Analyses on molecular mechanisms of activation of intravascular Tissue Factor:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
2007
|
| Schlagworte: | |
| Online-Zugang: | kostenfrei https://nbn-resolving.org/urn:nbn:de:bvb:19-77216 Inhaltsverzeichnis |
| Beschreibung: | München, Univ., Diss., 2007 |
| Beschreibung: | 103 S. Ill., graph. Darst. |
Internformat
MARC
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| 100 | 1 | |a Reinhardt, Christoph |d 1978- |e Verfasser |0 (DE-588)133754499 |4 aut | |
| 245 | 1 | 0 | |a Analyses on molecular mechanisms of activation of intravascular Tissue Factor |c von Christoph Reinhardt |
| 264 | 1 | |c 2007 | |
| 300 | |a 103 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a München, Univ., Diss., 2007 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 776 | 0 | 8 | |i Erscheint auch als |n Online-Ausgabe |o urn:nbn:de:bvb:19-77216 |
| 856 | 4 | 1 | |u http://edoc.ub.uni-muenchen.de/7721/ |x Verlag |z kostenfrei |3 Volltext |
| 856 | 4 | |u https://nbn-resolving.org/urn:nbn:de:bvb:19-77216 |x Resolving-System | |
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| 912 | |a ebook | ||
| 999 | |a oai:aleph.bib-bvb.de:BVB01-016393259 | ||
Datensatz im Suchindex
| _version_ | 1804137487009841152 |
|---|---|
| adam_text | Contents
i. Table of Contents
I. Introduction
1.1 Tissue Factor - the principal initiator of coagulation 1
1.2 Regulation of blood coagulation 3
1.3 The structural biology of TF 4
1.4 Tissue Factor Pathway Inhibitor-1 - the physiologic inhibitor of the
coagulation start 5
1.5 Proteolytic cleavage of TFPI 7
1.6 The procoagulant platelet-neutrophil microenvironment 8
1.7 Cellular microparticles 10
1.8 Tissue specific expression pattern of TF 10
1.9 Intravascular TF 11
1.10 The encrypted or latent state of TF 14
I.I 1 Potential role of disulfide switching in human TF 16
1.12 Aims of the investigation 17
II. Materials and Methods
II.1 Materials 19
II. 1.1 Instruments 19
II. 1.2 Reagents, Pharmaceuticals and general material 19
II.1.3 Cell culture materials 21
II. 1.4 Enzymes and proteins 21
11.1.5 Antibodies 22
11.1.6 Kits 23
Contents
11.1.7 Phagmid 23
11.1.8 PCR-primers 23
11.1.8.1 Cloning primers 24
II. 1.8.2 Site-directed mutagenesis primers 24
II. 1.9 Bacterial strains and cell lines 24
II. 1.10 Bacterial and cell culture media 25
II. 1.11 Buffers and solutions 25
IL2 Methods -.28
11.2.1 Cell isolation techniques 28
H.2.1.1 Blood recovery 28
11.2.1.2 Isolation of platelets 28
H.2.1.3 Preparation of platelet supernatant 29
II.2.1.4 Isolation of peripheral blood monocytes (PBM) 29
H.2.1.5 Isolation of polymorphonuclear neutrophils (PMN) 30
H.2.1.6 Stimulation of isolated blood cells 31
II.2.1.7 Isolation of microparticles derived from stimulated blood cells 31
11.2.2 Cell culture techniques 32
H.2.2.1 Bacterial cell cultures 32
11.2.2.2 Preparation of competent DH5a-cells 32
11.2.2.3 Transformation of competent bacteria 32
H.2.2.4 Cultivation of Chinese Hamster Ovary cells 33
H.2.2.5 Transfection of Chinese Hamster Ovary cells 33
11.2.3 DNA techniques 34
H.2.3.1 ElectrophoresisofDNAonagarosegels 34
II.2.3.2 Isolation of DNA from agarose gels (Qiagen gel extraction kit) 34
Contents
11.2.3.3 Purification of plasmid DNA (QIAquick PCR purification kit) 35
11.2.3.4 Maxi-preparation of plasmid DNA (Qiagen plasmid maxi kit) 35
H.2.3.5 Measurement of DNA concentration 36
11.2.3.6 DNA sequencing 36
11.2.3.7 Polymerase Chain Reaction (PCR) 36
11.2.3.8 Restriction digests of DNA fragments 37
H.2.3.9 Ligation of DNA fragments 38
11.2.3.10 Construction of the protein expression phagmid pBK-CMV-TF 38
H.2.3.11 Site-directed mutagenesis of TF C49S, TF C57S, TF C186S,
TF C209S, TF C49S/C57S and TF C186S/C209S in pBK-CMV-TF 38
11.2.4 Protein analyses 39
H.2.4.1 Determination of protein concentrations 39
H.2.4.2 UV-Spectroscopy 40
11.2.4.3 Circular Dichroism-Spectroscopy 40
11.2.4.4 Sodiumdodecylsulfate polyacrylamide gel electrophoresis
(SDS-PAGE) 41
H.2.4.5 Immunoblot 42
11.2.4.6 Na-(3-maleimidylpropionyl)biocytin-labelling of the reduced cysteine
residues in recombinant sTFi.219 and the extracellular protein domains
ofmonocytes 43
11.2.4.7 Biochemical detection of protein S-glutathionylation in
membrane proteins 44
11.2.4.8 EUman s assay 44
11.2.5 Functional assays 45
11.2.5.1 Factor Xa formation assay 45
11.2.5.2 Two-stage factor Xa formation assay 46
H.2.5.3 Thrombelastography (TEG) 46
H.2.5.4 Statistics 47
Contents
III. Results
HLA The procoagulant activity of platelet TF 48
III.A. 1 Collagen-stimulated platelets expose TF procoagulant activity 48
III.A.2 The TF procoagulant activity in collagen-activated platelets is
largely encrypted 52
III.B Decomposition of platelet TFPI by neutrophil serine proteases .55
III B 1 Human polymorphonuclear neutrophils and isolated human neutrophil
elastase (NE) evoke TF procoagulant activity 55
I1I.B.2 NE is surface associated on myeloid blood cells and their microparticles... 56 ;
III.B.3 Cell surface association of serine proteases results from polar interactions
with glycosaminoglycans and with nucleic acids 57
III.B.4 Platelet TFPI is degraded by NE in platelet-neutrophil conjugates 59
III.C A disulfide switch in the TF molecule regulates its procoagulant
activity 62
III.C.l TF contains a labile disulfide that is essential for its procoagulant
function 62
III.C.2 Protein Disulfide Isomerase oxidizes the C186/C209pair 64
III.C.3 Glutathionylation of C186/C209 vicinal thols of TF 67
III.C.4 In vitro protein S-glutathionylation of TF is reversible 69
IV. Discussion
IV.l TF procoagulant activity of activated platelets 70
IV.2 Encryption of platelet TF activity 71
IV.3 Neutrophil surface proteases trigger the TF procoagulant activity in
platelet-neutrophil conjugates 72
i
Contents
1V.4 Characterization of the procoagulant microenvironment formed
between activated platelets and polymorphonuclear neutrophils 74
IV.5 Disulfide switch of TF regulates initiation of intravascular coagulation
on monocytes - potential role for TF encryption 76
IV.6 Protein S-glutathionylation of TF - a potential safety device 78
I V.7 Model for the redox regulation of intravascular TF activity 80
V.I Summary 82
V.2 Zusammenfassung 83
VI. References „ 85
VII. Appendix .98
VILA UV-spectrum and Circular Dichroism-spectra of the recombinant TF
extracellular domain 98
VIII. Acknowledgements 99
Curriculum Vitae „ 101
|
| adam_txt |
Contents
i. Table of Contents
I. Introduction
1.1 Tissue Factor - the principal initiator of coagulation 1
1.2 Regulation of blood coagulation 3
1.3 The structural biology of TF 4
1.4 Tissue Factor Pathway Inhibitor-1 - the physiologic inhibitor of the
coagulation start 5
1.5 Proteolytic cleavage of TFPI 7
1.6 The procoagulant platelet-neutrophil microenvironment 8
1.7 Cellular microparticles 10
1.8 Tissue specific expression pattern of TF 10
1.9 Intravascular TF 11
1.10 The encrypted or latent state of TF 14
I.I 1 Potential role of disulfide switching in human TF 16
1.12 Aims of the investigation 17
II. Materials and Methods
II.1 Materials 19
II. 1.1 Instruments 19
II. 1.2 Reagents, Pharmaceuticals and general material 19
II.1.3 Cell culture materials 21
II. 1.4 Enzymes and proteins 21
11.1.5 Antibodies 22
11.1.6 Kits 23
Contents
11.1.7 Phagmid 23
11.1.8 PCR-primers 23
11.1.8.1 Cloning primers 24
II. 1.8.2 Site-directed mutagenesis primers 24
II. 1.9 Bacterial strains and cell lines 24
II. 1.10 Bacterial and cell culture media 25
II. 1.11 Buffers and solutions 25
IL2 Methods -.28
11.2.1 Cell isolation techniques 28
H.2.1.1 Blood recovery 28
11.2.1.2 Isolation of platelets 28
H.2.1.3 Preparation of platelet supernatant 29
II.2.1.4 Isolation of peripheral blood monocytes (PBM) 29
H.2.1.5 Isolation of polymorphonuclear neutrophils (PMN) 30
H.2.1.6 Stimulation of isolated blood cells 31
II.2.1.7 Isolation of microparticles derived from stimulated blood cells 31
11.2.2 Cell culture techniques 32
H.2.2.1 Bacterial cell cultures 32
11.2.2.2 Preparation of competent DH5a-cells 32
11.2.2.3 Transformation of competent bacteria 32
H.2.2.4 Cultivation of Chinese Hamster Ovary cells 33
H.2.2.5 Transfection of Chinese Hamster Ovary cells 33
11.2.3 DNA techniques 34
H.2.3.1 ElectrophoresisofDNAonagarosegels 34
II.2.3.2 Isolation of DNA from agarose gels (Qiagen gel extraction kit) 34
Contents
11.2.3.3 Purification of plasmid DNA (QIAquick PCR purification kit) 35
11.2.3.4 Maxi-preparation of plasmid DNA (Qiagen plasmid maxi kit) 35
H.2.3.5 Measurement of DNA concentration 36
11.2.3.6 DNA sequencing 36
11.2.3.7 Polymerase Chain Reaction (PCR) 36
11.2.3.8 Restriction digests of DNA fragments 37
H.2.3.9 Ligation of DNA fragments 38
11.2.3.10 Construction of the protein expression phagmid pBK-CMV-TF 38
H.2.3.11 Site-directed mutagenesis of TF C49S, TF C57S, TF C186S,
TF C209S, TF C49S/C57S and TF C186S/C209S in pBK-CMV-TF 38
11.2.4 Protein analyses 39
H.2.4.1 Determination of protein concentrations 39
H.2.4.2 UV-Spectroscopy 40
11.2.4.3 Circular Dichroism-Spectroscopy 40
11.2.4.4 Sodiumdodecylsulfate polyacrylamide gel electrophoresis
(SDS-PAGE) 41
H.2.4.5 Immunoblot 42
11.2.4.6 Na-(3-maleimidylpropionyl)biocytin-labelling of the reduced cysteine
residues in recombinant sTFi.219 and the extracellular protein domains
ofmonocytes 43
11.2.4.7 Biochemical detection of protein S-glutathionylation in
membrane proteins 44
11.2.4.8 EUman's assay 44
11.2.5 Functional assays 45
11.2.5.1 Factor Xa formation assay 45
11.2.5.2 Two-stage factor Xa formation assay 46
H.2.5.3 Thrombelastography (TEG) 46
H.2.5.4 Statistics 47
Contents
III. Results
HLA The procoagulant activity of platelet TF 48
III.A. 1 Collagen-stimulated platelets expose TF procoagulant activity 48
III.A.2 The TF procoagulant activity in collagen-activated platelets is
largely encrypted 52
III.B Decomposition of platelet TFPI by neutrophil serine proteases .55
III B 1 Human polymorphonuclear neutrophils and isolated human neutrophil
elastase (NE) evoke TF procoagulant activity 55
I1I.B.2 NE is surface associated on myeloid blood cells and their microparticles. 56 ;
III.B.3 Cell surface association of serine proteases results from polar interactions
with glycosaminoglycans and with nucleic acids 57
III.B.4 Platelet TFPI is degraded by NE in platelet-neutrophil conjugates 59
III.C A disulfide switch in the TF molecule regulates its procoagulant
activity 62
III.C.l TF contains a labile disulfide that is essential for its procoagulant
function 62
III.C.2 Protein Disulfide Isomerase oxidizes the C186/C209pair 64
III.C.3 Glutathionylation of C186/C209 vicinal thols of TF 67
III.C.4 In vitro protein S-glutathionylation of TF is reversible 69
IV. Discussion
IV.l TF procoagulant activity of activated platelets 70
IV.2 Encryption of platelet TF activity 71
IV.3 Neutrophil surface proteases trigger the TF procoagulant activity in
platelet-neutrophil conjugates 72
i
Contents
1V.4 Characterization of the procoagulant microenvironment formed
between activated platelets and polymorphonuclear neutrophils 74
IV.5 Disulfide switch of TF regulates initiation of intravascular coagulation
on monocytes - potential role for TF encryption 76
IV.6 Protein S-glutathionylation of TF - a potential safety device 78
I V.7 Model for the redox regulation of intravascular TF activity 80
V.I Summary 82
V.2 Zusammenfassung 83
VI. References „ 85
VII. Appendix .98
VILA UV-spectrum and Circular Dichroism-spectra of the recombinant TF
extracellular domain 98
VIII. Acknowledgements 99
Curriculum Vitae „ 101 |
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| indexdate | 2024-07-09T21:13:04Z |
| institution | BVB |
| language | English |
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| spelling | Reinhardt, Christoph 1978- Verfasser (DE-588)133754499 aut Analyses on molecular mechanisms of activation of intravascular Tissue Factor von Christoph Reinhardt 2007 103 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Univ., Diss., 2007 (DE-588)4113937-9 Hochschulschrift gnd-content Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:19-77216 http://edoc.ub.uni-muenchen.de/7721/ Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:19-77216 Resolving-System HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016393259&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Reinhardt, Christoph 1978- Analyses on molecular mechanisms of activation of intravascular Tissue Factor |
| subject_GND | (DE-588)4113937-9 |
| title | Analyses on molecular mechanisms of activation of intravascular Tissue Factor |
| title_auth | Analyses on molecular mechanisms of activation of intravascular Tissue Factor |
| title_exact_search | Analyses on molecular mechanisms of activation of intravascular Tissue Factor |
| title_exact_search_txtP | Analyses on molecular mechanisms of activation of intravascular Tissue Factor |
| title_full | Analyses on molecular mechanisms of activation of intravascular Tissue Factor von Christoph Reinhardt |
| title_fullStr | Analyses on molecular mechanisms of activation of intravascular Tissue Factor von Christoph Reinhardt |
| title_full_unstemmed | Analyses on molecular mechanisms of activation of intravascular Tissue Factor von Christoph Reinhardt |
| title_short | Analyses on molecular mechanisms of activation of intravascular Tissue Factor |
| title_sort | analyses on molecular mechanisms of activation of intravascular tissue factor |
| topic_facet | Hochschulschrift |
| url | http://edoc.ub.uni-muenchen.de/7721/ https://nbn-resolving.org/urn:nbn:de:bvb:19-77216 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016393259&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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