Phosphoproteomic analysis of platelet signalling cascades by flow cytometry

Spurgeon, Benjamin E. J.

Abstract

The activation of blood platelets is a critical haemostatic response that serves to prevent haemorrhage, but unregulated platelet activation is associated with arterial thrombosis. Endothelial-derived inhibitors prostacyclin (PGI₂) and nitric oxide (NO) activate protein kinase-mediated signalling cascades to regulate platelet function and prevent vascular thrombosis. These signalling cascades involve a number of complex protein phosphorylation reactions, which regulate different aspects of platelet function. Dissecting the signalling events that regulate platelet function could facilitate the development of novel antiplatelet agents. Intraplatelet protein phosphorylation is commonly measured by immunoblotting, which is not conducive to whole blood analysis and therefore may not provide an accurate representation of signalling events in a (patho)physiological context. Therefore, the major aim of this thesis was to develop methodologies that could examine platelet signalling events in a more physiological context. In particular, we wanted to develop methodologies that could evaluate the ability of PGI₂ to modulate blood platelet activity. Using whole blood flow cytometry, PGI₂ was found to inhibit platelet fibrinogen binding and P-selectin expression, two independent markers of platelet activation. The inhibition of platelet function by PGI₂ corresponded with increased phosphorylation of proteins known to be targeted by PGI₂-mediated signalling cascades including vasodilator-stimulated phosphoprotein (VASP). In the next series of experiments, we developed an assay to evaluate these signalling events in whole blood. This phosphoflow assay was sensitive enough to accurately and reproducibly detect subtle dose- and time-dependent changes in protein phosphorylation in whole blood that were consistent with immunoblotting protocols with washed platelets. The application of fluorescent barcoding protocols to this assay enabled the simultaneous staining and acquisition of 24-96 samples in a single analysis tube. To exploit the high-throughput nature of the method and demonstrate its value as a drug discovery platform, we screened a library of 70 prostaglandins for their ability to stimulate intraplatelet VASP phosphorylation. The screen revealed three previously uncharacterised molecules that stimulated cAMP formation, induced VASP phosphorylation, and inhibited platelet aggregation. Because whole blood samples could be processed after cold storage, the method could be performed on samples obtained at remote locations such as clinical sites. To this end, we showed that the method could be used to measure signalling events in patients with polycystic ovary syndrome (PCOS), an endocrine disorder associated with platelet dysfunction. We envisage that the method will be useful for basic scientists, clinicians, and pharmacologists seeking novel therapies.

Citation

Spurgeon, B. E. J. (2014). Phosphoproteomic analysis of platelet signalling cascades by flow cytometry. (Thesis). Hull York Medical School, the University of Hull and the University of York. Retrieved from https://hull-repository.worktribe.com/output/4216373

• Thesis Type: Thesis
• Deposit Date: Mar 5, 2015
• Publicly Available Date: Feb 23, 2023
• Keywords: Medicine
• Public URL: https://hull-repository.worktribe.com/output/4216373
• Additional Information: Hull York Medical School, The University of Hull and the University of York
• Award Date: Oct 1, 2014