E-Thesis 24 views
Molecular Analysis of the Role of Shear and Glycosylation in von Willebrand Factor Degradation by ADAMTS13 / VICTORIA HIGGINBOTHAM
Swansea University Author: VICTORIA HIGGINBOTHAM
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...
| Published: |
Swansea, Wales, UK
2024
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| 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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| 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. |
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| Keywords: |
vWF, ADAMTS13, VAD, shear, glycosylation |
| College: |
Faculty of Medicine, Health and Life Sciences |
| Funders: |
EPSRC; Calon Cardio-Technology Ltd |