PACSIN2 Orchestrates Actin Reorganization during Megakaryocyte Maturation

Thrombocytopenia, defined by a platelet count below 150,000/µL, poses significant mortality risks, primarily due to bleeding. Blood platelets are produced by bone marrow megakaryocytes (MKs) in unique cellular processes that require polyploidization and extensive intracellular membrane rearrangements leading to the formation of the demarcation membrane system (DMS), the surface-connected membrane reservoir for future platelets. The membrane-deforming F-BAR protein PACSIN2 is one of the most abundant and conserved BAR proteins in platelets, and exomechip analysis and genome-wide association studies have associated PACSIN2 single nucleotide variants with platelet count and mean platelet volume in humans. Our previous work has shown that PACSIN2 localizes with the initiating DMS in MKs in a process regulated by the cytoskeletal and scaffolding protein filamin A (FlnA). Recently, we have found that Pacsin2 ^-/- mice display mild thrombocytopenia with slightly enlarged platelets and severe thrombus formation defects due to increased activation of platelet integrin β1. Here we investigated the role of PACSIN2 in MK maturation and platelet production. Cultured fetal liver cell-derived Pacsin2 ^-/- MKs formed proplatelets normally compared to controls. Pacsin2 ^-/- bone marrow sections displayed MK count comparable to control mice, and freshly isolated bone marrow Pacsin2 ^-/- MKs had normal ploidy, indicating normal MK maturation. The observed effects of PACSIN2 deficiency led to a defective formation of the DMS within MKs. In PACSIN2 deficient MKs, the DMS exhibited a less distinct structure, making it challenging to delineate clear platelet territories. Interestingly, the lack of PACSIN2 also impacted the spatial distribution of GPIbα and integrin β1 membrane receptors. These receptors, typically dispersed within the cytoplasm in normal MKs, were concentrated at the cell periphery in Pacsin2 ^-/-MKs. A similar relocation was observed for polymerized actin. This finding underscores the pivotal role of PACSIN2 in actin reorganization within MKs, which in turn influences the rearrangement of GPIbα and integrin β1. The data imply that receptor and actin cytoskeleton rearrangements are critical to DMS formation and subsequent platelet production in vivo.