α-辅肌动蛋白-1的缺失致整合素αIIbβ3信号上调在血小板减少症中的作用及机制研究

先天性α-辅肌动蛋白-1（ACTN1）的基因突变会导致遗传性血小板减少症。但目前对ACTN1在血小板生成中的作用及机制了解甚少。本项目组前期的工作提示：ACTN1缺失能抑制巨核细胞在固相纤维蛋白原上的前血小板生成；抑制整合素αIIbβ3信号能促进血小板的生成。因此，我们提出假说：ACTN1缺失引起血小板生成减少与整合素αIIbβ3信号上调有关。本研究拟使用敲除ACTN1的Dami、SET-2、Meg-01细胞和特异性敲除ACTN1的小鼠，在细胞和小鼠活体层面观察巨核细胞成熟及血小板生成过程，评估整合素αIIbβ3双向信号转导情况及观察ACTN1、肌动蛋白及整合素αIIbβ3等蛋白在巨核细胞中的表达、定位，探讨分子机制。观察整合素αIIbβ3抑制剂、活化剂或信号转导途径中关键激酶的抑制剂能否逆转或加剧ACTN1缺失导致的血小板生成减少。研究结果有望为阐述血小板减少症的发病机制提供理论基础。

Recently, mutations in α-ACTININ-1 were found to be the cause of inherited thrombocytopenia. α-ACTININ-1 is the gene that encodes α-actinin-1 (ACTN1), but little is known about the exact role that α-actinin-1 plays in thrombocytopoiesis and its underlying mechanism. Our previous studies confirmed that α-actinin-1 deficiency inhibits proplatelet formation of megakaryocyte on immobilized fibrinogen. Furthermore, inhibition of integrin αIIbβ3 signaling can enhance thrombocytopoiesis. Therefore, we hypothesized that α-actinin-1 deficiency-induced thrombocytopenia is related to the upregulation of integrin αIIbβ3 signal transduction. In this study, CRISPR/Cas9 technology will be used to knock out α-actinin-1 in Dami, SET-2 and Meg-01 cells. These knockout cells, as well as α-actinin-1-deficient mouse hematopoietic stem cells, will be used to observe megakaryocyte maturation and thrombocytopoiesis at the cellular level. A mouse model of megakaryocytic/platelet lineage-specific α-actinin-1 knockout will be adopted to investigate megakaryocyte maturation and thrombocytopoiesis. To investigate the molecular mechanism underlying the role of α-actinin-1 in thrombocytopenia, we will examine the expression of α-actinin-1, actin and integrin αIIbβ3 and determine their localization in megakaryocyte. We will also investigate the bidirectional signal transduction of integrin αIIbβ3, which has been previously established to play a critical role in the process of thrombocytopoiesis. We will also investigate whether the inhibition of either integrin αIIbβ3 outside-in signaling or protein kinases related to the bidirectional signal transduction of integrin αIIbβ3 can potentially reverse the effects of α-actinin-1 deficiency on thrombocytopenia. Similarly, we will also establish whether integrin αIIbβ3 agonists can potentially enhance the effects of α-actinin-1 deficiency on thrombocytopenia. Through this project, we hope to further elucidate the causes of inherited thrombocytopenia on a theoretical basis and further explore the role and underlying mechanism of α-actinin-1 in thrombocytopoiesis.