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Metals and the conformation of fibrin
The carboxy terminal of the γ-chain of human fibrinogen contains at least three biologically. important functional domains: (i) the fibrinogen γ-chain polymerisation centre, (ii) the platelet receptor domain and (iii) the site for staphyloccocal clumping. The nature of the site specificity of these...
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| Format: | Thesis |
| Language: | English |
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Division of Chemical Pathology
2017
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| _version_ | 1867613252241850368 |
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| access_status_str | Open Access |
| author | Naidoo, Dhesigen P |
| author2 | Purvis, Langley R |
| author_browse | Naidoo, Dhesigen P Purvis, Langley R |
| author_facet | Purvis, Langley R Naidoo, Dhesigen P |
| author_sort | Naidoo, Dhesigen P |
| collection | Thesis |
| description | The carboxy terminal of the γ-chain of human fibrinogen contains at least three biologically. important functional domains: (i) the fibrinogen γ-chain polymerisation centre, (ii) the platelet receptor domain and (iii) the site for staphyloccocal clumping. The nature of the site specificity of these interactions necessitates the existence of a preferred conformation for this region, the nature of which has yet to be clearly established. A novel zinc metalloproteinase isolated from puff adder venom (PAV protease) capable of specifically cleaving the di-γ-chain of transglutaminase (Factor XIIIa) catalysed crosslinked plasmin derived D-dimer into apparently symmetrical monomers has been described. The activity is fibrin specific and displays an unusual site specificity for the γ-carboxy terminal domains within the crosslink region. The activity was reported to be potentiated by zinc. The effect of zinc on the digestion of D-dimer by PAV protease was evaluated by SDS-PAGE and by a fluorimetric technique utilising a fluorescent dansylcadaverine conjugate of the substrate (f-D-dimer). A differential zinc binding study determined that the potentiation of activity by zinc was due to a zinc-substrate rather than a zinc-enzyme interaction. The binding constant for zinc to D-dimer was determined by Scatchard analysis of zinc titration data. The interaction of zinc and f-D-dimer was confirmed by fluorescence anisotropy determinations. The nature of the coordination capsule around the metal cation was determined by examining a cobalt-fibrin-D-dimer complex and characterising the difference visible absorption spectrum thereof. The donor ligands from the D-dimer fragment for the metal ion were determined as histidines by examining zinc(II) and cobalt(II) binding to diethylpyrocarbonate modified fibrin-D-dimer and hydroxylamine treated DEPC-fibrin-D-dimer. Through this study it has been established that the PAV protease cleavage of the di-γ-chain of the plasmin derived D-dimer fragment is potentiated by zinc(II) ions through the formation of a novel zinc determined conformation of fibrin-D-dimer. This presents the possibility of a fibrinspecific neo-epitope being manifested in the presence of zinc ions that could provide a means to determine fibrin degradation products more specifically. A model for the neo-epitope has been proposed. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/26555 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:33:10.259Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2017 |
| publishDateRange | 2017 |
| publishDateSort | 2017 |
| publisher | Division of Chemical Pathology |
| publisherStr | Division of Chemical Pathology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/26555 Metals and the conformation of fibrin Naidoo, Dhesigen P Purvis, Langley R Fibrin - chemistry Metals - Analysis Zinc - analysis The carboxy terminal of the γ-chain of human fibrinogen contains at least three biologically. important functional domains: (i) the fibrinogen γ-chain polymerisation centre, (ii) the platelet receptor domain and (iii) the site for staphyloccocal clumping. The nature of the site specificity of these interactions necessitates the existence of a preferred conformation for this region, the nature of which has yet to be clearly established. A novel zinc metalloproteinase isolated from puff adder venom (PAV protease) capable of specifically cleaving the di-γ-chain of transglutaminase (Factor XIIIa) catalysed crosslinked plasmin derived D-dimer into apparently symmetrical monomers has been described. The activity is fibrin specific and displays an unusual site specificity for the γ-carboxy terminal domains within the crosslink region. The activity was reported to be potentiated by zinc. The effect of zinc on the digestion of D-dimer by PAV protease was evaluated by SDS-PAGE and by a fluorimetric technique utilising a fluorescent dansylcadaverine conjugate of the substrate (f-D-dimer). A differential zinc binding study determined that the potentiation of activity by zinc was due to a zinc-substrate rather than a zinc-enzyme interaction. The binding constant for zinc to D-dimer was determined by Scatchard analysis of zinc titration data. The interaction of zinc and f-D-dimer was confirmed by fluorescence anisotropy determinations. The nature of the coordination capsule around the metal cation was determined by examining a cobalt-fibrin-D-dimer complex and characterising the difference visible absorption spectrum thereof. The donor ligands from the D-dimer fragment for the metal ion were determined as histidines by examining zinc(II) and cobalt(II) binding to diethylpyrocarbonate modified fibrin-D-dimer and hydroxylamine treated DEPC-fibrin-D-dimer. Through this study it has been established that the PAV protease cleavage of the di-γ-chain of the plasmin derived D-dimer fragment is potentiated by zinc(II) ions through the formation of a novel zinc determined conformation of fibrin-D-dimer. This presents the possibility of a fibrinspecific neo-epitope being manifested in the presence of zinc ions that could provide a means to determine fibrin degradation products more specifically. A model for the neo-epitope has been proposed. 2017-12-12T10:55:32Z 2017-12-12T10:55:32Z 1992 Master Thesis Masters MSc (Med) http://hdl.handle.net/11427/26555 eng application/pdf Division of Chemical Pathology Faculty of Health Sciences University of Cape Town |
| spellingShingle | Fibrin - chemistry Metals - Analysis Zinc - analysis Naidoo, Dhesigen P Metals and the conformation of fibrin |
| thesis_degree_str | Master's |
| title | Metals and the conformation of fibrin |
| title_full | Metals and the conformation of fibrin |
| title_fullStr | Metals and the conformation of fibrin |
| title_full_unstemmed | Metals and the conformation of fibrin |
| title_short | Metals and the conformation of fibrin |
| title_sort | metals and the conformation of fibrin |
| topic | Fibrin - chemistry Metals - Analysis Zinc - analysis |
| url | http://hdl.handle.net/11427/26555 |
| work_keys_str_mv | AT naidoodhesigenp metalsandtheconformationoffibrin |