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The fibrinolytic potential of proteolytic enzymes on plasma microclots: a comparative analysis of long COVID patients using fluorescence microscopy and imaging flow cytometry
Usher, Thomas James. 2024. The fibrinolytic potential of proteolytic enzymes on plasma microclots: a comparative analysis of long COVID patients using fluorescence microscopy and imaging flow cytometry. Unpublished masters dissertation. Stellenbosch : Stellenbosch University [online]. Available: htt...
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Stellenbosch : Stellenbosch University
2025
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| _version_ | 1867613953808400384 |
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| access_status_str | Open Access |
| author | Usher, Thomas James |
| author2 | Pretorius, Etheresia |
| author_browse | Pretorius, Etheresia Usher, Thomas James |
| author_facet | Pretorius, Etheresia Usher, Thomas James |
| author_sort | Usher, Thomas James |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Usher, Thomas James. 2024. The fibrinolytic potential of proteolytic enzymes on plasma microclots: a comparative analysis of long COVID patients using fluorescence microscopy and imaging flow cytometry. Unpublished masters dissertation. Stellenbosch : Stellenbosch University [online]. Available: https://scholar.sun.ac.za/handle/10019.1/131941
Thesis (MSc)--Stellenbosch University, 2024. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/131941 |
| institution | Stellenbosch University (South Africa) |
| last_indexed | 2026-06-10T12:44:20.637Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/131941 The fibrinolytic potential of proteolytic enzymes on plasma microclots: a comparative analysis of long COVID patients using fluorescence microscopy and imaging flow cytometry Usher, Thomas James Pretorius, Etheresia Venter, Chantelle Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences. Post COVID-19 condition (Disease) Blood platelets -- Receptors Biofilms Blood -- Coagulation Proteolytic enzymes -- Mechanism of action Serratiopeptidase Nattokinase Microclots -- Treatment UCTD Usher, Thomas James. 2024. The fibrinolytic potential of proteolytic enzymes on plasma microclots: a comparative analysis of long COVID patients using fluorescence microscopy and imaging flow cytometry. Unpublished masters dissertation. Stellenbosch : Stellenbosch University [online]. Available: https://scholar.sun.ac.za/handle/10019.1/131941 Thesis (MSc)--Stellenbosch University, 2024. ENGLISH ABSTRACT: Long COVID patients experience a range of debilitating symptoms attributed to several pathological processes primarily stemming from fibrinolytic-resistant amyloid microclots. The disease’s complex pathophysiology has hindered the development of targeted treatments. Recently, novel enzyme treatments have garnered global attention for their potential in treating inflammatory conditions; however, these potential treatment avenues have not been explored in Long COVID. This study evaluates the effects of two proteolytic enzymes, nattokinase (NK) and serrapeptase (SP), on Long COVID-associated coagulopathies. The analysis incorporated platelet-poor plasma (PPP) samples from Long COVID patients (n = 50) and healthy controls (n = 45). Fluorescence microscopy and imaging flow cytometry were utilised to assess amyloid microclot formation, platelet activation, biofilm adherence, and the enzymatic digestion of microclots following in vitro treatment with NK or SP. Furthermore, a three-week in vivo treatment regimen was conducted on two patients: Patient A received NK alone, while Patient B received a combination of NK and SP. Weekly blood samples were analysed using fluorescence microscopy to assess microclot digestion. During the in vitro assessment, fluorescence microscopy revealed significant fibrinolysis post-treatment, with a sevenfold reduction in percentage amyloid area following SP treatment and a sixfold reduction with NK treatment (p < .0001). Imaging flow cytometry showed varied results. SP treatment led to a threefold decrease in microclot counts and objects per mL (p < .05), while NK treatment showed no significant change. Detailed analysis of microclot size distributions indicated that SP was most effective in the 0-100 μm² and 100-400 μm² size ranges, whereas NK primarily targeted smaller microclots in the 0-100 μm² range. Results from in vivo assessment showed that the percentage amyloid area of Patient A decreased tenfold over the treatment duration. A more pronounced 28-fold decrease was observed in Patient B, who was treated with the combination, highlighting the potential synergism of using both enzymes. Additional analyses of the coagulopathies in Long COVID patients showed significant fibrinogen misfolding, evidenced by an amyloid area 16 times larger than in controls. Flow cytometry analyses revealed a 33-fold increase in microclot counts and objects per mL in Long COVID samples compared with controls. The study also identified significant platelet hyperactivation and biofilm presence among Long COVID samples. The findings demonstrate that both NK and SP exhibit remarkable fibrinolytic and anti-biofilm capabilities in vitro and in vivo. Imaging flow cytometry provided valuable quantitative data on the size and quantity of microclots, offering promise for therapeutic applications in diagnostics and treatment evaluations. These results showcased the potential of NK and SP to significantly reduce amyloid microclots, hinder biofilm persistence, and promote endothelial recovery, supporting their use as therapeutic agents in Long COVID. AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar. Masters 2025-04-30T07:10:47Z 2025-04-30T07:10:47Z 2024-12 Thesis https://scholar.sun.ac.za/handle/10019.1/131941 Stellenbosch University xiv, 183 pages : illustrations (some color) application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Post COVID-19 condition (Disease) Blood platelets -- Receptors Biofilms Blood -- Coagulation Proteolytic enzymes -- Mechanism of action Serratiopeptidase Nattokinase Microclots -- Treatment UCTD Usher, Thomas James The fibrinolytic potential of proteolytic enzymes on plasma microclots: a comparative analysis of long COVID patients using fluorescence microscopy and imaging flow cytometry |
| title | The fibrinolytic potential of proteolytic enzymes on plasma microclots: a comparative analysis of long COVID patients using fluorescence microscopy and imaging flow cytometry |
| title_full | The fibrinolytic potential of proteolytic enzymes on plasma microclots: a comparative analysis of long COVID patients using fluorescence microscopy and imaging flow cytometry |
| title_fullStr | The fibrinolytic potential of proteolytic enzymes on plasma microclots: a comparative analysis of long COVID patients using fluorescence microscopy and imaging flow cytometry |
| title_full_unstemmed | The fibrinolytic potential of proteolytic enzymes on plasma microclots: a comparative analysis of long COVID patients using fluorescence microscopy and imaging flow cytometry |
| title_short | The fibrinolytic potential of proteolytic enzymes on plasma microclots: a comparative analysis of long COVID patients using fluorescence microscopy and imaging flow cytometry |
| title_sort | fibrinolytic potential of proteolytic enzymes on plasma microclots a comparative analysis of long covid patients using fluorescence microscopy and imaging flow cytometry |
| topic | Post COVID-19 condition (Disease) Blood platelets -- Receptors Biofilms Blood -- Coagulation Proteolytic enzymes -- Mechanism of action Serratiopeptidase Nattokinase Microclots -- Treatment UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/131941 |
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