Actn1 deficiency in megakaryocytes causes thrombocytopenia and platelet dysfunction

Recently, Actn1 mutations have been reported in patients with congenital macrothrombocytopenia. However, the role and underlying mechanism of Actn1 in thrombocytopoiesis and platelet function remain elusive. Using MK-specific Actn1 knockout 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. HE staining and flow cytometry revealed a decrease in the number of MKs in the bone marrow of PF4-Actn1-/- mice. The absence of Actn1 increased the proportion of 2 N-4 N MKs and decreased the proportion of 8 N-32 N MKs. CFU-MK colony formation, the ratio of proplatelet formation-bearing MKs, and MK migration in response to SDF-1 signaling were inhibited in PF4-Actn1-/- mice. Platelet spreading, clot retraction, aggregation, integrin activation, and P-selectin exposure in response to various agonists were decreased in PF4-Actn1-/- platelets. Notably, PF4-Actn1-/- platelets inhibited calcium mobilization, ROS generation, and actin polymerization in response to collagen and thrombin. Furthermore, the PF4-Actn1-/- mice demonstrated impaired hemostasis and thrombosis. Mechanistically, proteomic analysis of low- and high-ploidy PF4-Actn1-/- MKs revealed that Actn1 deletion reduced platelet activation and mitochondrial function. PF4-Actn1-/- platelets and Actn1 KO 293T cells exhibited reduced mitochondrial membrane potential, mitoROS generation, mitochondrial calcium mobilization, and mitochondrial bioenergetics. Overall, in this study, we report that mice with Actn1 deficiency in MKs recapitulate the features of thrombocytopenia and exhibit impaired platelet function, thrombosis, and mitochondrial bioenergetics.