Abstract 3261

Megakaryocyte (MK) maturation and platelet formation are associated with extensive intracellular membrane rearrangements. However, the cellular proteins and mechanisms responsible for the formation of the platelet open canalicular system (OCS) are unknown. PACSIN 1–3 belong to the novel family of F-BAR domain-containing proteins, which bind and deform membranes, promoting tubular invaginations. Blood platelets and MKs contain the ubiquitously expressed PACSIN 2, but not PACSIN 1 or 3, as evidenced by intracellular flow cytometry and immunoblot analysis. Spinning disk laser fluorescence confocal microscopy revealed a specific association between PACSIN 2 and tubular structures in human platelets. PACSIN 2 did not colocalize with granule or lysosome markers, i.e. CD62P, CD63 and LAMP1, suggesting that it coats the OCS. In mouse MKs, PACSIN 2 was distributed through the cell body and colocalized with the membrane marker, CD61. Immunoprecipitation of PACSIN 2 from human platelet lysates pulled down filamin A (FlnA), a major platelet cytoskeletal protein. The interaction required PACSIN 2 F-BAR domain, as evidenced by in vitro binding assays using recombinant proteins. Mice lacking FlnA in the MK lineage have a severe thrombocytopenia with large platelets (Falet et al, J Exp Med 2010; Jurak Begonja et al, Blood 2011). In contrast to wild type platelets, FlnA-null platelets had diffuse PACSIN 2 staining and abnormal intracellular membrane structures, suggesting that the PACSIN 2-FlnA interaction is necessary for the integrity of the platelet intracellular membrane systems. PACSIN 2-null mice had a mild thrombocytopenia, enlarged spleens and increased spleen megakaryopoiesis. Together, the data shows that PACSIN 2 is expressed in platelets and MKs, where it specifically coats the anastomosing intracellular membrane systems in cooperation with FlnA.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.