αIIb(R990W), a Gain-of Function Mutation of αIIbβ3, Knock-in Mice Show Moderately Impaired Thrombopoiesis
Authors: Akuta Keigo , Kiyomizu Kazunobu , Kashiwagi Hirokazu , Kunishima Shinji , Banno Fumiaki , Kokame Koichi , Nishiura Nobuko , Kato Hisashi , Honda Shigenori , Kanakura Yuzuru , Miyata Toshiyuki , Tomiyama Yoshiaki 
Published: October 2019

Abstract
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<0.05 compared with WT)]. Absolute reticulated platelet numbers were significantly decreased in Homo mice compared with WT [WT: 102 ± 21.9 (x103/μL), Hetero: 84.0 ± 23.7, Homo: 66.6 ± 12.3* (*p<0.05 compared with WT)]. Although serum TPO level tended to be high in Homo mice [WT: 374 ± 155 (pg/mL), hetero: 380 ± 83, homo: 430 ±283], there were no statistically significant differences. Platelet increase after TPO injection was significantly delayed and peak platelet counts were lower in Homo mice compared to WT and Hetero mice. Recovery from anti-GPIb antibody induced thrombocytopenia was mildly impaired on 4 days after injection in KI mice. These results suggest that platelet production is impaired in KI mice. In addition, the half-life of platelets was slightly but significantly shortened in KI mice compared with WT [WT: 28.99 ± 1.76 (hours), Hetero: 24.90 ± 4.73, Homo: 19.54 ± 1.01* (*p<0.05 compared with WT)], which may also contribute to thrombocytopenia in KI mice. Between Homo mice and WT, there was no difference in the number of MgK in femur bone marrow [WT: 68.7 ± 5.04 (/10 visual fields), Homo: 75.7 ± 6.74] and spleen [WT: 24.7 ± 5.21 (/slide), Homo: 28.3 ± 5.55]. MgK ploidy in KI mice were similar to WT. However, PPF of MgK derived from fetal liver cells was significantly decreased in Homo mice [WT: 24.94 ± 3.11 (%), hetero: 22.21 ± 1.55, homo: 15.65 ± 1.49* (*p<0.05 compared with WT)].
Conclusion:
Our results suggest that the αIIbβ3 gain-of-function mutation, αIIb(R990W), leads to thrombocytopenia via mainly impaired thrombopoiesis. There was no difference in number and ploidy of MgK. However, Homo KI mice show marked reduction of PPF, indicating impaired proplatelet formation appears to be a main cause of impairment of thrombopoiesis by the mutation.
Disclosures:
No relevant conflicts of interest to declare.

Disclosures
Kanakura: Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Tomiyama:Kyowa Hakko Kirin Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sysmex Corporation: Consultancy.