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Structural and functional characterisation of dioxolane A3, a new eicosanoid generated by cycooxygenase-1 in platelets

Hinz, Christine 2016. Structural and functional characterisation of dioxolane A3, a new eicosanoid generated by cycooxygenase-1 in platelets. PhD Thesis, Cardiff University.
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Abstract

Platelet-derived eicosanoids play important signalling roles in haemostasis and innate immunity and have major medical implications in cardiovascular disease, inflammation and cancer. Therefore, identification and characterisation of novel platelet lipids is an important goal. Prior to my PhD, our group identified a novel cyclooxygenase-1 (COX-1) product generated by platelets upon agonist activation in an aspirin–sensitive manner. Preliminary structural data suggested this lipid to be 8-hydroxy-9,11-dioxolane eicosatrienoic acid (DXA3) (Aldrovandi, unpublished). However, this required further investigation and its biology was unknown. In this study, I undertook a detailed structural characterisation of DXA3. Using GC/MS, high resolution LC/MSn and UV enabled structural characterisation of the lipid. Through pharmacological studies on isolated platelets and using mutant COX-2 enzymes, I revealed a distinct enzymatic mechanism of COX where the dioxolane forms within the COX catalytic site, then a radical escapes from the enzyme to form DXA3 possibly via a peroxidase. I developed a quantitative assay and showed that DXA3 is generated in ng amounts by thrombin-activated platelets (human and murine) with 77% of DXA3 esterified to phosphatidylethanolamine (PE). In addition to platelets, I detected the lipid in human serum derived from whole blood following clot formation in vitro and the macrophage cell line RAW246.7 in response to calcium ionophore A23187 stimulation. Last, DXA3 stimulated expression of the integrin Mac-1 on human neutrophils. In conclusion, DXA3 represents a novel eicosanoid, generated by activated platelets that activates neutrophils and may play a role in early immune responses.

Item Type: Thesis (PhD)
Date Type: Publication
Status: Unpublished
Schools: Medicine
Subjects: R Medicine > R Medicine (General)
Date of First Compliant Deposit: 13 September 2016
Last Modified: 26 Sep 2017 01:30
URI: http://orca-mwe.cf.ac.uk/id/eprint/94475

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