shinji Kunishima , Takanori Teshima 30 31 32 33 34 35 1Deparatment of Hematology, Hokkaido University Faculty of Medicine, Graduate School of Medicine, 36 37 38 Sapporo, Japan 39 40 41 2Division of Laboratory and Transfusion Medicine, Hokkaido University Hospital, Sapporo, Japan 42 43 44 3 45 Division of Clinical Genetics, Hokkaido University Hospital, Sapporo, Japan 46 47 4 48 Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, 49 50 51 Tokyo, Japan 52 53 54 5Department of Advanced Diagnosis, Clinical Research Center, National Hospital Organization Nagoya 55 56 57 Medical Center, Nagoya, Japan 58 59 60 61 1 62 63 64 65 1 2 3 4 5 6 Corresponding author 7 8 9 10 Masahiro Onozawa, MD, PhD 11 12 13 Department of Hematology, Hokkaido University Faculty of Medicine, Graduate School of Medicine, 14 15 16 Sapporo, Japan 17 18 19 Kita 15, Nishi 7, Kita-ku, Sapporo, JAPAN 0608638 20 21 22 Phone: +81-11-706-7214 23 24 25 Fax: +81-11-706-7823 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 2 62 63 64 65 1 2 Acknowledgments 3 4 5 We thank Professor Kazuhiko Matsuno (Rakuno Gakuen University, Ebetsu) for kind advice for flow 6 7 8 cytometry. 9 10 11 12 13 14 Funding 15 16 17 There is no financial relationship with other people or organizations that could inappropriately influence 18 19 20 21 their work. 22 23 24 25 26 27 Disclosure of conflict of interest 28 29 30 The authors declare that they have no conflict of interest. 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 3 62 63 64 65 1 2 3 Abstract 4 5 6 We identified a novel heterozygous ITGB3 p.T720del mutation in a pedigree with 7 8 9 10 macrothrombocytopenia exhibiting aggregation dysfunction. Platelet aggregation induced by ADP and 11 12 13 collagen was significantly reduced, while ristocetin aggregation was normal. Integrin αIIbβ3 was partially 14 15 16 activated in a resting status, but platelet expression of αIIbβ3 was downregulated. Functional analysis 17 18 19 using a cell line showed spontaneous phosphorylation of FAK in αIIb/β3 (p.T720del)-transfected 293T 20 21 22 cells in suspension conditions. Abnormal cytoplasmic protrusions, membrane ruffling, and cytoplasmic 23 24 25 localization of αIIbβ3 were observed in αIIb/β3 (p.T720del)-transfected CHO cells. Such morphological 26 27 28 29 changes were reversed by treatment with an FAK inhibitor. These findings imply spontaneous, but partial, 30 31 32 activation of αIIbβ3 followed by phosphorylation of FAK as the initial mechanism of abnormal 33 34 35 thrombopoiesis. Internalization and decreased surface expression of αIIbβ3 would contribute to 36 37 38 aggregation dysfunction. We reviewed the literature of congenital macrothrombocytopenia associated 39 40 41 with heterozygous ITGA2B or ITGB3 mutations. Reported mutations were highly clustered at the 42 43 44 45 membrane proximal region of αIIbβ3, which affected the critical interaction between αIIb R995 and β3 46 47 48 D723, resulting in a constitutionally active form of the αIIbβ3 complex. Macrothrombocytopenia caused 49 50 51 by a heterozygous activating mutation of ITGA2B or ITGB3 at the membrane proximal region forms a 52 53 54 distinct entity of rare congenital thrombocytopenia. 55 56 57 58 59 60 61 4 62 63 64 65 1 2 3 Keywords 4 5 6 Congenital macrothrombocytopenia, FAK phosphorylation, ITGB3, integrin αIIbβ3, platelet 7 8 9 10 11 12 13 Introduction 14 15 16 Congenital macrothrombocytopenia is a group of rare platelet disorders characterized by a decreased 17 18 19 platelet count with macrothrombocytes showing a varied bleeding tendency. Mutations causing 20 21 22 congenital macrothrombocytopenia have been reported in more than 12 genes including MYH9, which is 23 24 25 responsible for autosomal dominant MYH9 disorders, and GP1BA, GP1BB and GP9, which are 26 27 28 29 responsible for Bernard-Soulier syndrome [1-4]. Variants of integrin αIIb, coded by ITGA2B, and integrin 30 31 32 β3, coded by ITGB3, have also been identified in patients with congenital macrothrombocytopenia [5-12]. 33 34 35 The integrin αIIbβ3 complex is expressed on the surface of platelets and megakaryocytes. αIIbβ3 plays 36 37 38 essential roles in the processes of platelet aggregation, thrombus formation, and thrombopoiesis through 39 40 41 actin remodeling [13-15]. These reported variants are associated with spontaneous, but partial, activation 42 43 44 45 of αIIbβ3 and macrothrombocytopenia with aggregation dysfunction of platelets. The molecular 46 47 48 consequences after spontaneous αIIbβ3 activation contributing to macrothrombocytopenia and 49 50 51 aggregation dysfunction have not been fully elucidated. Here, we report a novel heterozygous ITGB3 52 53 54 p.T720del variatnt in a pedigree of macrothrombocytopenia and its causative mecahnisms. We reviewed 55 56 57 58 59 60 61 5 62 63 64 65 1 2 3 the literature and illustrated the distinct clinical entity of this rare congenital macrothrombocytopenia with 4 5 6 platelet aggregation dysfunction. 7 8 9 10 11 12 13 Materials and Methods 14 15 16 Patients 17 18 19 The patients were a 56-year-old Japanese woman (i2 in Fig 1A) and her two sons (ii2 and ii3 in Fig 20 21 22 1A). They had no bleeding tendencies and near-normal bleeding time evaluated by Duke’s method (Table 23 24 25 1). Hematological examination revealed mildly decreased platelet counts (58-75 x 109/L) with an increase 26 27 28 29 of mean platelet volume (13.4-14.5 fl, Table 1). 30 31 32 Written informed consent was obtained from all family members in accordance with the Declaration of 33 34 35 Helsinki. This study was approved by the Institutional Review Board of Hokkaido University Faculty of 36 37 38 Medicine. 39 40 41 42 43 44 45 Genetic analysis 46 47 48 Genomic DNA was obtained from the three affected family members (i2, ii2, and ii3 in Fig 1A) and a 49 50 51 non-affected member (ii1 in Fig 1A) in the pedigree. Whole exome sequencing (WES) was performed by 52 53 54 HiSeq1500/2500 (Illumina, San Diego, CA) using SureSelect - Human ALL ExomeV6 (Agilent 55 56 57 Technologies, Santa Clara, CA). The ITGB3 gene was amplified using the primer set 5’- 58 59 60 61 6 62 63 64 65 1 2 3 CTCCTGCTTCTTCACAACC-3’ and 5’-GGTCTGAGACTCTTAAGTGGAAG-3’. The identified 4 5 6 ITGB3 p.T720del variatnt was confirmed by direct sequencing using the primer 5’- 7 8 9 10 CTCCTGCTTCTTCACAACC-3’. 11 12 13 14 15 16 Structural analysis 17 18 19 Interaction between αIIb and β3 molecules was evaluated by in silico model construction using 20 21 22 SWISS-MODEL and Cn3D 4.3.1. [16,17]. 23 24 25 26 27 28 29 Platelet glycoprotein analysis 30 31 32 Surface expression of αIIb, β3, αIIbβ3, and GPIb was evaluated by mean fluorescent intensity (MFI) of 33 34 35 CD41 (5B12; Dako, Glostrup, Denmark), CD61 (SZ21; Beckman-Coulter, Fullerton, CA), CD41 (P2; 36 37 38 Beckman-Coulter, Fullerton, CA), and CD42b (AN51; Dako, Glostrup, Denmark), respectively. 39 40 41 The activation status of αIIbβ3 was evaluated by binding of the ligand-mimetic antibody PAC-1 (BD 42 43 44 45 Biosciences, Franklin Lakes, NJ) with or without 1 mM of an Arg-Gly-Asp-Ser (RGDS) mimetic 46 47 48 antagonist (Sigma-Aldrich, St. Louis, MO) [18]. Platelets were discriminated from other blood cells by 49 50 51 GPIb staining. MFI ratio was calculated by dividing MFI of PAC-1 binding on platelets by that of PAC-1 52 53 54 binding in the presence of the RGDS mimetic antagonist. MFI ratio was also calculated by dividing MFI 55 56 57 of PAC-1 binding on platelets in the presence of 1 µM adenosine diphosphate (ADP) by MFI of PAC-1 58 59 60 61 7 62 63 64 65 1 2 3 binding in the absence of ADP. Activation index is defined as (Fx-Fmin) / (Fmax-Fmin). Fx is the MFI of 4 5 6 PAC-1 binding to the platelets, F is the MFI of PAC-1 binding in the presence of the RGDS mimetic 7 min 8 9 10 antagonist, and Fmax is the MFI of PAC-1 binding in the presence of 200 mM phorbol-12-myristate-13- 11 12 13 acetate (PMA; Wako, Osaka, Japan).
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