Release Reaction Of Human Platelets Research Articles

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

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

β2-glycoprotein-I (apo-H) inhibits the release reaction of human platelets during ADP-induced aggregation

In order to further characterize the modulation of the ADP-induced aggregation of gelfiltered human platelets by β 2-glycoprotein-I 02G-1), the influence of this glycoprotein upon the serotonin (5-HT) release during aggregation was measured. The following results were obtained: β 2-G-I completely inhibits the 5-HT release during ADP-induced platelet activation. The inhibition is correlated with the inhibition of the second wave of the ADP-induced aggregation. This effect of β 2-G-I is not dose-dependent and appears above a threshold concentration of 0.1-0.15 mg/ml in the assay. The specificity of the β 2-G-I interaction with the ADP-activation is supported by results obtained with collagen or thrombin as aggregating agents. In these cases neither the aggregation nor the release is influenced by the glycoprotein. In respect to the results obtained, β 2-G-I is a potent candidate to be a modulator of ADP-induced platelet activation in vivo.

Triggering by Paf-acether and adrenaline of cyclo-oxygenase-independent platelet aggregation.

Platelet-activating factor (Paf-acether, 1-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) induced full aggregation and a limited release reaction of human platelets in plasma or in blood. Cyclo-oxygenase inhibition with aspirin only reduced aggregation when induced by threshold amounts of Paf-acether, whereas higher concentrations surmounted inhibition whether tested in citrated or in heparinized platelet-rich plasma or blood. Aspirin-induced inhibition of platelet secretion by Paf-acether was insurmountable and independent of the anti-coagulant used. Paf-acether and adrenaline acted synergistically in inducing aggregation in citrate and heparin. Aspirin in vitro or after oral ingestion at doses that suppressed aggregation induced by arachidonic acid alone, failed to reduce significantly the synergized aggregation induced by Paf-acether alone or combined with adrenaline. Twenty-four hours after the oral ingestion of aspirin, when aggregation by arachidonic acid remained blocked, a slight inhibitory activity on the effect of Paf-acether noted 4 h after aspirin, had ceased. This was probably accounted for by the synthesis of thromboxane A2 by newly formed platelets, since the in vitro addition of aspirin, or of the thromboxane/endoperoxide receptor inhibitor 13-azaprostanoic acid caused the 24 h platelets to behave in a manner similar to platelets collected 4 h after aspirin. The alpha 2-adrenoceptor inhibitor, yohimbine, blocked the direct effect of adrenaline as well as its synergism with Paf-acether. Since the synergistic effect of Paf-acether and adrenaline was maintained when thrombin-degranulated platelets were used, and aspirin remained ineffective against it, it is clear that the augmented platelet responsiveness is not accounted for by the platelet release reaction. 6 Paf-acether and adrenaline act synergistically and stimulate platelets by cyclo-oxygenaseindependent mechanisms, which may be relevant in human physiopathological conditions.

Effects of adenosine 3′:5′-monophosphate and platelet aggregation on thromboxane biosynthesis in human platelets

The prostaglandin (PC) endoperoxides, PGGs and PGHz [l], and thromboxane (TX)A, [2] cause rapid aggregation and induce the release reaction in human platelets. Aggregating agents, such as collagen and thrombin, stimulate release of arachidonic acid from platelet phospholipids. This leads to synthesis of endoperoxides, thromboxanes, prostaglandins and hydroxy acids [3-51. Elevation of adenosine 3’: 5’-monophosphate (cyclic AMP) levels in platelets prevents aggregation [6-8] and the simultaneous production of TXBz [9-121. Two mechanisms for the inhibition of thromboxane formation have been proposed: (1) Inhibition of prostaglandin endoperoxide synthase (EC 1.14.99.1) [9]; (2) Inhibition of arachidonic acid release from platelet phospholipids [lo121. In the latter reports no inhibition of prostaglandin endoperoxide synthase was observed [lo-121. We have therefore reinvestigated the effects of cyclic AMP on arachidonic acid release and on the conversion of arachidonic acid to TXBs. The results suggest that elevation of cyclic AMP levels inhibits both of these reactions in platelets and that the inhibitory effect of cyclic AMP on TXBz formation from arachidonic acid is not direct but secondary to inhibition of platelet aggregation.

Thrombin-platelet interactions investigated with the calcium electrode

The thrombin-induced release reaction of human platelets is studied using a calcium ion electrode to follow the release of calcium. A continuous tracing of the time course of calcium release shows three phases: 1) an initial lag phase of variable length, which is dependent on the thrombin concentration; 2) an exponential phase during which most of the calcium is released; 3) a final linear phase of slow calcium release which starts 90–120 sec after the addition of thrombin. The calcium yield (the amount of calcium released 90 sec after thrombin addition) is directly related to the thrombin concentration and platelet number. The release of calcium is inhibited by preincubation of the platelets with dibutyryl cyclic AMP, but this inhibition is overcome by additional thrombin. Results obtained with varying platelet number and thrombin concentration suggest that platelets stimulated by a low concentration of thrombin release only a portion of their releasable calcium. If thrombin is added in two increments rather than in a single increment, a second release of calcium is observed; the final calcium yield, however, decreases as the time interval between thrombin additions increases. The indication that platelets may undergo a partial release reaction without complete impairment of their hemostatic effectiveness suggests that platelets may be perceived as homeostatic as well as hemostatic cells, with implications for the maintenance of vascular integrity by circulating platelets.

Membrane alterations in connection with the release reaction in human platelets as studied by the lactoperoxidase-iodination technique and by agglutination with bovine factor VIII-related protein

Changes in the platelet membrane surface as a result of the thrombin-induced release reaction have been examined by lactoperoxidase-catalyzed iodination and agglutination of the platelets with bovine factor VIII-related protein. The distribution of labeled substances after iodination was studied after sodium dodecyl sulphate polyacrylamide gel electrophoresis of platelet proteins. The major radioactive peaks in the control samples were associated with (glyco)polypeptides of estimated molecular weights about 100 000 and 120 000, and a smaller peak was observed at 147 000 daltons. Platelets labeled after the release reaction had been performed incorporated markedly less radioactive iodine into the membrane proteins than control platelets, i.e. 57.7% ± 9.2 (147 000), 26.6% ± 13.0 (120 000) and 20.6% ± 5.5 (100 000) of the corresponding peaks of the control. These platelets agglutinated poorly with bovine factor VIII-related protein. These effects were not seen when the platelets were preincubated with metabolic inhibitors to prevent secretion. After sodium dodecyl sulphate polyacrylamide gel electrophoresis of the soluble fraction from released platelets, the main glycoprotein band (GPS), when examined in unreduced form, showed slower migration than the corresponding band in the control sample. It is concluded, that the surface structure of thrombin-secreted platelets is different from that of untreated cells. This change is independent of the direct action of thrombin on the membrane, but may be a result of conformational alterations during the secretion process and/or adsorption of released material to the external membrane surface.

Factors responsible for ADP-induced release reaction of human platelets.

Extensive aggregation of human platelets can be induced by ADP without secondaryaggregation or release of granule contents. This occurs with washed platelets in Tyrode solution containing 0.35% albumin, human fibrinogen, and apyrase, and in platelet-rich, heparin- or hirudin-plasma. Conditions that caused release during ADP-inducedaggregation were-citrate as the anticoagulant in platelet-rich plasma; addition of citrate (11-15 mM) to a suspension of washed platelets, or to hirudin-plasma or heparin-plasma; suspension of platelets in a medium containing magnesium but no calcium;and the presence of trace amounts of thrombin or aggregated gamma globulin in the platelet suspensions. Acetylsalicylic acid, phenylbutazone, or sulfinpyrazone inhibited secondary aggregation and release in all these circumstances. Heparin or hirudin inhibited ADP-INDUCED SECONDARY AGGREGATION AND RELEASE PROMOTED BY TRACES OF THROMBIN. Although fibrinogen is required for ADP-induced primary aggregation, it does not support secondary aggregation and release, provided that it has no clot-promoting activity. The main agent responsible for ADP-induced secondary aggregation and release in human, citrated, platelet-rich plasma appears to be sodium citrate. Suspending washed human platelets in a medium without calcium mimics the effect of citrate.

Induction of aggregation and of the release reaction in human platelets by polylysine.

Polylysine aggregates human blood platelets and, within a concentration range which is typical for a given preparation, can elicit the release reaction. No evidence for a relation between the active concentration range of different polylysine preparations and their structure or molecular weight could be established. ADP stimulates the release at ub- or superoptimal concentrations of polylysine. These results support the hypothesis that cell contact, here facilitated by the polycation, ADP, fibrinogen and calcium ions are required for the induction of the release reaction. Once these components are available in adequate amounts within a polylysine-induced aggregate, the release reaction will be propagated automatically throughout the aggregate.

Proteolytic Inhibitors, Contact and Other Variables in the Release Reaction of Human Platelets

SummaryPlatelet-rich plasma (PRP) containing platelets labelled with 14C-serotonin was used to characterize the release reaction. Heparin promotes release, and the reduced release response to epinephrine observed in heparinized compared to citrated PRP is probably due to the earlier reaction. The release reaction can occur in citrated PRP at pH 7.0, is marked between pH 7.2 and 7.6, and decreases above pH 8.0. It is retained better when PRP is stored below pH 7.7. Adenosine, prostaglandin E1, and theophylline inhibit both aggregation and release, probably because they elevate cyclic AMP. D- and L-tosyl-phenylalanine chloromethylketone (TPCK) also inhibit both aggregation and release at a concentration of 0.1 mM whereas 0.1 mM D- and l-L-chloro-2-tosylamido-7-amino-2-heptanone (TLCK) and 5 mM diisopropylfluoro-phosphate (DFP), like aspirin, inhibit only release. Colchicine (0.1 mM) alters platelet shape but does not affect release or aggregation. Centrifugation of PRP at 37° C causes release which responds to inhibitors and pH changes in a manner similar to release induced by aggregation with ADP or epinephrine. Release occurs in thromb-asthenic platelets centrifuged at 37° C and is increased by epinephrine. Release of ATP and ADP correlates significantly with release of 14C-serotonin. Thrombin plus CaCl2 causes release of β-glucuronidase but not of zinc.