Measurement of extracellular vesicles by a novel fluorescent imaging method

Price, Joshua M.J. ORCID: 0000-0001-9950-7992 (2024). Measurement of extracellular vesicles by a novel fluorescent imaging method. University of Birmingham. Ph.D.

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Abstract

Extracellular vesicles (EV) are small phospholipid delimited particles released by virtually all cells that cannot replicate. EV are difficult to measure, partially due to their small size and isolation contaminants including lipoproteins. EV isolation and measurement are not standardised and can be associated with bias in measured populations. An instrument that can measure EV in complex biofluids without the need for isolation may improve standardisation of EV research. The use of immunoaffinity capture combined with fluorescent labelling would allow for a greater depth of phenotypic characterisation and could have use diagnostically with EV increasingly being identified as biomarkers of health and disease. Here I test whether ExoView, a new chip-based immunocapture platform for EV measurement, can measure EV without the need for purification.
I generated platelet enhanced plasma (PEP) a platelet EV (PEV) enriched plasma with high procoagulant activity, as measured through thrombin generation and clotting assays. I designed a protocol for measurement of phosphatidylserine (PS) using ExoView which confirmed high PS expression on EV in PEP compared to platelet free plasma (PFP). I also investigate whether PEP may hold potential as a novel resuscitation fluid for use in austere environments where platelets are often unavailable.
Having found ExoView to be capable of measuring PS on EV, I next moved to a trace protein that is also involved in clotting: tissue factor (TF). TF is a lowly expressed protein that can be EV associated. In addition to historic EV measurement problems, TF has its own historic and ongoing controversies. However, a reliable method of TF EV quantification would be useful clinically in ovarian cancer, pancreatic cancer and venous thromboembolism. I utilised custom capture with ExoView to enrich TF expressing EV from plasma followed by fluorescent labelling with a separate anti-TF clone and found ExoView can distinguish unstimulated vs lipopolysaccharide stimulated PFP. However, my TF EV method could not distinguish healthy PFP from clinical cohorts (cancer or trauma).
PEV and megakaryocyte (MK) derived EV are thought to be the largest EV population in circulation, however due to their similar parental lineage unique markers of either are difficult to identify and validate. They are interesting to distinguish as a change in the ratio of PEV:MK EV may indicate an increased risk of cardiovascular disease or may reflect bleeding risk in patients on antithrombotic therapeutics. I utilised ExoView and theoretically unique markers of PEV and MK EV on purified platelet supernatants and MK cell culture supernatants to try and identify unique signatures of PEV and MK EV which were then applied to PFP. The set of markers I used and the conditions I tested did not reveal a unique signature of PEV or MK EV that could be used to determine whether PEV or MK EV are more the more dominant EV source in circulation.
In conclusion, this thesis shows that ExoView has great capacity for EV measurement both in basic characterisation of EV-enriched markers such as tetraspanins, but also has potential for measurement of clinically relevant, rare subpopulations of EV.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):