NO-cGMP and ROS pathways in regulation of platelet function and megakaryocyte maturation [Elektronische Ressource] / vorgelegt von Antonija Jurak Begonja

Antonija

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2007
• Nombre de lectures: 56
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

NO/cGMP and ROS Pathways in Regulation
of Platelet Function and Megakaryocyte
Maturation








Dissertation

zur Erlangung des naturwissenschaftlichen Doktorgrades der
Julius-Maximilians-Universität Würzburg


vorgelegt von
Antonija Jurak Begonja
aus Zadar, Kroatien


Würzburg 2007

Eingereicht am: ...........................................
bei der Fakultät für Chemie und Pharmazie




1. Gutachter: Prof. Dr. Ulrich Walter
2. Gutachter: Prof. Dr. Friedrich Grummt
der Dissertation




1. Prüfer: Prof. Dr. Ulrich Walter
2. Prüfer: Prof. Dr. Friedrich Grummt
3. Prüfer: ...........................................
des öffentlichen Promotionskolloquiums




Tag des öffentlichen Promotionskolloquiums: ..........................................


Doktorurkunde ausgehändigt am: .............................................................


CONTENTS

SUMMARY .........................................................................................................................1
ZUSAMMENFASSUNG .......................................................................................................3
INTRODUCTION .................................................................................................................5
1. PLATELETS...................................................................................................................6
1.1. Morphology ....................................................................................................................... 7
1.2. Adhesive Platelet Receptors........................................................................................... 9
1.3. Platelet Receptors for Soluble Agonists....................................................................... 15
1.3. The Role of Platelets in Haemostasis and Thrombosis ............................................... 18
2. REACTIVE OXYGEN SPECIES ..................................................................................21
2.1. NAD(P)H Oxidases .......................................................................................................... 22
2.2. Regulation of NOX..........................................................................................................23
2.3. ROS and Platelets.. 24
3. BIOGENESIS OF PLATELETS......................................................................................25
3.1. Development of Megakaryocytes from Haematopoietic Stem Cells ................... 25
3.2. Platelet Formation........................................................................................................... 26
3.3. Thrombopoietin and Signals Involved in Thrombopoiesis......................................... 27
3.4. Other Cytokines and Transcription Factors................................................................. 28
AIMS OF THE STUDY30
MATERIALS AND METHODS ............................................................................................31
4. MATERIALS...............................................................................................................31
4.1. Cell culture....................................................................................................................... 31
4.2. Animals ............................................................................................................................. 31
4.3. Plasmids ............................................................................................................................ 31
4.4. Markers.......... 31
4.5. Kits ..................................................................................................................................... 31
4.6. Antibodies ........................................................................................................................ 32
4.7. Chemicals ........................................................................................................................ 33
4.8. Special Materials and Equipment................................................................................ 34
4.9. Software ........................................................................................................................... 35
5. METHODS36
5.1. Cell Culture ...................................................................................................................... 36
5.2. Blood Preparations .........................................................................................................37
5.3. Culturing and Differentiation of Mouse Megakaryocytes........................................ 39
5.4. Cell Count........................................................................................................................ 40
5.5. ROS Measurement.......................................................................................................... 40
5.6. Flow Cytometry ............................................................................................................... 41
5.7. Serotonin Secretion......................................................................................................... 42
5.8. Thromboxane Synthase Activity ................................................................................... 42
5.9. Aggregometry................................................................................................................. 43
5.10. Adhesion Under Flow Conditions (Flow Chamber) ................................................. 43
5.11. Protein Analysis.............................................................................................................. 43



5.12. Pull Down Assays........................................................................................................... 46
5.13. Immunofluorescence of Megakaryocytes ............................................................... 48
5.14. Platelet Formation Assay ............................................................................................. 48
5.15. Data Analysis ................................................................................................................. 49
RESULTS ............................................................................................................................50
6. Role of ROS in Platelets..........................................................................................50
6.1. Platelets Produce Intracellular ROS after Stimulation with Different Agonists....... 50
6.2. NAD(P)H Oxidase and COX are the Source of ROS Production in Platelets......... 52
6.3. NAD(P)H Oxidase Subunits in Platelets ........................................................................ 55
6.4. ROS Production Affects αIIb β3 Activation .................................................................. 56
6.5. P-Selectin, CD40L Expression, Serotonin Secretion and TxA Production............... 59 2
6.6. ROS and NO Interaction................................................................................................ 60
6.7. Small GTPases Rap1 and Rac1..................................................................................... 61
6.8. Platelet Activation and Thrombus Formation in NOX1 Knockout Mice ................. 63
7. Role of Protein Kinase G I in Platelets65
7.1. Stimulation of sGC and PKG I Does not Activate ERK or p38 Kinases .................... 65
7.2. p38 Has no Effect on Platelet Aggregation and P-selectin Expression .................. 67
7.3. Activation of p38 MAP Kinase by ADP and TxA Secretion...................................... 68 2
8. Role of NO/cGMP and cAMP Pathways in Megakaryocytes ...........................71
8.1. In vitro Development of Megakaryocytes.................................................................. 71
8.2. Increased PKG, PKA, VASP and β sGC Expression During Megakaryocyte
Differentiation ......................................................................................................................... 72
8.3. Modulation of Megakaryocyte Differentiation by cGMP and cAMP.................... 75
8.4. Platelet Formation........................................................................................................... 79
DISCUSSION ....................................................................................................................81
9. Intracellular ROS Production from NOX in Platelets ...........................................81
9.1. NOX is Involved in Integrin Activation.......................................................................... 83
9.2. ROS is not Scavenging NO in Platelets ........................................................................ 85
9.3. Possible Mechanisms of ROS Action ............................................................................ 86
10. Inhibitory Role of NO/cGMP in Platelets............................................................89
10.1. p38 is Activated by Thrombin Induced ADP and TxA2 Secretion.......................... 91
11. Functionally Active cGMP/cAMP Pathway in Megakaryocytes.................