Signaling pathways regulating LIM-kinase-1 activation and cofilin phosphorylation in activated platelets:
Gespeichert in:
| Beteilige Person: | |
|---|---|
| Format: | Hochschulschrift/Dissertation Buch |
| Sprache: | Englisch |
| Veröffentlicht: |
2007
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| Schlagwörter: | |
| Links: | http://edoc.ub.uni-muenchen.de/8222/ https://nbn-resolving.org/urn:nbn:de:bvb:19-82227 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016541377&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| Umfang: | 133 S. graph. Darst. |
Internformat
MARC
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| 245 | 1 | 0 | |a Signaling pathways regulating LIM-kinase-1 activation and cofilin phosphorylation in activated platelets |c von Dharmendra Pandey |
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Datensatz im Suchindex
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| adam_text | Table of contents [
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-lkinase 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 jU.
3.7.1. Work with E.coli 49
3.7.2. Sub-cloning of cofilin and GFPcDNA 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
4.1.2. LIMK-1 59
4.2. Platelet shape change induced by thrombin 61
4.2.1. Shape change studied by decrease in light transmission 61
4.2.2. Shape change studied by confocal microscopy 62
4.2.3. F-actin increase in thrombin-stimulated platelets 63
4.2.4. Activation of Rho-kinase (MYPT phosphorylation) 64
4.2.5. LIMK-1 and cofilin phosphorylation 65
4.2.6. Rapid association of cofilin with F-actin during shape change 67
4.3. Platelet secretion and aggregation induced by thrombin 69
4.3.1. Effect of Y-27632 on platelet secretion and aggregation 69
4.3.2. Rho-kinase activation (MYPT phosphorylation) 70
4.3.3. LIMK-1 phosphorylation and activation 71
4.3.4. Reversible cofilin dephosphorylation during thrombin-induced secretion and
aggregation 74
4.3.5. Inhibition of cofilin rephosphorylation by Rho-kinase inhibitors 74
4.3.6. F-actin increase in thrombin-stimulated platelet secretion/aggregation 75
4.3.7. Cofilin association with F-actin 76
4.4. Platelet shape change induced by LPA 78
4.4.1. LPA-stimulated platelet shape change and actin polymerization are Rho-kinase
dependent 78
4.4.2. Activation of Rho-kinase during LPA-induced shape change 79
4.4.3. Regulation of LIMK-1 and cofilin phosphorylation during shape change 80
Table of contents jv
4.4.4. LPA-induced PAK phosphorylation does not regulate LIMK-1 activation during shape
change 82
4.4.5. LPA induced a rapid association of cofrlin with actin cytoskeleton 83
4.5. LPA-mediated platelet secretion and aggregation 83
4.5.1. Platelet secretion and aggregation studied in Lumi-aggregometer 83
4.5.2. MYPT phosphorylation during LPA-stimulated platelet secretion and aggregation... 85
4.5.3. Regulation of LPA-induced cofilin de-and rephosphorylation 86
4.6. Signaling for cofilin dephosphorylation 87
4.6.1. Effect of phosphatase inhibitors on cofilin dephosphorylation 87
4.6.2. Inhibition of cofilin dephosphorylation by calcineurin inhibitor 88
4.6.3. Inhibition of LPA-induced cofilin dephosphorylation by BAPTA-AM 89
4.6.4. Inhibition of cofilin dephosphorylation by PI3-kinase inhibitor 90
4.7. Peptide or protein transfection into platelets 91
4.7.1. Poly-arginine based peptide transfection 91
4.7.2. Protein transfection by Chariot™ 92
5. Discussion 93
5.1. Role ofRho-kinase in activated platelets 93
5.1.1. Activation of Rho-kinase 93
5.1.2. Rho-kinase activation mediates F-actin increase during shape change 94
5.1.3. Rho-kinase is involved in secretion and platelet aggregation 95
5.2. Identification and regulation of LIMK-1 in human platelets 97
5.2.1. LIMK-1 but not LIMK-2 expressed in platelets 97
5.2.2. Rho-kinase activation leads to LIMK-1 phosphorylation 98
5.3. Regulation of cofilin activities 99
5.3.1. Cofilin association with F-actin 102
5.3.2. Possible factors regulating cofilin dephosphorylation 103
5.4. Two-step model for cofilin phospho-cycle 107
6. Summary 109
7. Zusammenfassung Ill
8. References 114
Table of contents v
Acknowledgements 130
List of Publications 131
Curriculum vitae 138
|
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| dewey-full | 616.1 573.159 |
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| dewey-ones | 616 - Diseases 573 - Specific physiological systems in animals |
| dewey-raw | 616.1 573.159 |
| dewey-search | 616.1 573.159 |
| dewey-sort | 3616.1 |
| dewey-tens | 610 - Medicine and health 570 - Biology |
| discipline | Biologie Medizin |
| format | Thesis Book |
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| illustrated | Illustrated |
| indexdate | 2024-12-20T13:14:25Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-016541377 |
| oclc_num | 246675951 |
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| publishDate | 2007 |
| publishDateSearch | 2007 |
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| spellingShingle | Pandey, Dharmendra 1978- Signaling pathways regulating LIM-kinase-1 activation and cofilin phosphorylation in activated platelets |
| subject_GND | (DE-588)4113937-9 |
| title | Signaling pathways regulating LIM-kinase-1 activation and cofilin phosphorylation in activated platelets |
| title_auth | Signaling pathways regulating LIM-kinase-1 activation and cofilin phosphorylation in activated platelets |
| title_exact_search | Signaling pathways regulating LIM-kinase-1 activation and cofilin phosphorylation in activated platelets |
| title_full | Signaling pathways regulating LIM-kinase-1 activation and cofilin phosphorylation in activated platelets von Dharmendra Pandey |
| title_fullStr | Signaling pathways regulating LIM-kinase-1 activation and cofilin phosphorylation in activated platelets von Dharmendra Pandey |
| title_full_unstemmed | Signaling pathways regulating LIM-kinase-1 activation and cofilin phosphorylation in activated platelets von Dharmendra Pandey |
| title_short | Signaling pathways regulating LIM-kinase-1 activation and cofilin phosphorylation in activated platelets |
| title_sort | signaling pathways regulating lim kinase 1 activation and cofilin phosphorylation in activated platelets |
| topic_facet | Hochschulschrift |
| url | http://edoc.ub.uni-muenchen.de/8222/ https://nbn-resolving.org/urn:nbn:de:bvb:19-82227 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016541377&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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