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HDAC6 controls the kinetics of platelet activation

Karin Sadoul, Jin Wang, Boubou Diagouraga, Anne-Laure Vitte, Thierry Buchou, Thérèse Rossini, Benoît Polack, Xiaodong Xi, Patrick Matthias and Saadi Khochbin

Article Figures & Data

Figures

  • Figure 1

    Dynamic changes of tubulin acetylation in activated platelets. (A) Human platelets were fixed after different spreading periods (as indicated, ON indicates overnight) on glass surfaces and stained using an antiacetylated tubulin antibody (clone 6-11B-1, Sigma-Aldrich, T6793; green, top panels) or an antitubulin antibody (clone B-5-1-2, Sigma-Aldrich, T5168; green, bottom panels) detected with AlexaFluor-488 goat anti–mouse IgGs (Invitrogen; A11029). Colabeling of the actin cytoskeleton with phalloidin-rhodamine (Sigma-Aldrich, P1951; red) served as an indicator for the extent of platelet spreading. Fluorescent images were acquired using an upright microscope (Olympus BX41) equipped with a color camera DP70 using a 100× oil immersion objective and the software analySIS. (B) Human platelets were incubated for 40 minutes (left panels) or 9 hours (right panels) on glass cover slips coated with 20 μg/mL collagen (Col), fibrinogen (Fg), fibronectin (Fn), or poly-L-lysine (PLL) and blocked with 3% BSA. Platelets were then fixed and stained using an antiacetylated tubulin antibody and phalloidin-rhodamine for the actin cytoskeleton. (C) Platelets in human PRP were induced or not (no) to aggregate with 1.5mM arachidonic acid (AA), 10μM adenosine diphosphate (ADP), or 10 μg/mL collagen (Col). Aggregation was followed using an APACT 4004/LABiTec aggregometer. The PRP was then centrifuged and the platelet pellet lysed and analyzed by Western blot using an acetylated tubulin and an antitubulin antibody (inset). (D) Human platelets were allowed to spread on glass Petri dishes (6 × 107/10 mL/dish) for the indicated periods of time and then scrapped and analyzed by Western blot using an acetylated tubulin antibody. The same membrane was stained with Coomassie as a loading control. (E) Human PRP was incubated for 20 minutes on ice or at room temperature and then centrifuged. The platelet pellet was lysed and analyzed by Western blot using an acetylated tubulin and an antitubulin antibody. (F) Human platelets were incubated for 30 minutes at room temperature with 15 μg/mL nocodazole, 25μM taxol, or without drug and then either fixed in suspension (top panel) or allowed to spread on glass coverslips for 60 minutes (bottom panel). Platelets were then stained with the mouse monoclonal antiacetylated tubulin antibody and a monoclonal rabbit antitubulin antibody (clone EP1332Y, Millipore, 04-1117) detected with AlexaFluor-546 goat antirabbit IgGs (Invitrogen; A11035) as indicated. Scale bars represent 10 μm.

  • Figure 2

    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.