Novel mechanisms underlying arterial thrombus formation: in vivo studies in (genetically modified) mice
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2011
|
| Schlagworte: | |
| Online-Zugang: | kostenfrei Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. und engl. Sprache. - Online erschienen: 2014 |
| Beschreibung: | VIII, 142 Bl. Ill. |
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| 245 | 1 | 0 | |a Novel mechanisms underlying arterial thrombus formation |b in vivo studies in (genetically modified) mice |c von Ina Hagedorn |
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Datensatz im Suchindex
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|---|---|
| adam_text | NOVEL MECHANISMS UNDERLYING ARTERIAL THROMBUS FORMATION
IN VIVO STUDIES IN (GENETICALLY MODIFIED) MICE
NEUE MECHANISMEN DER ARTERIELLEN THROMBUSBILDUNG:
IN WVO-STUDIEN IN (GENETISCH VERANDERTEN) MAUSEN
DOCTORAL THESIS FOR A DOCTORAL DEGREE
AT THE GRADUATE SCHOOL OF LIFE
SCIENCES,
BAYERISCHE JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG,
SECTION BIOMEDICINE
SUBMITTED BY
INA HAGEDORN
FROM
NORTHEIM
WURZBURG, 2011
HTTP://D-NB.INFO/1048252035
TABLE OF CONTENTS
TABLE OF CONTENTS
1 INTRODUCTION 1
1.1 PLATELETS 1
1.2 PLATELET ACTIVATION AND THROMBUS FORMATION 2
1.3 THE GPIB-V-IX COMPLEX 5
1.4 PHOSPHOLIPASE D1 6
1.5 GPVI 7
1.6 CLEC-2 8
1.7 SMALL GTPASES OF THE RHO FAMILY 9
1.7.1 RHOA 10
1.8 BLOOD COAGULATION 12
1.9 MURINE ARTERIAL THROMBOSIS MODELS 15
2 AIM OF THE STUDY 17
3 MATERIALS AND METHODS 18
3.1 MATERIALS 18
3.1.1 CHEMICALS AND KITS 18
3.1.2 ANTIBODIES 20
3.1.2.1 PURCHASED PRIMARY AND SECONDARY ANTIBODIES 20
3.1.2.2 MONOCLONAL ANTIBODIES (MABS) 20
3.1.3 HUMAN SERINE PROTEASES 21
3.1.4 BUFFERS 21
3.1.5 ANIMALS 24
3.2 METHODS 26
3.2.1 MOUSE GENOTYPING 26
3.2.1.1 ISOLATION OF GENOMIC DNA FROM MOUSE EARS 26
3.2.1.2 DETECTION OF THE RHOA FLOXED ALLELE BY PCR 26
3.2.1.3 DETECTION OF THE PF4-CRE TRANSGENE BY PCR 27
3.2.2 IN VITRO ANALYSIS OF PLATELET FUNCTION 27
3.2.2.1 PLATELET ISOLATION AND WASHING 27
3.2.2.2 PLATELET COUNTING 28
3.2.2.3 WESTERN BLOTTING 28
3.2.2.4 FLOW CYTOMETRIC ANALYSIS 28
3.2.2.5 AGGREGATION 29
3.2.2.6 AGGREGATION + ATP RELEASE 30
3.2.2.7 INTRACELLULAR CALCIUM-MEASUREMENTS 30
3.2.2.8 DETERMINATION OF PLATELET FILAMENTOUS (F)-ACTIN CONTENT 30
3.2.2.9 CLOT RETRACTION 31
V
TABLE OF CONTENTS
3.2.2.10 STATIC ADHESION ON HUMAN FIBRINOGEN 31
3.2.2.11 ADHESION UNDER FLOW CONDITIONS 31
3.2.3 COAGULATION AND FIBRINOLYSIS ASSAYS 32
3.2.3.1 PREPARATION OF MOUSE PLASMA 32
3.2.3.2 DETERMINATION OF APTT AND PT 32
3.2.3.3 CHROMOGENIC ASSAYS 32
3.2.3.4 D-DIMER CONCENTRATION MEASUREMENTS 33
3.2.4 IN VIVO MURINE MODELS 33
3.2.4.1 DETERMINATION OF PLATELET LIFE SPAN 33
3.2.4.2 FECI
3
-INDUCED THROMBUS FORMATION IN SMALL MESENTERIC ARTERIOLES 33
3.2.4.3 MECHANICAL INJURY OF THE ABDOMINAL AORTA 33
3.2.4.4 CAROTID ARTERY THROMBOSIS MODEL 34
3.2.4.5 BLEEDING TIME ASSAYS 34
3.2.4.6 TRANSIENT OCCLUSION MODEL OF THE MIDDLE CEREBRAL ARTERY 34
3.2.5 STATISTICS 34
3.2.6 ELECTRON MICROSCOPY 35
3.2.6.1 TRANSMISSION ELECTRON MICROSCOPY (TEM) OF PLATELETS IN
SUSPENSION 35
3.2.6.2 SCANNING ELECTRON MICROSCOPY (SEM) OF PLATELETS 35
3.2.7 FLUORESCENCE MICROSCOPY OF PLATELETS 36
3.2.7.1 STAINING OF SPREAD PLATELETS FOR CONFOCAL MICROSCOPY 36
3.2.7.2 STIMULATED EMMISION DEPLETION (STED) MICROSCOPY OF SPREAD
PLATELETS.. 36
3.2.8 HISTOLOGY 36
3.2.8.1 PREPARATION OF PARAFFIN SECTIONS 36
3.2.8.2 HEMATOXYLIN/EOSIN STAINING OF PARAFFIN SECTIONS 37
RESULTS 38
4.1 RELEVANCE OF THE GPIBA-VWF INTERACTION AND DOWNSTREAM SIGNALING IN
THROMBUS
FORMATION 38
4.1.1 TARGETING OF GPIBA PROTECTS MICE FROM ARTERIAL THROMBOSIS, BUT
CAUSES A
HEMOSTATIC DEFECT 38
4.1.2 PHOSPHOLIPASE D1 IS A CRITICAL MEDIATOR OF SHEAR-DEPENDENT
THROMBUS FORMATION
DOWNSTREAM OF GPIB 40
4.1.2.1 PLD1PLATELETS FAIL TO FORM STABLE AGGREGATES ON COLLAGEN UNDER
FLOW
CONDITIONS 41
4.1.2.2 PLD1-DEFICIENT PLATELETS DISPLAY DEFECTIVE ADHESION ON VWF UNDER
FLOW
CONDITIONS 42
4.1.2.3 PLD1-DEFICIENT MICE ARE PROTECTED FROM OCCLUSIVE ARTERIAL
THROMBUS
FORMATION 43
VI
TABLE OF CONTENTS
4.2 GENETIC AND ANTIBODY-INDUCED GPVI DEFICIENCY SIMILARLY PROTECTS MICE
FROM FECI
3
-
AND MECHANICALLY INDUCED THROMBOSIS 45
4.3 THE PLATELET ACTIVATING RECEPTOR CLEC-2 IS ESSENTIALLY INVOLVED IN
THROMBOSIS AND
HEMOSTASIS 48
4.3.1 CLEC-2 CAN SPECIFICALLY BE DOWNREGULATED FROM CIRCULATING
PLATELETS BY THE
MONOCLONAL ANTIBODY INU1 48
4.3.2 CLEC-2 DEFICIENCY LEADS TO THE FORMATION OF UNSTABLE THROMBI UNDER
FLOW 49
4.3.3 CLEC-2-DEFICIENT MICE DISPLAY DEFECTIVE THROMBUS FORMATION IN A
FECL3
-INJURY
MODEL AND PROLONGED BLEEDING TIMES 51
4.4 SEVERELY DEFECTIVE ARTERIAL THROMBUS FORMATION IN MICE LACKING GPVI
AND CLEC-2 54
4.5 THE SMALL GTPASE RHOA IS A CRUCIAL MEDIATOR OF PLATELET ACTIVATION
IN HEMOSTASIS
AND THROMBOSIS 58
4.5.1 RHOAMICE DISPLAY A MARKED THROMBOCYTOPENIA 58
4.5.2 RHOA
/
MICE DISPLAY A REDUCED PLATELET LIFE SPAN 59
4.5.3 REDUCED INTEGRIN ACTIVATION AND RELEASE OF A GRANULES IN
RHOAPLATELETS UPON
G
I
3
- AND
GQ
-STIMULATION 60
4.5.4 DEFECTIVE SHAPE CHANGE OF RHOAPLATELETS AFTER LOW AGONIST
STIMULATION 62
4.5.5 RHOA IS DISPENSABLE FOR AGONIST-INDUCED CA
2+
MOBILIZATION IN PLATELETS 65
4.5.6 RHOAPLATELETS SPREAD NORMALLY ON FIBRINOGEN BUT EXHIBIT ABOLISHED
INTEGRIN-
DEPENDENT CLOT-RETRACTION 66
4.5.7 RHOAPLATELETS SHOW SLIGHTLY REDUCED AGGREGATE FORMATION ON
COLLAGEN AND
ADHESION ON VWF UNDER HIGH SHEAR FLOW 70
4.5.8 RHOA IS CRITICAL FOR THROMBUS STABILIZATION IN VIVO 71
4.5.9 RHOAMICE ARE PROTECTED FROM ISCHEMIC BRAIN INFARCTION 73
4.6 FXIIA INHIBITOR RHA-LNFESTIN-4 ABOLISHES OCCLUSIVE ARTERIAL THROMBUS
FORMATION WITHOUT
AFFECTING BLEEDING 75
4.6.1 RHA-LNFESTIN-4 SPECIFICALLY INHIBITS THE COAGULATION FACTOR XLLA
75
4.6.2 RHA-LNFESTIN-4 DISPLAYS A SIGNIFICANT IMPACT ON FIBRINOLYSIS 77
4.6.3 PREVENTION OF ARTERIAL THROMBOSIS IN RHA-LNFESTIN-4 TREATED MICE
77
4.6.4 RHA-LNFESTIN-4 DOES NOT REDUCE RESIDUAL THROMBUS FORMATION IN F1Z
1
MICE 80
4.6.5 RHA-LNFESTIN-4 PROTECTS MICE FROM ISCHEMIC BRAIN INFARCTION 81
DISCUSSION 83
5.1 STUDIES ON THE FUNCTIONAL ROLE OF THE GPIBA-VWF INTERACTION AND
DOWNSTREAM
SIGNALING IN SHEAR-DEPENDENT THROMBUS FORMATION 84
5.1.1 THE GPIBA-VWF INTERACTION IS ESSENTIAL FOR IN VIVO THROMBUS
STABILIZATION AT HIGH
SHEAR 84
VII
TABLE OF CONTENTS
5.1.2 PLD1 MEDIATES ALLBP3 INTEGRIN ACTIVATION UNDER HIGH SHEAR AND IS
CRITICAL FOR
PATHOLOGICAL THROMBUS FORMATION BUT DISPENSABLE FOR HEMOSTASIS 87
5.2 STUDIES ON THE FUNCTIONAL ROLE OF THE (HEM)ITAM-COUPLED RECEPTORS
GPVI AND
CLEC-2 IN HEMOSTASIS AND THROMBOSIS 90
5.2.1 GPVI IS A CRITICAL MEDIATOR OF ARTERIAL THROMBOSIS UPON FECI
3
- AND MECHANICALLY
INDUCED VASCULAR INJURY 90
5.2.2 CLEC-2 IS IMPORTANT FOR THROMBUS STABILIZATION IN HEMOSTASIS AND
THROMBOSIS 94
5.2.3 FUNCTIONALLY REDUNDANCY OF GPVI AND CLEC-2 DURING IN VIVO THROMBUS
FORMATION 99
5.3 RHOA IS INVOLVED IN G
13
-AND G
Q
-MEDIATED PLATELET ACTIVATION AND ESSENTIAL FOR IN VIVO
THROMBUS FORMATION 101
5.4 THE FXIIA INHIBITOR RHA-LNFESTIN-4 IS A POTENT ANTITHROMBOTIC AGENT
WITHOUT CAUSING
BLEEDING SIDE EFFECTS 107
5.5 CONCLUDING REMARKS AND FUTURE PLANS 112
6 REFERENCES 114
7 APPENDIX 134
VIII
|
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| spelling | Hagedorn, Ina 1982- Verfasser (DE-588)1047080486 aut Novel mechanisms underlying arterial thrombus formation in vivo studies in (genetically modified) mice von Ina Hagedorn Neue Mechanismen der arteriellen Thrombusbildung: in vivo-Studien in (genetisch veränderten) Mäusen 2011 VIII, 142 Bl. Ill. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. und engl. Sprache. - Online erschienen: 2014 Würzburg, Univ., Diss., 2011 Maus (DE-588)4169148-9 gnd rswk-swf Gerinnungsfaktor (DE-588)4123279-3 gnd rswk-swf Zielstruktur (DE-588)1028794711 gnd rswk-swf Thrombus (DE-588)7606612-5 gnd rswk-swf Arterielles Blut (DE-588)4431626-4 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Thrombus (DE-588)7606612-5 s Arterielles Blut (DE-588)4431626-4 s Gerinnungsfaktor (DE-588)4123279-3 s Zielstruktur (DE-588)1028794711 s Maus (DE-588)4169148-9 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-85752 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-85752 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=027060206&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Hagedorn, Ina 1982- Novel mechanisms underlying arterial thrombus formation in vivo studies in (genetically modified) mice Maus (DE-588)4169148-9 gnd Gerinnungsfaktor (DE-588)4123279-3 gnd Zielstruktur (DE-588)1028794711 gnd Thrombus (DE-588)7606612-5 gnd Arterielles Blut (DE-588)4431626-4 gnd |
| subject_GND | (DE-588)4169148-9 (DE-588)4123279-3 (DE-588)1028794711 (DE-588)7606612-5 (DE-588)4431626-4 (DE-588)4113937-9 |
| title | Novel mechanisms underlying arterial thrombus formation in vivo studies in (genetically modified) mice |
| title_alt | Neue Mechanismen der arteriellen Thrombusbildung: in vivo-Studien in (genetisch veränderten) Mäusen |
| title_auth | Novel mechanisms underlying arterial thrombus formation in vivo studies in (genetically modified) mice |
| title_exact_search | Novel mechanisms underlying arterial thrombus formation in vivo studies in (genetically modified) mice |
| title_full | Novel mechanisms underlying arterial thrombus formation in vivo studies in (genetically modified) mice von Ina Hagedorn |
| title_fullStr | Novel mechanisms underlying arterial thrombus formation in vivo studies in (genetically modified) mice von Ina Hagedorn |
| title_full_unstemmed | Novel mechanisms underlying arterial thrombus formation in vivo studies in (genetically modified) mice von Ina Hagedorn |
| title_short | Novel mechanisms underlying arterial thrombus formation |
| title_sort | novel mechanisms underlying arterial thrombus formation in vivo studies in genetically modified mice |
| title_sub | in vivo studies in (genetically modified) mice |
| topic | Maus (DE-588)4169148-9 gnd Gerinnungsfaktor (DE-588)4123279-3 gnd Zielstruktur (DE-588)1028794711 gnd Thrombus (DE-588)7606612-5 gnd Arterielles Blut (DE-588)4431626-4 gnd |
| topic_facet | Maus Gerinnungsfaktor Zielstruktur Thrombus Arterielles Blut Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-85752 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027060206&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT hagedornina novelmechanismsunderlyingarterialthrombusformationinvivostudiesingeneticallymodifiedmice AT hagedornina neuemechanismenderarteriellenthrombusbildunginvivostudieningenetischverandertenmausen |