C0188 The acetylation of tubulin regulates dense granule release in human platelets

Abstract

Background: Tubulin is present in the platelet microtubule cytoskeleton and plays a role in platelet shape change and release of granules through polymerization of the protein. Tubulin could undergo several post-traductional modifications including lysine acetylation, with a presently unknown role in platelet function. Acetylation of proteins is regulated by acetilase/deacetylase activities, the latter including HDAC and Sirt, although the role of these enzymes in platelet function remains unidentified. Aim. To study the functional implications of tubulin acetylation in platelet responses. Methods: Methods. For the detection of acetylated tubulin, platelets lysates were immunodetected with an specific antibody against the acetylated formof tubulin. For studies of dense granule release platelets were pre-incubated with 14C-5HT. The aggregation of washed platelet was stimulated with a panel of physiological agonists (collagen, thrombin, TXA2 analogue U46619, ADP) and determined by optical aggregometry. The role of deacetylases in tubulin acetylation was assayed by incubating platelets with specific inhibitors: trichostatin A (HDAC inhibitor) and cambinol (Sirt inhibitor). Results: Results. In resting platelets tubulin appears in an acetylated form. However, when platelets were stimulated and aggregated, tubulin acetylationwas strongly decreasedwith all the platelet agonists used. Inhibition of the deacetylase Sirt with cambinol (100 nM) markedly reduced platelet aggregation and dense granule release reaction, which is concomitant with the blockade of the agonistinduced tubulin deacetylation. This indicates a link between the level of acetylation of tubulin and these platelet responses; in contrast, the inhibition of the other deacetylases, HDAC with trichostatin A (10 μM) had a non significant effect, suggesting that Sirt is the main deacetylase responsible of tubulin deacetylation in platelets. Finally, when washed platelets were aspirin-treated (100 μM) previously to activation, the level of acetylation of tubulin remained as in non-stimulated samples, independently of the platelet agonist used; this was parallel with a marked inhibition of dense granule release in aspirin-treated platelets. Comment: Conclusion. We describe here for the first time that the acetylation of tubulin is strikingly relatedwith the platelet release-reaction in human platelets, and that the activation-dependent deacetylation of tubulin in platelets is regulated by Sirt, and specifically inhibited by aspirin. Grants: PI07/0463;Retics06/0026