Interferon-α2b-Induced Thrombocytopenia Is Caused by Inhibition of Cytoplasmic Maturation and Platelet Production, but Not Proliferation and Endomitosis in Human Megakaryocytes.

Abstract

Human interferon (IFN)-α is a standard treatment for the patients with chronic hepatitis C. It is well known that chronic hepatitis C can transform into liver cirrhosis and hepatocellular carcinoma, and the risk of progression to liver cirrhosis is around 40% in 5 years. Thus, it is important for chronic hepatitis C patients to receive IFN-α treatment to prevent its malignant transformation. Thrombocytopenia is one of the major adverse effects of IFN-α and often leads to a dose reduction or discontinuation of IFN-α therapy. However, there is little information how IFN-α inhibits human megakaryopoiesis. In this study, we demonstrated that IFN-α does not inhibit colony formation of megakaryocytes (CFU-MK) from human CD34-positive hematopoietic stem cells. IFN-α also does not inhibit endomitosis (DNA duplication without cytokinesis), but inhibits cytoplasmic maturation of megakaryocytes and platelet production in vitro. The inhibitory effects of IFN-α on the development of demarcation membrane in human megakaryocytes were visualized by staining primary megakaryocytes with di-8-ANEPPS dye under a confocal laser microscope. Loss of platelet production by IFN-α was analyzed by measuring proplatelet formation (PPF) and counting the human platelets produced in the supernatant of human primary megakaryocytes by flow cytometry. The expression of transcription factors regulating late stage megakaryopoiesis such as GATA-1, p45 NF-E2 and MafG was suppressed by IFN-α. To confirm these in vitro observations in vivo system, we transplanted human CD34-positive hematopoietic cells into the immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice (hu-NOG) after 2.4 Gy irradiation. Recombinant human(rh) IFN-α significantly reduced the number of human platelets but did not decrease the number of human megakaryocytes in hu-NOG mice. rhIFN-α did not change murine platelet counts in hu-NOG mice, as IFN-α has species specificity. The DNA ploidy of human megakaryocytes in the bone marrow of hu-NOG mice was not altered after treatement with rhIFN-α. We also confirmed the life time of human platelets was not shortend by rhIFN-α in hu-NOG mice, indicating IFN-α does not promote the clearance of human platelets. We have also confirmed that a novel thrombopoietin mimetics, NIP-004, prevented rhIFN-α-induced thrombotytopenia and increased the number of polyploid human megakaryocytes in hu-NOG mice, suggesting NIP-004 might be useful for the patients with hepatitis C to avoid IFN-α-induced thrombocytopenia. In conclusion, these results clarified that IFN-α induces thrombocytopenia by inhibiting the cytoplasmic maturation of megakaryocytes but not proliferation and endomitosis in human megakaryocytes.