The Investigation of the anti-platelet properties of Cucurbitacins

Khalifa, B (2020) The Investigation of the anti-platelet properties of Cucurbitacins. Masters by Research thesis (MSc), Manchester Metropolitan University.

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Abstract

Background: Cucurbitacin’s are dietary compounds that have been shown to cause anti-tumour, anti-inflammatory and anti-atherosclerotic activities. Mechanisms of action include dysregulation of the actin cytoskeleton and disruption of integrin function. Integrin outside-in signalling and cytoskeletal rearrangements are important for stable thrombus formation and clot retraction after platelet adhesion at the site of vessel damage. Aims: To identify the effect of cucurbitacin’s B, E and I on platelet function and thrombus formation. Methods: Human washed platelets, platelet rich plasma and whole blood in vitro platelet function assays were used including real time and end point platelet aggregation, platelet adhesion assay with DiOC6 and phalloidin staining, thrombus formation, flow cytometry and western blots to determine actin polymerisation activity and integrin function. Results: Anti-platelet and anti-thrombotic effects following treatment with cucurbitacin’s B, E and I were observed. Treatment of platelets with cucurbitacin B, E or I lead to reduction of platelet aggregation and fibrinogen binding following stimulation with ADP and collagen. Cucurbitacin B, E and were found to inhibit other integrin mediated events, including adhesion and spreading on adhesive surfaces such as fibrinogen. Additional investigation of cytoskeletal dynamics found treatment with cucurbitacin’s B, E and I increased actin polymerisation and myosin light chain phosphorylation which has been shown to disrupt integrin activation and platelet spreading. Treatment with cucurbitacins was also found to disrupt stable thrombus formation, with an increase in the formation of unstable thrombi and a reduction in thrombus density compared to control. Conclusions: Cucurbitacins have anti-platelet and anti-thrombotic activity, which appear to be linked to dysregulation of actin cytoskeletal dynamics and inhibition of integrin activity.