Abstract
A dichotomy in platelet activation has been described, where along with well-known thrombotic and haemostatic responses, platelets can be activated via alternate pathways leading to inflammatory responses. Such activation pathways are believed to be involved in allergic inflammatory diseases such as asthma, where platelets can migrate extravascularly into lung airway walls and parenchyma in response to allergen. Understanding the mechanisms of platelet recruitment and migration in allergic inflammation could lead to novel drug approaches for the treatment of allergic conditions.The purpose of this research was to firstly develop and advance in-vitro and in-vivo models of platelet migration in response to allergen sensitization and challenge. Moreover, the mechanisms behind the allergic inflammatory actions of platelets by investigating the roles of platelet chemokine receptors.
Platelet CCR1, CCR3, CCR4 and CXCR4 chemokine receptor expression was confirmed by flow cytometry and western blot analysis, using antibodies specific to each receptor. Several in-vitro chemotaxis assays were studied and developed for measuring human and mouse platelet motility. Platelet chemotaxis was recorded in-vitro towards eotaxin, fMLP, MDC and SDF-1α. Furthermore, increased platelet chemotaxis to eotaxin was recorded when platelets were harvested from mice sensitized to the experimental allergen ovalbumin, compared to platelets harvested from sham-sensitized mice.
In other experiments, a house dust mite extract (HDM) sensitisation and exposure protocol was optimised to sensitise mice to allergen, to then be used for: 1. intravital-microscopy preparations of mouse cremaster muscle; 2. lung immunohistochemistry assays. Intravital microscopy of fluorescently labelled platelets in the mouse cremaster muscle enabled recordings of platelet-endothelial wall interactions. After HDM challenge in the cremaster muscle, increased platelet rolling and adhesion events were recorded in post capillary venules of HDM-sensitised mice compared with sham-sensitised mice. Immunohistochemical staining of extravascular platelets in cremaster muscle sections, and lungs similarly displayed an increased number of platelets in mice that were HDM-sensitised and HDM challenged.
The roles of platelet chemokine CCR1, CCR3, CCR4, and CXCR4 receptors on platelet accumulation were investigated using antagonists to these receptors in-vivo. In response to allergen challenge, CCR3 receptor antagonism attenuated localized platelet adhesion and accumulation; whilst CXCR4 receptor antagonism decreased leukocyte migration.
These results demonstrate that platelet migration can occur in response to a chemokine stimulus in-vitro and allergen exposure in-vivo, and suggests novel roles of platelet chemokine receptors in allergic inflammatory platelet function.
| Date of Award | 2018 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Simon Pitchford (Supervisor) & Clive Page (Supervisor) |