α-Actinin-1 deficiency in megakaryocytes causes low platelet count, platelet dysfunction, and mitochondrial impairment
Xiangjie Lin, Hanchen Gao, Min Xin, Jian Huang, Xia Li, Yutong Zhou, Keyu Lv, Xin Huang, Jinghan Wang, Yulan Zhou, Dawei Cui, Chao Fang, Lanlan Wu, Xiaofeng Shi, Zhixin Ma, Yu Qian, Hongyan Tong, Jing Dai, Jie Jin, Jiansong Huang
Abstract
ABSTRACT: Cytoskeletal remodeling and mitochondrial bioenergetics play important roles in thrombocytopoiesis and platelet function. Recently, α-actinin-1 mutations have been reported in patients with congenital macrothrombocytopenia. However, the role and underlying mechanism of α-actinin-1 in thrombocytopoiesis and platelet function remain elusive. Using megakaryocyte (MK)-specific α-actinin-1 knockout (KO; PF4-Actn1-/-) mice, we demonstrated that PF4-Actn1-/- mice exhibited reduced platelet counts. The decreased platelet number in PF4-Actn1-/- mice was due to defects in thrombocytopoiesis. Hematoxylin and eosin staining and flow cytometry revealed a decrease in the number of MKs in the bone marrow of PF4-Actn1-/- mice. The absence of α-actinin-1 increased the proportion of 2 N-4 N MKs and decreased the proportion of 8 N-32 N MKs. Colony-forming unit-MK colony formation, the ratio of proplatelet formation-bearing MKs, and MK migration in response to stromal cell-derived factor-1 signaling were inhibited in PF4-Actn1-/- mice. Platelet spreading, clot retraction, aggregation, integrin αIIbβ3 activation, and CD62P exposure in response to various agonists were decreased in PF4-Actn1-/- platelets. Notably, PF4-Actn1-/- platelets inhibited calcium mobilization, reactive oxygen species (ROS) generation, and actin polymerization in response to collagen and thrombin. Furthermore, the PF4-Actn1-/- mice exhibited impaired hemostasis and thrombosis. Mechanistically, proteomic analysis of low-ploidy (2-4 N) and high-ploidy (≥8 N) PF4-Actn1-/- MKs revealed that α-actinin-1 deletion reduced platelet activation and mitochondrial function. PF4-Actn1-/- platelets and Actn1 KO 293T cells exhibited reduced mitochondrial membrane potential, mitochondrial ROS generation, mitochondrial calcium mobilization, and mitochondrial bioenergetics. Overall, in this study, we report that mice with α-actinin-1 deficiency in MKs exhibit low platelet count and impaired platelet function, thrombosis, and mitochondrial bioenergetics.