αIIb(R990W), a Gain-of Function Mutation of αIIbβ3, Knock-in Mice Show Moderately Impaired Thrombopoiesis

Introduction: Integrin αIIbβ3 plays an essential role in platelet aggregation, and quantitative or qualitative defects in αIIbβ3 lead to severe bleeding diathesis, which is known as Glanzmann thrombasthenia (GT). Although platelet counts and morphology are usually normal in GT patients, recent several reports demonstrate that constitutive gain-of-function mutations around transmembrane regions of αIIb or β3 are associated with congenital macrothrombocytopenia, which is referred to as αIIbβ3-related thrombocytopenia. αIIb(R995W) is the most prevalent mutation in αIIbβ3-related thrombocytopenia among Japanese (Kunishima, Kashiwagi, et al. Blood. 2011;117:5479-84). We also found reduction of surface expression of αIIbβ3 on platelets in patients with αIIbβ3-rerated thrombocytopenia, suggesting that activating mutations may affect platelet function via not only activating state of αIIbβ3 but also its expression (Kashiwagi, et al. Mol Genet Genomic Med. 2013;1:77-86). However, the pathogenesis of thrombocytopenia caused by αIIbβ3 gain-of-function mutations is not fully understood. We have generated αIIb(R990W), which is corresponding to human αIIb(R995W), knock-in (KI) mice and previously reported that αIIb(R990W) KI mice showed mild macrothrombocytopenia and Homo KI mice revealed marked reduction of surface αIIbβ3 expression in platelet and impaired platelet function like GT (ASH meeting 2014). In this study, we further analyzed αIIb(R990W) KI mice to elucidate the mechanism causing macrothrombocytopenia. Methods: αIIb(R990W) mutation was introduced to C57BL/6-derived ES cells by homologous recombination and KI mice were generated. Reticulated platelets were detected by flow cytometry after thiazole orange staining. Serum thrombopoietin (TPO) levels were measured by ELISA. Platelet life span was assessed by flow cytometry after injection with NHS-biotin. Platelet counts and reticulated platelets were monitored after injection of murine TPO or anti-GPIb antibody. We counted megakaryocytes (MgK) in tissue preparation of femur and spleen. MgK ploidy was analyzed by flow cytometry. We analyzed proplatelet formation (PPF) of cultured fetal liver cells from embryonic day 14.5 embryos. Results: Platelet counts were decreased in KI mice [WT: 1.22 ± 0.11 (x106/μL), Hetero: 1.10 ± 0.14*, Homo: 0.91 ± 0.11* (*p