HDAC6 mediates tubulin deacetylation during platelet activation. (A) Human platelets were fixed after 30 minutes of spreading in the absence or presence of 100 ng/mL TSA, 10mM Na-butyrate, or 5mM nicotinamide and stained using an antiacetylated tubulin antibody and phalloidin-rhodamine for the actin cytoskeleton. (B) Western blot of a human platelet lysate and a lysate of the human lung carcinoma cell line A549 (cultured in RPMI 1640/10% FCS; 70 μg/lane) revealed with an anti-HDAC6 antibody (Santa Cruz Biotechnology; sc-11420), followed by Coomassie staining of the transfer membrane. (C) Anti-HDAC6 antibodies were used for immunoprecipitation from a human platelet lysate, and unspecific IgGs were used as control. Immune complexes were incubated with an acetylated tubulin peptide (MW 1893), which was then analyzed by mass spectrometry for loss of acetylation (loss of 42 Da). Incubations with control IgGs resulted in 0% deacetylation versus 55.1% ± 2.9% deacetylation for incubations with HDAC6 immune complexes (n = 3). (D) HDAC6 WT and KO platelets in the resting state or spread for 60 and 90 minutes on glass surfaces were fixed and stained with phalloidin-rhodamine and an acetylated tubulin antibody as indicated. (E) Quantification of the surface area occupied by the actin cytoskeleton using images taken as in Figure 2D for phalloidin-rhodamine stainings after 60 minutes of spreading. The histogram represents the percentage of platelets present in different size categories as indicated on the x-axis; ∼ 150 platelets were counted for each condition of a typical experiment repeated 4 times. Scale bars represent 10 μm.