Signaling pathways regulating LIM-kinase-1 activation and cofilin phosphorylation in activated platelets [Elektronische Ressource] / vorgelegt von Dharmendra Pandey

Pandey Dharmendra

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Aus dem Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten
der Ludwig-Maximilians-Universität München
Direktor: Prof. Dr. med. P. C. Weber





Signaling pathways regulating LIM-kinase-1
activation and cofilin phosphorylation in
activated platelets



Dissertation
zum Erwerb des Doktorgrades der Humanbiologie
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität zu München



vorgelegt von
Dharmendra Pandey


aus
Ahmednagar, Indien

2007

Mit Genehmigung der Medizinischen Fakultät
der Universität München






1. Berichterstatter: Prof. Dr. Med. Wolfgang Siess
2. Bericht Prof. Dr. M. Schleicher

Mitberichterstatter: Dr. B. Walzog
Priv. Doz. Dr. M. Weis

Dekan: Prof. Dr. med. D. Reinhardt

Tag der mündlichen Prüfung: 05.11.2007








Dedicated To My Grandparents















Jai Kisan, Jai Jawan, Jai Vigyan



Table of contents i
Table of contents
Table of contents ............................................................................................................................... i
Abbreviations and units ................................................................................................................... vi
1. Introduction .................................................................................................................................. 1
1.1. Overview ............................................................................................................................... 1
1.2. Platelets ................................................................................................................................. 1
1.2.1. Morphology of platelets ................................................................................................. 2
1.2.2. Platelet responses during hemostasis ............................................................................. 4
1.2.3. Signaling pathways for platelet activation ..................................................................... 5
1.3. Regulation of platelet function by the cytoskeleton .............................................................. 7
1.3.1. The actin cytoskeleton in platelets ................................................................................. 8
1.3.2. Resting platelets ............................................................................................................. 9
1.3.3. Stimulated platelets ...................................................................................................... 10
1.4. Actin dynamics .................................................................................................................... 12
1.4.1. Actin filament assembly and disassembly .................................................................... 12
1.4.2. Proteins regulating actin dynamics ............................................................................... 13
1.4.3. Signaling for actin dynamics ........................................................................................ 15
1.4.4. Effectors of Rho-, Rac- and Cdc42-like GTPases ........................................................ 15
1.4.5. Rho-kinase .................................................................................................................... 17
1.4.6. LIM-kinases .................................................................................................................. 19
1.5. Cofilin .................................................................................................................................. 20
1.5.1. Structure of cofilin ........................................................................................................ 20
1.5.2. Properties and functions of cofilin ............................................................................... 22
1.5.3. Regulation of cofilin activities ..................................................................................... 24
1.5.4. Cell biological functions of cofilin 26
2. Aim of the study ......................................................................................................................... 27
3. Materials and methods ................................................................................................................ 28
3.1. General equipments ............................................................................................................. 28
3.2. Materials .............................................................................................................................. 29
Table of contents ii
3.2.1. Chemicals ..................................................................................................................... 29
3.2.2. Enzymes and reagents for molecular biology .............................................................. 30
3.2.3. Antibodies and fluorescent probes ............................................................................... 31
3.2.4. Inhibitors, blockers and agonists .................................................................................. 33
3.2.5. Commercial kits and other materials ............................................................................ 34
3.2.6. Softwares ........ 34
3.3. In vitro studies of human platelets ...................................................................................... 35
3.3.1. Isolation of washed human platelets 35
3.3.2. Platelet shape change and aggregation by turbidimetric method ................................. 36
3.3.3. Measurement of ATP secretion .................................................................................... 37
3.4. Flow cytometric analysis of human platelets ...................................................................... 37
3.4.1. Shape change measurement .......................................................................................... 38
3.4.2. F-actin measurement using flow cytometry ................................................................. 39
3.5. Biochemical analysis of human platelets ............................................................................ 39
3.5.1. Platelet lysates for measuring phosphorylation of proteins .......................................... 39
3.5.2. Isolation of the total F-actin and actin cytoskeleton from platelets ............................. 39
3.5.3. Immunoprecipitation of LIMK-1 from platelets .......................................................... 41
3.5.4. LIMK-1 kinase assay .................................................................................................... 41
3.5.5. Measurement of protein concentration ......................................................................... 42
3.5.6. SDS-PAGE ................................................................................................................... 43
3.5.7. Isoelectric focusing (IEF) ............................................................................................. 44
3.5.8. Detection of protein on gel ........................................................................................... 45
3.5.9. Immunoblotting ............................................................................................................ 46
3.5.10. Densitometric analysis of immunoblots ..................................................................... 47
3.6. Microscopic study of human platelets ................................................................................. 48
3.6.1. Principle of confocal microscopy ................................................................................. 48
3.6.2. Preparation of poly-lysine coated coverslips ................................................................ 48
3.6.3. F-Actin staining of human platelets ............................................................................. 48
3.7. Expression and purification of recombinant cofilins .......................................................... 49
Table of contents iii
3.7.1. Work with E.coli .......................................................................................................... 49
3.7.2. Sub-cloning of cofilin and GFP cDNA ........................................................................ 51
3.7.3. In vitro expression of His-tagged cofilin ...................................................................... 55
3.7.4. Purification by Ni-NTA affinity chromatography ........................................................ 55
3.8. Peptide and protein delivery into platelets .......................................................................... 57
4. Results ........................................................................................................................................ 59
4.1. Identification of cofilin and LIMK-1 in human platelets .................................................... 59
4.1.1. Cofilin ........................................................................................................................... 59
4.1