CLINICAL OBSERVATIONS IN HEPATOLOGY | Hepatology, VOL. 0, NO. 0, 2019 Regression of Idiopathic Thrombocytopenic Purpura in a Patient With Eradication of Hepatitis C Virus by Direct-Acting Antivirals Yusuke Johira,1* Haruna Hatooka,2* Nami Mori,1 Takayuki Fukuhara,1 Shintaro Takaki,1 Yumi Shimomura,2 Michio Imamura,2 C. Nelson Hayes,2 Keiji Tsuji,1 and Kazuaki Chayama2 I diopathic thrombocytopenic purpura (ITP) is an autoimmune disorder that is characterized by antibody-mediated platelet destruction and decreased platelet production. ITP is associated with high morbidity in hepatitis C virus (HCV)–infected patients,(1) but the relationship between these diseases is unclear. Although interferon therapy was hampered due to thrombocytopenia, recent direct-acting antiviral (DAA) therapies permit HCV eradication in patients with chronic hepatitis complicated with ITP. Here, we report a case of successful regression of ITP following HCV eradication by DAA therapy. A 42-year-old man visited our hospital with purpura in March 2014. Blood testing revealed a platelet count of 2 × 103/μL and a serum alanine aminotransferase (ALT) level of 142 IU/mL. He was diagnosed with ITP by bone marrow examination and chronic genotype 2a HCV infection without any evidence of cirrhosis assessed by computed tomography, blood examination, and liver fibrosis marker (total bilirubin, 0.6 mg/dL; albumin, 4.7 g/dL; mac-2 binding protein, 1.41 cutoff index). He received a platelet transfusion and immunoglobulin and was treated with prednisolone and eltrombopag, a thrombopoietin receptor agonist (Fig. 1). Although the platelet count increased to 30 × 103/μL, eltrombopag was switched to romiplostim because ALT levels elevated to approximately 700 IU/L. Subsequently, the serum ALT level decreased, and the bleeding symptoms disappeared with weekly administration of romiplostim. Then from August 2015 he received 12 weeks of sofosbuvir plus ribavirin therapy for HCV infection. Serum HCV RNA became negative during the therapy, and he achieved sustained virological response (SVR) 12 and 24 (serum HCV RNA-negative 12 and 24 weeks after treatment completion, respectively). Although 5 mg/day of prednisolone and 1,750 μg/month of romiplostim were required to maintain a platelet count of >50 × 103/μL at the start of anti-HCV therapy, these requirements were gradually relaxed. Despite withdrawal of prednisolone and romiplostim 12 and 32 months after achievement of SVR, respectively, his platelet counts have been maintained at >100 × 103/μL for 1 year. Abbreviations: ALT, alanine aminotransferase; HCV, hepatitis C virus; ITP, idiopathic thrombocytopenic purpura; NS, nonstructural (protein); WES, capillary western immunoassay. Received February 22, 2019; accepted July 25, 2019. *These authors contributed equally to this work. Supported by the Research Program on Hepatitis from the Japan Agency for Medical Research and Development (17fk0210104h0001). Part of this work was carried out at the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. © 2019 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.30900 Potential conflict of interest: Dr. Chayama has received honoraria from Bristol-Myers Squibb and MSD and research funding from Dainippon Sumitomo Pharma and AbbVie. Dr. Imamura has received research funding from Bristol-Myers Squibb. 1 JOHIRA, HATOOKA, ET AL. Hepatology, Month 2019 EHIRUH DIWHU [ GD\V ,9,J PJGD\ 3UHGQLVRORQH PJGD\ 5RPLSORVWLP ȝJPRQWK +&951$ ORJ,8P/ 6RIRVEXYLUULEDYLULQ 3ODWHOHW ȝ/ FIG. 1. Clinical course of the patient treated for ITP and HCV infection. Dotted line indicates serum HCV RNA titers. Arrows indicate the time points when pretreatment and posttreatment antibodies were tested by western blotting. Abbreviation: IVIg, intravenous immunoglobulin. Because regression of ITP was achieved by HCV eradication, we hypothesized that HCV antibody caused platelet destruction. HCV RNA was extracted from patient serum and complementary DNA (cDNA) of HCV core; nonstructural protein 3 (NS3), NS4, NS5A, and NS5B were amplified by PCR (Fig. 2A); then HCV proteins were synthesized using an in vitro protein synthesis kit. Capillary western immunoassay (WES) analysis was performed on a WES system (004-600; ProteinSimple, San Jose, CA) to clarify the relationship between HCV antibodies and ITP. When synthesized HCV proteins were incubated with patient serum, anticore, anti-NS3, anti-NS4, anti-NS5A, and anti-NS5B antibodies were detected before anti-HCV therapy (Fig. 2B). These antibodies had been reduced, and ARTICLE INFORMATION: From the 1 Department of Gastroenterology, Hiroshima Red Cross Hospital and Atomic-Bomb Survivors Hospital, Naka-ku, Hiroshima, Japan; 2 Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Sciences, Hiroshima University, Minami-ku, Hiroshima, Japan. ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: Nami Mori, M.D., Ph.D. Department of Gastroenterology Hiroshima Red Cross Hospital and Atomic-Bomb Survivors Hospital 2 1-9-6 Sendamachi, Naka-ku Hiroshima 734-8619, Japan E-mail: [email protected] Tel.: +81-82-241-3111 Hepatology, Vol. 0, No. 0, 2019 A JOHIRA, HATOOKA, ET AL. B C N'D ES FRUH 16 16 16$ FRUH 16 16 16$ 16% EHIRUH DIWHU EHIRUH DIWHU EHIRUH DIWHU EHIRUH DIWHU EHIRUH DIWHU EHIRUH DIWHU 16% FIG. 2. Relationship between HCV antibodies and platelet proteins. (A) The cDNA of HCV core, NS3, NS4, NS5A, and NS5B was amplified by PCR. In vitro transcription and translation were performed using the TNT T7 Coupled Wheat Germ Extract System (Promega, WI). Synthesized HCV proteins, core (19 kDa), NS3 (67 kDa), NS4 (27 kDa), NS5A (50 kDa), and NS5B (65 kDa) (B) or extracted platelet proteins (57 kDa) (C) were reacted with patient serum obtained before and 24 weeks after antiviral therapy and analyzed by WES. Arrow indicates HCV and platelet proteins. Molecular markers are in kilodaltons. anti-NS3 antibody disappeared 24 weeks after antiviral therapy. Next, platelet proteins were extracted and reacted with the serum. WES analysis showed the presence of a 57-kDa band before treatment and disappearance 24 weeks after antiviral therapy (Fig. 2C). These results suggest that anti-NS3 antibody might cross-react as an antiplatelet antibody to platelet protein and, therefore, that regression of ITP might be achieved by disappearance of anti-NS3 antibody by HCV eradication. Chronic HCV infection is associated with the development of several extrahepatic alterations, including thrombocytopenia. Pathogenesis of ITP is gradually being elucidated, and autoantibodies to glycoprotein IIb/IIIa on the platelet surface have been found to be associated with platelet destruction.(2) HCV infection may lead to development of secondary ITP by several mechanisms, such as cross-reaction of antibody against HCV with platelet protein, infection to platelet and megakaryocyte, low serum thrombopoietin level, or autoantibody to the thrombopoietin receptor.(3-5) Our results indicate a possible mechanism in which anti-NS3 was related to ITP, and regression of ITP was achieved by loss of anti-NS3 antibody following HCV eradication. Acknowledgment: We thank Mitsuhiro Itagaki, Shinya Katsutani, and Hideki Asaoku from the Department of Hematology of Hiroshima Red Cross Hospital and Atomic-Bomb Survivors Hospital for their special treatment for ITP of the patient. 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