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Molecular Analysis of the Role of Shear and Glycosylation in von Willebrand Factor Degradation by ADAMTS13 / VICTORIA HIGGINBOTHAM

Swansea University Author: VICTORIA HIGGINBOTHAM

  • E-Thesis – open access under embargo until: 3rd July 2029

DOI (Published version): 10.23889/SUthesis.67189

Abstract

Background: Heart failure affects ~26 million people worldwide. While heart transplantation is an effective surgical treatment, donor availability is limited. Ventricular assist devices (VADs) have been used as a bridge-to-transplant or destination treatment for heart failure. Although VADs are effe...

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Published: Swansea, Wales, UK 2024
Institution: Swansea University
Degree level: Doctoral
Degree name: Ph.D
Supervisor: Kanamarlapudi, Venkateswarlu
URI: https://cronfa.swan.ac.uk/Record/cronfa67189
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fullrecord <?xml version="1.0" encoding="utf-8"?><rfc1807 xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:xsd="http://www.w3.org/2001/XMLSchema"><bib-version>v2</bib-version><id>67189</id><entry>2024-07-25</entry><title>Molecular Analysis of the Role of Shear and Glycosylation in von Willebrand Factor Degradation by ADAMTS13</title><swanseaauthors><author><sid>8246ae1e9243a688cfd89e063af64fad</sid><firstname>VICTORIA</firstname><surname>HIGGINBOTHAM</surname><name>VICTORIA HIGGINBOTHAM</name><active>true</active><ethesisStudent>false</ethesisStudent></author></swanseaauthors><date>2024-07-25</date><abstract>Background: Heart failure affects ~26 million people worldwide. While heart transplantation is an effective surgical treatment, donor availability is limited. Ventricular assist devices (VADs) have been used as a bridge-to-transplant or destination treatment for heart failure. Although VADs are effective in supporting weakened hearts in pumping blood, post-operative gastrointestinal (GI) bleeding remains a significant obstacle in their long-term usage. High Molecular Weight von Willebrand Factor (HMW vWF) is a blood-circulating glycoprotein that functions in haemostasis. An increase in the breakdown of HMW vWF multimers by ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 3) under high shear stress generated by the implanted VAD is one of the causes for GI bleeding in VAD patients. The overall objective of this study is to investigate the role of shear stress generated by VAD and N-linked glycosylation of vWF and ADAMTS13 on their functionality.Hypothesis and aims: We hypothesised that ADAMTS13 proteolysis of HMW vWF is mediated by shear stress and N-glycosylation of the proteins. The hypothesis was investigated by analysing the HMW vWF degradation and ADAMTS13 activity under VAD-like shear stress (aim 1), assessing the functional characteristics of vWF and ADAMTS13 shear effect-mimicking and functional mutants (aim 2), and studying the role of N-linked deglycosylation on the functional properties of vWF and ADAMTS13 (aim 3). Methods: Blood components with various treatments were subjected to VAD-like shear stress, comparable to in vivo VAD HMW vWF degradation. ADAMTS13 and vWF antigen levels were estimated using enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The functional and N-glycosylation mutants were generated using site-directed mutagenesis. Immunoblotting under non-reducing conditions was used to analyse vWF multimerisation. The proteolytic activity of ADAMTS13 was measured using the A2 domain vWF fragments. The function of vWF was tested using collagen- and multimer-based assays. Results: HMW vWF in platelet-poor plasma (PPP) was degraded under in vitro, in vivo and VAD-like shear stress. Treatment of PPP with ADAMTS13 inhibitors prevented HMW vWF degradation under shear stress. ADAMTS13 cleaved XS-vWF and FRET-vWF73 substrates in a shear stress-independent manner. Molecular analysis of vWF and ADAMTS13 by using site-directed mutagenesis revealed that the GofF (gain of function) mutant of ADAMTS13 exhibits increased functionality with reduced binding affinity for vWF, whereas the Open mutant of vWF generated fewer multimers with lower functionality. Although total N-deglycosylation decreased its function, no single N-linked glycosylation site of vWF was shown to play a role in its structure and function. In contrast, one of the N-linked glycosylation sites on the metalloprotease domain (N146) of ADAMTS13 is found to be critical for its function. Conclusion: ADAMTS13 activity is shear-independent, whereas the breakdown of HMW vWF is shear- and ADAMTS13-dependent. While N-glycosylation is critical for their activity, no single N-glycosylation site and glycosylation at N146 are critical for the function of vWF and ADAMTS13, respectively.</abstract><type>E-Thesis</type><journal/><volume/><journalNumber/><paginationStart/><paginationEnd/><publisher/><placeOfPublication>Swansea, Wales, UK</placeOfPublication><isbnPrint/><isbnElectronic/><issnPrint/><issnElectronic/><keywords>vWF, ADAMTS13, VAD, shear, glycosylation</keywords><publishedDay>3</publishedDay><publishedMonth>7</publishedMonth><publishedYear>2024</publishedYear><publishedDate>2024-07-03</publishedDate><doi>10.23889/SUthesis.67189</doi><url/><notes/><college>COLLEGE NANME</college><CollegeCode>COLLEGE CODE</CollegeCode><institution>Swansea University</institution><supervisor>Kanamarlapudi, Venkateswarlu</supervisor><degreelevel>Doctoral</degreelevel><degreename>Ph.D</degreename><degreesponsorsfunders>EPSRC; Calon Cardio-Technology Ltd</degreesponsorsfunders><apcterm/><funders>EPSRC; Calon Cardio-Technology Ltd</funders><projectreference/><lastEdited>2024-07-25T16:19:10.3746336</lastEdited><Created>2024-07-25T16:02:10.9547958</Created><path><level id="1">Faculty of Medicine, Health and Life Sciences</level><level id="2">Swansea University Medical School - Biomedical Science</level></path><authors><author><firstname>VICTORIA</firstname><surname>HIGGINBOTHAM</surname><order>1</order></author></authors><documents><document><filename>Under embargo</filename><originalFilename>Under embargo</originalFilename><uploaded>2024-07-25T16:09:55.4636849</uploaded><type>Output</type><contentLength>22546533</contentLength><contentType>application/pdf</contentType><version>E-Thesis – open access</version><cronfaStatus>true</cronfaStatus><embargoDate>2029-07-03T00:00:00.0000000</embargoDate><documentNotes>Copyright: The Author, Victoria Higginbotham, 2024.</documentNotes><copyrightCorrect>true</copyrightCorrect><language>eng</language></document></documents><OutputDurs/></rfc1807>
spelling v2 67189 2024-07-25 Molecular Analysis of the Role of Shear and Glycosylation in von Willebrand Factor Degradation by ADAMTS13 8246ae1e9243a688cfd89e063af64fad VICTORIA HIGGINBOTHAM VICTORIA HIGGINBOTHAM true false 2024-07-25 Background: Heart failure affects ~26 million people worldwide. While heart transplantation is an effective surgical treatment, donor availability is limited. Ventricular assist devices (VADs) have been used as a bridge-to-transplant or destination treatment for heart failure. Although VADs are effective in supporting weakened hearts in pumping blood, post-operative gastrointestinal (GI) bleeding remains a significant obstacle in their long-term usage. High Molecular Weight von Willebrand Factor (HMW vWF) is a blood-circulating glycoprotein that functions in haemostasis. An increase in the breakdown of HMW vWF multimers by ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 3) under high shear stress generated by the implanted VAD is one of the causes for GI bleeding in VAD patients. The overall objective of this study is to investigate the role of shear stress generated by VAD and N-linked glycosylation of vWF and ADAMTS13 on their functionality.Hypothesis and aims: We hypothesised that ADAMTS13 proteolysis of HMW vWF is mediated by shear stress and N-glycosylation of the proteins. The hypothesis was investigated by analysing the HMW vWF degradation and ADAMTS13 activity under VAD-like shear stress (aim 1), assessing the functional characteristics of vWF and ADAMTS13 shear effect-mimicking and functional mutants (aim 2), and studying the role of N-linked deglycosylation on the functional properties of vWF and ADAMTS13 (aim 3). Methods: Blood components with various treatments were subjected to VAD-like shear stress, comparable to in vivo VAD HMW vWF degradation. ADAMTS13 and vWF antigen levels were estimated using enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The functional and N-glycosylation mutants were generated using site-directed mutagenesis. Immunoblotting under non-reducing conditions was used to analyse vWF multimerisation. The proteolytic activity of ADAMTS13 was measured using the A2 domain vWF fragments. The function of vWF was tested using collagen- and multimer-based assays. Results: HMW vWF in platelet-poor plasma (PPP) was degraded under in vitro, in vivo and VAD-like shear stress. Treatment of PPP with ADAMTS13 inhibitors prevented HMW vWF degradation under shear stress. ADAMTS13 cleaved XS-vWF and FRET-vWF73 substrates in a shear stress-independent manner. Molecular analysis of vWF and ADAMTS13 by using site-directed mutagenesis revealed that the GofF (gain of function) mutant of ADAMTS13 exhibits increased functionality with reduced binding affinity for vWF, whereas the Open mutant of vWF generated fewer multimers with lower functionality. Although total N-deglycosylation decreased its function, no single N-linked glycosylation site of vWF was shown to play a role in its structure and function. In contrast, one of the N-linked glycosylation sites on the metalloprotease domain (N146) of ADAMTS13 is found to be critical for its function. Conclusion: ADAMTS13 activity is shear-independent, whereas the breakdown of HMW vWF is shear- and ADAMTS13-dependent. While N-glycosylation is critical for their activity, no single N-glycosylation site and glycosylation at N146 are critical for the function of vWF and ADAMTS13, respectively. E-Thesis Swansea, Wales, UK vWF, ADAMTS13, VAD, shear, glycosylation 3 7 2024 2024-07-03 10.23889/SUthesis.67189 COLLEGE NANME COLLEGE CODE Swansea University Kanamarlapudi, Venkateswarlu Doctoral Ph.D EPSRC; Calon Cardio-Technology Ltd EPSRC; Calon Cardio-Technology Ltd 2024-07-25T16:19:10.3746336 2024-07-25T16:02:10.9547958 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Biomedical Science VICTORIA HIGGINBOTHAM 1 Under embargo Under embargo 2024-07-25T16:09:55.4636849 Output 22546533 application/pdf E-Thesis – open access true 2029-07-03T00:00:00.0000000 Copyright: The Author, Victoria Higginbotham, 2024. true eng
title Molecular Analysis of the Role of Shear and Glycosylation in von Willebrand Factor Degradation by ADAMTS13
spellingShingle Molecular Analysis of the Role of Shear and Glycosylation in von Willebrand Factor Degradation by ADAMTS13
VICTORIA HIGGINBOTHAM
title_short Molecular Analysis of the Role of Shear and Glycosylation in von Willebrand Factor Degradation by ADAMTS13
title_full Molecular Analysis of the Role of Shear and Glycosylation in von Willebrand Factor Degradation by ADAMTS13
title_fullStr Molecular Analysis of the Role of Shear and Glycosylation in von Willebrand Factor Degradation by ADAMTS13
title_full_unstemmed Molecular Analysis of the Role of Shear and Glycosylation in von Willebrand Factor Degradation by ADAMTS13
title_sort Molecular Analysis of the Role of Shear and Glycosylation in von Willebrand Factor Degradation by ADAMTS13
author_id_str_mv 8246ae1e9243a688cfd89e063af64fad
author_id_fullname_str_mv 8246ae1e9243a688cfd89e063af64fad_***_VICTORIA HIGGINBOTHAM
author VICTORIA HIGGINBOTHAM
author2 VICTORIA HIGGINBOTHAM
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publishDate 2024
institution Swansea University
doi_str_mv 10.23889/SUthesis.67189
college_str Faculty of Medicine, Health and Life Sciences
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hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Biomedical Science{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Biomedical Science
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description Background: Heart failure affects ~26 million people worldwide. While heart transplantation is an effective surgical treatment, donor availability is limited. Ventricular assist devices (VADs) have been used as a bridge-to-transplant or destination treatment for heart failure. Although VADs are effective in supporting weakened hearts in pumping blood, post-operative gastrointestinal (GI) bleeding remains a significant obstacle in their long-term usage. High Molecular Weight von Willebrand Factor (HMW vWF) is a blood-circulating glycoprotein that functions in haemostasis. An increase in the breakdown of HMW vWF multimers by ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 3) under high shear stress generated by the implanted VAD is one of the causes for GI bleeding in VAD patients. The overall objective of this study is to investigate the role of shear stress generated by VAD and N-linked glycosylation of vWF and ADAMTS13 on their functionality.Hypothesis and aims: We hypothesised that ADAMTS13 proteolysis of HMW vWF is mediated by shear stress and N-glycosylation of the proteins. The hypothesis was investigated by analysing the HMW vWF degradation and ADAMTS13 activity under VAD-like shear stress (aim 1), assessing the functional characteristics of vWF and ADAMTS13 shear effect-mimicking and functional mutants (aim 2), and studying the role of N-linked deglycosylation on the functional properties of vWF and ADAMTS13 (aim 3). Methods: Blood components with various treatments were subjected to VAD-like shear stress, comparable to in vivo VAD HMW vWF degradation. ADAMTS13 and vWF antigen levels were estimated using enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The functional and N-glycosylation mutants were generated using site-directed mutagenesis. Immunoblotting under non-reducing conditions was used to analyse vWF multimerisation. The proteolytic activity of ADAMTS13 was measured using the A2 domain vWF fragments. The function of vWF was tested using collagen- and multimer-based assays. Results: HMW vWF in platelet-poor plasma (PPP) was degraded under in vitro, in vivo and VAD-like shear stress. Treatment of PPP with ADAMTS13 inhibitors prevented HMW vWF degradation under shear stress. ADAMTS13 cleaved XS-vWF and FRET-vWF73 substrates in a shear stress-independent manner. Molecular analysis of vWF and ADAMTS13 by using site-directed mutagenesis revealed that the GofF (gain of function) mutant of ADAMTS13 exhibits increased functionality with reduced binding affinity for vWF, whereas the Open mutant of vWF generated fewer multimers with lower functionality. Although total N-deglycosylation decreased its function, no single N-linked glycosylation site of vWF was shown to play a role in its structure and function. In contrast, one of the N-linked glycosylation sites on the metalloprotease domain (N146) of ADAMTS13 is found to be critical for its function. Conclusion: ADAMTS13 activity is shear-independent, whereas the breakdown of HMW vWF is shear- and ADAMTS13-dependent. While N-glycosylation is critical for their activity, no single N-glycosylation site and glycosylation at N146 are critical for the function of vWF and ADAMTS13, respectively.
published_date 2024-07-03T16:19:09Z
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score 11.017776

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