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Title: The role and regulation of Rasa3 in platelets and platelet cell models
Author: Battram, Anthony Matthew
ISNI:       0000 0004 6059 0399
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2016
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Upon vascular insult, platelets are recruited to the site of injury, are activated, and form a haemostatic plug to prevent bleeding. However, inappropriate platelet activation can lead to the formation of occluding thrombi in arteries and veins in a process known as thrombosis. The GTPase Rap1 plays a critical role in platelet activation by regulating the affinity state of the fibrinogen receptor, integrin allbb3. The aim of this thesis was to ascertain the role and regulation of a Rap1-targeting GTPase-activating protein (GAP), Rasa3 (previously GAP1lP4BP), in platelet signal transduction and function. I first investigated possible mechanisms of Rasa3 regulation in platelets, and I found that platelet Rasa3 bound to phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), suggesting a role of PI3 kinase. Although PI(3,4,5)P3 binding had no direct effect on Rasa3 Rap1 GAP activity in vitro, Pl3 kinase did regulate Rasa3 subcellular localisation and activation of Rasa3 substrate, Rap1. Rasa3 also underwent thrombin-induced phosphorylation and associated with integrin b3. Next, to assess potential effects of Rasa3 on platelet function, I used a CHO cell model of integrin allbb3 signalling, C19 cells. I demonstrated that Rasa3 Rap1GAP activity was responsible for inhibiting integrin allbb3-mediated C19 cell spreading. I then characterised two Rasa3 mutants, H794L and G125V, found in thrombocytopaenic mouse models. I found that both mutations caused a loss of Rasa3 Rap1GAP and RasGAP activity, and Rasa3 inhibition of C19 cell spreading. Furthermore, platelets from mice expressing Rasa3 (H794L) exhibit increased spreading in a manner that is insensitive to PI3 kinase inhibition, suggesting that Pl3 kinase regulates platelet spreading by blocking Rasa3 Rap1GAP activity. I finally evaluated CMK cells as a possible cell line in which to study the effect of Rasa3 on platelet signalling and integrin allbb3 activation, and I found that they have some advantages compared to the C19 cell model. Overall, this thesis establishes a novel role for Rasa3 in platelet integrin allbb3 outside-in signalling and begins to explore the Pl3 kinase-dependent and -independent mechanisms of Rasa3 regulation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available