The Role of ADP in Platelet Activation and its Signalling in a Murine Model of Acute Allergic Inflammation

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Abstract Background: Clinical studies have demonstrated platelet activation in inflammatory disorders including asthma and allergic rhinitis. This is distinct from platelet aggregation involved in the maintenance of haemostasis. Whilst signalling involved in platelet activation in haemostasis is well known, signalling pathways in activation responding to inflammatory stimuli remains unclear. Objectives: Here I investigated whether purinergic activation of platelets in allergic inflammation is distinct from purine involvement in platelet aggregation. In the second part, stimulation of platelets with a large range of both inflammatory and haemostatic stimuli was used to investigate potential distinct differences in platelet function. Methods: Balb/c mice were sensitised to Ovalbumin (OVA) and subsequent OVA challenge. Bronchoalveolar lavage fluid was analysed for inflammatory cells and blood samples were collected and analysed for platelet activation. The role of platelet purinergic receptors and associated signalling mechanisms (RhoA) were also assessed. Additional in vitro experiments were performed on isolated human platelets to investigate the impact of a range of inflammatory and haemostatic stimuli on platelet function through measurements on P-selectin expression, platelet-leukocyte conjugation, aggregation and migration. Results: Allergen challenge induced significant increases in pulmonary leukocyte recruitment compared to sham controls (P<0.001).P2Y1 (P < 0.001), but not P2Y12 or P2X1 antagonism inhibited allergen induced pulmonary leukocyte recruitment. The formation of platelet-leukocyte conjugates in vivo and platelet/P-selectin dependent polymorphonuclear cell migration in vitro was exclusively P2Y1 dependent, furthermore allergen challenge induced significant increases in RhoA activity, a process which was inhibited exclusively through P2Y1 receptor antagonism. Leukocyte recruitment remained significantly suppressed in thrombocytopenic mice following reinfusion of platelets treated with a P2Y1 antagonist or a Rho-associated kinase inhibitor confirming a crucial role for RhoA activity downstream of platelet P2Y1 receptors. Secondly, stimulation of platelets with chemotactic stimuli such as macrophage-derived chemokine (P < 0.01) and stromal-cell derived factor 1α (P < 0.001) induced significant platelet migration without increases in P-selectin, platelet-leukocyte conjugates or aggregation. Conversely haemostatic stimuli induced increases in all measured parameters bar platelet migration Conclusion: RhoA signalling downstream of platelet P2Y1, but not P2Y12, represents a clear dichotomy in platelet activation during allergic inflammation versus haemostasis. Furthermore, platelet activation by different stimuli (inflammatory or haemostatic) can induce differences in platelet function further emphasising a dichotomy in platelet function.
Date of Award2014
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorClive Page (Supervisor) & Simon Pitchford (Supervisor)

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