Relevance of platelet count and ITAM-signalling pathway in murine models of haemostasis, thrombosis and thrombo-inflammation:
Gespeichert in:
1. Verfasser: | |
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Format: | Abschlussarbeit Buch |
Sprache: | English |
Veröffentlicht: |
2014
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Schlagworte: | |
Online-Zugang: | Volltext Inhaltsverzeichnis |
Beschreibung: | Zsfassung in dt. und engl. Sprache |
Beschreibung: | VIII, 105 Bl. Ill., graph. Darst. |
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adam_text | JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG
RELEVANCE OF PLATELET COUNT AND ITAM-SIGNALLING PATHWAY IN MURINE MODELS
OF
HAEMOSTASIS, THROMBOSIS AND THROMBO-INFLAMMATION
RELEVANZ DER THROMBOZYTENZAHL UND DES ITAM-SIGNALWEGS IN MAUSMODELLEN
DER HAMOSTASE, THROMBOSE UND THROMBOINFLAMMATION
DOCTORAL THESIS
FOR A DOCTORAL DEGREE
AT THE
GRADUATE SCHOOL
OF LIFE SCIENCES
SECTION BIOMEDICINE
SUBMITTED
BY
MARTINA MOROWSKI
FROM KASSEL
WURZBURG, 2014
HTTP://D-NB.INFO/1055055266
VI
TABLE OF CONTENTS
1 INTRODUCTION 1
1.1 ORIGIN OF PLATELETS 2
1.2 PLATELET ACTIVATION AND THROMBUS FORMATION 3
1.2.1 RHEOLOGICAL CONDITIONS IN THE VESSEL AND PLATELET TETHERING VIA
GPIB 3
1.2.2 THE GPVI/FCRY-CHAIN COMPLEX AND ITAM-SIGNALLING 4
1.2.3 PLATELET ACTIVATION THROUGH G PROTEIN-COUPLED RECEPTORS 7
1.2.4 FIRM PLATELET ADHESION AND THROMBUS GROWTH 7
1.3 PLATELET COUNT 8
1.3.1 THROMBOCYTOPENIA 8
1.3.1.1 PRIMARY IMMUNE THROMBOCYTOPENIA 8
1.3.1.2 SECONDARY IMMUNE THROMBOCYTOPENIA 10
1.3.1.3 NON-IMMUNE THROMBOCYTOPENIA 10
1.3.2 HAEMOSTASIS AND THROMBOSIS DURING THROMBOCYTOPENIA 11
1.3.3 YOUNG AND YOUNG STRESSED PLATELETS AND THEIR PROPERTIES 12
1.3.4 TREATMENT OF THROMBOCYTOPENIA 12
1.4 ARTERIAL THROMBOSIS AND HAEMOSTASIS MODELS IN MICE 13
1.4.1 ARTERIAL THROMBOSIS MODELS IN GENETICALLY MODIFIED MICE 16
1.4.1.1 CLP36 16
1.4.1.2 GRB2 17
1.4.1.3 SLAP AND SLAP2 17
1.4.1.4 EFHD2 18
1.5 AIM OF THIS STUDY 19
2 MATERIALS AND METHODS 20
2.1 MATERIALS 20
2.1.1 CHEMICALS AND REAGENTS 20
2.2 ANTIBODIES 22
2.2.1 PRIMARY AND SECONDARY ANTIBODIES 22
2.2.2 MONOCLONAL ANTIBODIES 22
2.2.3 BUFFERS 22
2.2.4 MICE 23
2.3 METHODS 24
2.3.1 GENOTYPING OF MICE 24
2.3.1.1 ISOLATION OF MURINE GENOMIC DNA 24
2.3.1.2 DETECTION OF THE EFHD2 ALLELE BY PCR 25
2.3.1.3 AGAROSE GEL ELECTROPHORESIS 26
2.3.2 IN VITRO ANALYSIS OF PLATELET FUNCTION 26
2.3.2.1 PREPARATION OF WASHED PLATELETS AND PLATELET-RICH PLASMA 26
2.3.2.2 CALCULATION OF THE PLATELET COUNT 26
2.3.2.2.1 SYSMEX CELL COUNTER 26
2.3.2.2.2 FLOW CYTOMETRIC ANALYSIS OF THE PLATELET COUNT 27
2.3.2.3 WESTERN BLOTTING 28
VII
2.3.2.3.1 WESTERN BLOTTING OF PLATELET LYSATES 28
2.3.2.3.2 WESTERN BLOTTING FOR TYROSINE PHOSPHORYLATION ASSAY 29
2.3.2.4 FLOW CYTOMETRIC ANALYSIS OF PLATELETS 29
2.3.2.5 AGGREGATION 30
2.3.2.6 PLATELET ADHESION UNDER FLOW CONDITIONS 31
2.3.2.7 PHOSPHATIDYLSERINE EXPOSURE 31
2.3.2.8 CLOT RETRACTION 31
2.3.2.9 PLATELET SPREADING ASSAY 32
2.3.2.10 INTRACELLULAR CA
2+
MEASUREMENTS 32
2.3.3 IN VIVO MODELS IN MICE 32
2.3.3.1 ANAESTHESIA 32
2.3.3.2 IN VIVO PLATELET DEPLETION IN MICE 33
2.3.3.3 DETERMINATION OF PLATELET LIFE SPAN 33
2.3.3.4 MECHANICAL INJURY OF THE ABDOMINAL AORTA 33
2.3.3.5 CAROTID ARTERY INJURY MODEL 33
2.3.3.6 FECU-INDUCED INJURY OF MESENTERIC ARTERIOLES 34
2.3.3.7 TAIL BLEEDING TIME ASSAY IN SALINE SOLUTION 34
2.3.3.8 TAIL BLEEDING TIME ASSAY WITH FILTER PAPER 34
2.3.3.9 TRANSIENT MIDDLE CEREBRAL ARTERY OCCLUSION MODEL 36
2.3.4 ELECTRON MICROSCOPY 36
2.3.4.1 TRANSMISSION ELECTRON MICROSCOPY 36
2.3.5 HISTOLOGY 37
2.3.5.1 PREPARATION OF PARAFFIN SECTIONS 37
2.3.5.2 HAEMATOXYLIN AND EOSIN STAINING 37
2.3.6 STATISTICS 37
RESULTS 38
3.1 ANALYSIS OF THE HAEMOSTATIC AND THROMBOTIC FUNCTION OF PLATELETS IN
THROMBOCYTOPENIC MICE 38
3.1.1 INJECTION OF ANTI-GPIBA ANTIBODY INDUCES THROMBOCYTOPENIA
DOSE-DEPENDENTLY
38
3.1.2 ANTI-GPIBA ANTIBODY TREATMENT DOES NOT AFFECT THE REMAINING
PLATELET
POPULATION 39
3.1.3 ONLY VERY LOW PLATELET COUNTS ARE REQUIRED TO MAINTAIN NORMAL
HAEMOSTASIS IN
MICE 41
3.1.4 ONLY PLATELET COUNT REDUCTIONS 70% SIGNIFICANTLY AFFECT OCCLUSIVE
THROMBUS
FORMATION IN THE AORTA 45
3.1.5 OCCLUSIVE THROMBUS FORMATION IN THE FECH-INJURED CAROTID ARTERY IS
LESS AFFECTED
BY REDUCED PLATELET COUNTS COMPARED WITH THE AORTA INJURY MODEL 46
3.1.6 PLATELET COUNTS OF 2.5% OF CONTROL ARE SUFFICIENT TO EFFICIENTLY
OCCLUDE FECU-
INJURED MESENTERIC ARTERIOLES 47
3.1.7 ONLY APPROXIMATELY 10% OF THE NORMAL PLATELET COUNT IS SUFFICIENT
TO INDUCE FULL
BRAIN INFARCTION FOLLOWING TMCAO 50
VIII
3.2 IN VITRO AND IN VIVO PLATELET FUNCTION DURING RECOVERY FROM
THROMBOCYTOPENIA 52
3.2.1 DETERMINATION OF THE PLATELET COUNT AND SIZE AFTER HIGH-DOSE
ANTI-GPIBA
ANTIBODY TREATMENT 52
3.2.2 PLATELET GLYCOPROTEIN EXPRESSION LEVELS ARE NORMAL IN HIGH-DOSE
ANTI-GPIBA
ANTIBODY-TREATED MICE 54
3.2.3 INCREASED NUMBER OF MKS IN BONE MARROW BUT NOT IN SPLEEN 55
3.2.4 PLATELETS OF HIGH-DOSE ANTI-GPIBA-TREATED MICE DISPLAY A SPECIFIC
ITAM-SIGNALLING
DEFECT 56
3.2.5 HIGH DOSE ANTI-GPIBA-TREATED MICE SHOW A DEFECTIVE THROMBUS
FORMATION IN VIVO
58
3.3 IN VIVO STUDIES IN GENETICALLY MODIFIED MICE WITH MODULATED
GPVI-SIGNALLING 59
3.3.1 GRBX
1
MICE DISPLAY IMPAIRED THROMBUS FORMATION AFTER ASA TREATMENT 60
3.3.2 CLP36
AUM
AND SLAP/SLAP2-DOUBLE-DEFICIENT MICE DISPLAY A PRO-THROMBOTIC
PHENOTYPE IN VIVO 61
3.4 IN VIVO AND IN VITRO ANALYSES OF EFHD2-DEFICIENT MICE 64
3.4.1 EFHD2 IS DISPENSABLE FOR PLATELET PRODUCTION 64
3.4.2 EFHD2-DEFICIENT MICE DISPLAY NORMAL HAEMATOLOGIC PARAMETERS 65
3.4.3 EFHD2-DEFICIENT PLATELETS SHOW UNALTERED ACTIVATION AND
AGGREGATION 65
3.4.4 CLOT RETRACTION AND SPREADING OF EFHDZ
1
PLATELETS IS UNALTERED 67
3.4.5 EFHD2*~ PLATELETS SHOW A SLIGHTLY INCREASED PS EXPOSURE BUT NORMAL
CA
2+
INFLUX
UPON STIMULATION 68
3.4.6 THROMBUS FORMATION UNDER FLOW IS UNALTERED IN EFHDZPLATELETS 70
3.4.7 THROMBOSIS AND HAEMOSTASIS ARE UNALTERED IN EFHD2-DEFICIENT MICE
71
4 DISCUSSION 73
4.1 ROBUST HAEMOSTATIC AND THROMBOTIC FUNCTION IN THROMBOCYTOPENIC MICE
74
4.2 PLATELET DEFECT IN ITAM-SIGNALLING AFTER HIGH-DOSE
ANTI-GPIBA-MEDIATED
THROMBOCYTOPENIA 80
4.3 ARTERIAL THROMBOSIS MODELS IN GENETICALLY MODIFIED MICE SHOWING
ALTERED ITAM-
SIGNALLING IN VITRO 82
4.3.1 GRBZ MICE SHOW REDUCED OCCLUSIVE THROMBUS FORMATION AFTER ASA
TREATMENT IN
VIVO 83
4.3.2 CIPSS*
1
BONE MARROW CHIMERIC MICE AND SLAP/SLAP2-DEFICIENT MICE SHOW
ENHANCED THROMBUS FORMATION IN VIVO 84
4.4 EFHD2 IS DISPENSABLE FOR
PLATELET FUNCTION IN VIVO AND IN VITRO 86
4.5 CONCLUDING REMARKS AND PERSPECTIVE 87
5 REFERENCES 89
6 APPENDIX 101
6.1 CURRICULUM VITAE 101
6.2 PUBLICATIONS 102
6.3 ACKNOWLEDGEMENTS 104
6.4 AFFIDAVIT 105
|
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spelling | Morowski, Martina 1985- Verfasser (DE-588)1053850182 aut Relevance of platelet count and ITAM-signalling pathway in murine models of haemostasis, thrombosis and thrombo-inflammation von Martina Morowski Relevanz der Thrombozytenzahl und des ITAM-Signalwegs in Mausmodellen der Hämostase, Thrombose und Thromboinflammation 2014 VIII, 105 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. und engl. Sprache Würzburg, Univ., Diss., 2014 Blutstillung (DE-588)4069555-4 gnd rswk-swf Maus (DE-588)4169148-9 gnd rswk-swf Thrombose (DE-588)4059963-2 gnd rswk-swf Thrombozyt (DE-588)4059964-4 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Thrombozyt (DE-588)4059964-4 s Maus (DE-588)4169148-9 s Blutstillung (DE-588)4069555-4 s Thrombose (DE-588)4059963-2 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-99193 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-99193 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027425308&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
spellingShingle | Morowski, Martina 1985- Relevance of platelet count and ITAM-signalling pathway in murine models of haemostasis, thrombosis and thrombo-inflammation Blutstillung (DE-588)4069555-4 gnd Maus (DE-588)4169148-9 gnd Thrombose (DE-588)4059963-2 gnd Thrombozyt (DE-588)4059964-4 gnd |
subject_GND | (DE-588)4069555-4 (DE-588)4169148-9 (DE-588)4059963-2 (DE-588)4059964-4 (DE-588)4113937-9 |
title | Relevance of platelet count and ITAM-signalling pathway in murine models of haemostasis, thrombosis and thrombo-inflammation |
title_alt | Relevanz der Thrombozytenzahl und des ITAM-Signalwegs in Mausmodellen der Hämostase, Thrombose und Thromboinflammation |
title_auth | Relevance of platelet count and ITAM-signalling pathway in murine models of haemostasis, thrombosis and thrombo-inflammation |
title_exact_search | Relevance of platelet count and ITAM-signalling pathway in murine models of haemostasis, thrombosis and thrombo-inflammation |
title_full | Relevance of platelet count and ITAM-signalling pathway in murine models of haemostasis, thrombosis and thrombo-inflammation von Martina Morowski |
title_fullStr | Relevance of platelet count and ITAM-signalling pathway in murine models of haemostasis, thrombosis and thrombo-inflammation von Martina Morowski |
title_full_unstemmed | Relevance of platelet count and ITAM-signalling pathway in murine models of haemostasis, thrombosis and thrombo-inflammation von Martina Morowski |
title_short | Relevance of platelet count and ITAM-signalling pathway in murine models of haemostasis, thrombosis and thrombo-inflammation |
title_sort | relevance of platelet count and itam signalling pathway in murine models of haemostasis thrombosis and thrombo inflammation |
topic | Blutstillung (DE-588)4069555-4 gnd Maus (DE-588)4169148-9 gnd Thrombose (DE-588)4059963-2 gnd Thrombozyt (DE-588)4059964-4 gnd |
topic_facet | Blutstillung Maus Thrombose Thrombozyt Hochschulschrift |
url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-99193 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027425308&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
work_keys_str_mv | AT morowskimartina relevanceofplateletcountanditamsignallingpathwayinmurinemodelsofhaemostasisthrombosisandthromboinflammation AT morowskimartina relevanzderthrombozytenzahlunddesitamsignalwegsinmausmodellenderhamostasethromboseundthromboinflammation |