Aus dem Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten der Ludwig-Maximilians-Universität München Direktor: Prof. Dr. med. C. Weber Role of the serotonin transporter and the 5-HT2A and 5-HT4 receptors for platelet function in blood Dissertation zum Erwerb des Doktorgrades der Medizin an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München Vorgelegt von Vasileios Bampalis aus Athen, Griechenland 2016 Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatter: Prof. Dr. Med. Wolfgang Siess Mitberichterstatter: Priv. Doz. Dr. Dirk Sibbing Prof. Dr. Dr. Christian Sommerhof Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 28.01.2016 [i] Dedicated to my parents [ii] Contents Contents ........................................................................................................................ iii 1. State of the art ............................................................................................................ 1 1.1 Introduction: Serotonin, the happiness hormone .................................................. 1 1.2 Biosynthesis and degradation of 5-HT .................................................................... 2 1.3 5-HT transporter (SERT) and 5-HT receptors .......................................................... 4 1.4 Platelets and platelet function ............................................................................... 5 1.4.1 Platelet origin and morphology ....................................................................... 5 1.4.2 Platelet adhesion ............................................................................................ 8 1.4.3 Platelet activation and aggregation ............................................................... 10 1.4.4 Secretion of platelet granule contents and amplification of platelet activation .................................................................................................................................... 11 1.4.5 Platelet aggregation ...................................................................................... 14 1.5 Uptake, storage and release of 5-HT by platelets ................................................. 15 1.6 The platelet 5-HT transporter (SERT) and selective serotonin reuptake inhibitors (SSRI) ............................................................................................................................... 16 1.6.1 The platelet 5-HT transporter (SERT) ............................................................. 16 1.6.2 Effects of selective serotonin reuptake inhibitors (SSRIs) on platelets ........... 17 1.7 Platelet protein serotonylation ............................................................................ 19 1.8 Platelet 5-HT receptors ........................................................................................ 21 1.8.1 The platelet 5-HT2A receptor ........................................................................ 21 1.8.2 Effects of 5-HT2A receptor antagonists on platelets ...................................... 21 1.8.3 The platelet 5-HT4 platelet receptor ............................................................. 25 1.9 Topic and question of the dissertation ................................................................. 25 2. Materials and methods ............................................................................................. 28 2.1 Materials ............................................................................................................. 28 2.1.1 Devices ......................................................................................................... 28 2.1.2 Disposable materials ..................................................................................... 28 2.1.3 Chemicals ..................................................................................................... 29 2.1.4 Buffers .......................................................................................................... 30 2.2 Methods .............................................................................................................. 30 2.2.1 Blood collection ............................................................................................ 30 2.2.2 Preparation and preservation of human atheromatous plaque homogenate 31 2.2.3 Whole blood platelet aggregation ................................................................. 32 [iii] 2.2.4 Measurement of ATP secretion ..................................................................... 34 2.2.5 Plaque-induced platelet adhesion and thrombus formation under flow ........ 35 2.2.6 Shear-induced platelet adhesion and aggregation ......................................... 37 2.2.7 Statistical analysis ......................................................................................... 38 3. Results ...................................................................................................................... 39 3.1 Role of the serotonin reuptake transporter (SERT) for platelet aggregation in blood ....................................................................................................................................... 39 3.1.1 5-HT potentiates the epinephrine-induced platelet aggregation ................... 39 3.1.2 The SERT-inhibitor fluoxetine inhibits the 5-HT-induced potentiation of platelet aggregation .................................................................................................... 41 3.1.3 No effect of fluoxetine on stimulus-dependent platelet aggregation ............. 45 3.1.4 Effects of the combination of acetylsalicylic acid and fluoxetine on platelet aggregation ................................................................................................................. 49 3.1.5 Role of different blood pre-incubation time periods on spontaneous and stimulus-induced platelet aggregation ......................................................................... 51 3.2 Role of the platelet 5-HT2A receptor for platelet adhesion, aggregation and secretion in blood............................................................................................................ 55 3.2.1 Role of 5-HT2A receptor antagonists on 5-HT induced potentiation of platelet aggregation ................................................................................................................. 55 3.2.2 Effect of ketanserin on stimulus-induced whole blood platelet aggregation .. 56 3.2.3 Effect of ketanserin on stimulus-induced ATP secretion in whole blood ........ 57 3.2.4 Effect of ketanserin on plaque-induced platelet aggregate formation under arterial flow ................................................................................................................. 59 3.2.5 Effect of 5-HT2A receptor antagonism on shear-induced platelet adhesion and aggregate formation .................................................................................................... 62 3.3 Role of the platelet 5-HT4 receptor for platelet aggregation in blood .................. 64 3.3.1 Role of 5-HT4 receptor agonism on platelet aggregation ............................... 64 3.3.2 Role of 5-HT4 receptor antagonism on platelet aggregation.......................... 66 4. Discussion ................................................................................................................. 69 4.1 Role of exogenous 5-HT on whole blood platelet aggregation .............................. 69 4.2 Role of the platelet 5-HT transporter for platelet aggregation ............................. 70 4.3 Effect of the combination of fluoxetine plus acetylsalicylic acid on inhibition of platelet aggregation ........................................................................................................ 73 4.4 Role of blood pre-incubation on stimulus-induced platelet aggregation ............... 74 4.5 Role of the platelet 5-HT2A receptor for platelet activation under static and flow conditions ....................................................................................................................... 75 4.6 Role of the platelet 5-HT4 receptor for platelet aggregation ................................ 79 4.7 Roles of platelet 5-HT in various diseases ............................................................ 81 5. Conclusion and perspectives ..................................................................................... 83 6. Summary ................................................................................................................... 85 [iv] 6.1 Summary ............................................................................................................. 85 6.2 Zusammenfassung ............................................................................................... 87 7. Own publications ...................................................................................................... 90 8. Literature .................................................................................................................. 91 9. Acknowledgements ................................................................................................. 103 10. Abbreviations and units ......................................................................................... 104 10.1 Abbreviations .................................................................................................. 104 10.2 Units...............................................................................................................