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C0195 The presence of the TXA2 receptor in lipid rafts of platelets is necessary for platelet activation by TXA2
Abstracts / Thrombosis Research 130 (2012) S100–S202
C0188 The acetylation of tubulin regulates dense granule release in human platelets Antonio Moscardó1, María Teresa Santos1, Ana Latorre1, Isabel Madrid2, Juana Vallés1 1 Research Center, University Hospital La fe - Avd Campanar 21 46009 Valencia, Spain; 2Intensive Care Unit - Avd Campanar 21 46009 Valencia, Spain 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 form of 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 acetylation was strongly decreased with 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 related with 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 doi:10.1016/j.thromres.2012.08.010
C0195 The presence of the TXA2 receptor in lipid rafts of platelets is necessary for platelet activation by TXA2 Antonio Moscardó1, Juana Vallés1, Isabel Madrid2, Ana Latorre1, Ángeles Dasí1, María Teresa Santos1 1 Research Center, University Hospital La fe - Avd Campanar 21 46009 Valencia, Spain; 2Intensive Care Unit - Avd Campanar 21 46009 Valencia, Spain Background: Thromboxane A2 (TXA2) synthesized by activated platelets plays an important role in the pathophysiology of thrombogenesis, by
S103
acting on its specific receptor (TXA2-R). This reinforces the activation of platelets that produce it and promotes platelet recruitment and thrombus growth. Lipid rafts are cholesterol-rich microdomains present in the plasma membrane which concentrate signaling molecules and receptors, and play an important role in signal transduction in platelets. Currently it has not been described the presence of TXA2-R associated with lipid rafts in human platelets. Aim. To analyze the presence of TXA2-R into platelet rafts and the possible effects of this location on the biochemical and functional responses of platelets to TXA2. Methods: Platelets from normal subjects were washed and aggregation was assessed by optical aggregometry, the release of adenine nucleotides by HPLC, the movements of calcium by fluorimetry and platelet activation (P-selectin and PAC-1) by flow cytometry. The isolation of rafts was performed by sucrose gradient ultracentrifugation of platelet lysates, analyzed for specific proteins by immunoblotting, and rafts were identifyed by the presence of CD36-positive fractions. The functional role of rafts was studied by disrupting them incubating platelets with methylb-cyclodextrin (MβCD). Results: Isolation of rafts allowed the detection in the fraction corresponding to the rafts (CD36+) of a significant percentage (38%) of total TXA2-R determined by densitometry. MβCD-treatment strongly reduced the presence of both CD36 and TXA2-R in the lipid rafts. By confocal microscopy, using cholera toxin-Alexa488 as a marker of rafts, we confirmed the colocalization of TXA2-R into platelet rafts. Pretreatment of platelets with MβCD decreased platelet aggregation induced by U46619 or IBOP (N85%), nucleotide release (N70%) and P-selectin exposure (N80%). This demonstrates the important role of lipid rafts for TXA2-induced platelet function. Disruption of lipid rafts also decreased the activation-induced calcium increase (−60%) and the activation of integrin IIbIIIa (PAC-1+), platelet responses directly linked to TXA2-R and independent of ADP receptor P2Y12, thus involving the lipid rafts in signal transduction mechanisms mediated by TXA2-R. Comment: The results demonstrate for the first time thatTXA2 receptor is associated with lipid rafts in platelets, and the biochemical and functional importance of their localization in these membrane microdomains for the platelet responses to TXA2 stimulation. Grants: PI07/0463; Retics06/0026. doi:10.1016/j.thromres.2012.08.011
C0270 Analysis of different mechanisms through hydrogen sulphide (H2S) inhibit platelet aggregation Emilse Bermejo1, Daniel Saenz2, Maria Fabiana Alberto1, Ruth Estela Rosenstein2, Maria Angela Lazzari1 1 Instituto de Investigaciones Hematologicas As, Academia Nacional de Medicina de BsHEMOSTASIA Y TROMBOSIS - Pacheco de Melo 3081, Argentina; 2Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina, Departamento de Bioquímica Humana, Paraguay 1812, CABA Background: Hydrogen sulphide (H2S) is the most recent of the gasotransmitters to be identified and investigated. H2S is an important endogenous modulator, which exhibits beneficial effects on the cardiovascular system, without producing toxic metabolites. Objective: In order to investigate H2S inhibitory pathways on platelet function we performed several tests: the opening of KATPchannels, the cGMP and cAMP accumulation, fibrinogen binding (B-Fg) and Ca2+ influx. Methods: Methods: Tests were performed using platelet rich plasma (PRP)(300 × 109 plat/L). Sodium hydrogen sulfide (NaHS) was used as H2S donor and 100 μM glibenclamide (Glib) as a selective K+-channel
C0188 The acetylation of tubulin regulates dense granule release in human platelets Antonio Moscardó1, María Teresa Santos1, Ana Latorre1, Isabel Madrid2, Juana Vallés1 1 Research Center, University Hospital La fe - Avd Campanar 21 46009 Valencia, Spain; 2Intensive Care Unit - Avd Campanar 21 46009 Valencia, Spain 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 form of 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 acetylation was strongly decreased with 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 related with 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 doi:10.1016/j.thromres.2012.08.010
C0195 The presence of the TXA2 receptor in lipid rafts of platelets is necessary for platelet activation by TXA2 Antonio Moscardó1, Juana Vallés1, Isabel Madrid2, Ana Latorre1, Ángeles Dasí1, María Teresa Santos1 1 Research Center, University Hospital La fe - Avd Campanar 21 46009 Valencia, Spain; 2Intensive Care Unit - Avd Campanar 21 46009 Valencia, Spain Background: Thromboxane A2 (TXA2) synthesized by activated platelets plays an important role in the pathophysiology of thrombogenesis, by
S103
acting on its specific receptor (TXA2-R). This reinforces the activation of platelets that produce it and promotes platelet recruitment and thrombus growth. Lipid rafts are cholesterol-rich microdomains present in the plasma membrane which concentrate signaling molecules and receptors, and play an important role in signal transduction in platelets. Currently it has not been described the presence of TXA2-R associated with lipid rafts in human platelets. Aim. To analyze the presence of TXA2-R into platelet rafts and the possible effects of this location on the biochemical and functional responses of platelets to TXA2. Methods: Platelets from normal subjects were washed and aggregation was assessed by optical aggregometry, the release of adenine nucleotides by HPLC, the movements of calcium by fluorimetry and platelet activation (P-selectin and PAC-1) by flow cytometry. The isolation of rafts was performed by sucrose gradient ultracentrifugation of platelet lysates, analyzed for specific proteins by immunoblotting, and rafts were identifyed by the presence of CD36-positive fractions. The functional role of rafts was studied by disrupting them incubating platelets with methylb-cyclodextrin (MβCD). Results: Isolation of rafts allowed the detection in the fraction corresponding to the rafts (CD36+) of a significant percentage (38%) of total TXA2-R determined by densitometry. MβCD-treatment strongly reduced the presence of both CD36 and TXA2-R in the lipid rafts. By confocal microscopy, using cholera toxin-Alexa488 as a marker of rafts, we confirmed the colocalization of TXA2-R into platelet rafts. Pretreatment of platelets with MβCD decreased platelet aggregation induced by U46619 or IBOP (N85%), nucleotide release (N70%) and P-selectin exposure (N80%). This demonstrates the important role of lipid rafts for TXA2-induced platelet function. Disruption of lipid rafts also decreased the activation-induced calcium increase (−60%) and the activation of integrin IIbIIIa (PAC-1+), platelet responses directly linked to TXA2-R and independent of ADP receptor P2Y12, thus involving the lipid rafts in signal transduction mechanisms mediated by TXA2-R. Comment: The results demonstrate for the first time thatTXA2 receptor is associated with lipid rafts in platelets, and the biochemical and functional importance of their localization in these membrane microdomains for the platelet responses to TXA2 stimulation. Grants: PI07/0463; Retics06/0026. doi:10.1016/j.thromres.2012.08.011
C0270 Analysis of different mechanisms through hydrogen sulphide (H2S) inhibit platelet aggregation Emilse Bermejo1, Daniel Saenz2, Maria Fabiana Alberto1, Ruth Estela Rosenstein2, Maria Angela Lazzari1 1 Instituto de Investigaciones Hematologicas As, Academia Nacional de Medicina de BsHEMOSTASIA Y TROMBOSIS - Pacheco de Melo 3081, Argentina; 2Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina, Departamento de Bioquímica Humana, Paraguay 1812, CABA Background: Hydrogen sulphide (H2S) is the most recent of the gasotransmitters to be identified and investigated. H2S is an important endogenous modulator, which exhibits beneficial effects on the cardiovascular system, without producing toxic metabolites. Objective: In order to investigate H2S inhibitory pathways on platelet function we performed several tests: the opening of KATPchannels, the cGMP and cAMP accumulation, fibrinogen binding (B-Fg) and Ca2+ influx. Methods: Methods: Tests were performed using platelet rich plasma (PRP)(300 × 109 plat/L). Sodium hydrogen sulfide (NaHS) was used as H2S donor and 100 μM glibenclamide (Glib) as a selective K+-channel