Investigating the role of P2Y12 in thrombosis, inflammation and infection in the zebrafish model

• Main Author: Bright, Rebecca
• Other Authors: Chico, Tim ; Storey, Robert
• Published: University of Sheffield 2014
• Subjects: 610
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.631422

The P2Y12 receptor is an important amplifier of thrombosis. P2Y12 is activated by ADP, inducing release of a and dense granules from the platelet. a-granules contain a myriad of proteins including pro-inflammatory mediators involved in vascular inflammation. P2Y12 antagonists are used in the treatment and prevention of arterial thrombosis and also reduce levels of inflammatory mediators, indicating that P2Y12 may play a role in inflammation. a granules also release antimicrobial peptides suggesting that P2Y12 may also be involved in resistance to infection. Zebrafish embryos represent an excellent model to study thrombosis, inflammation and infection in vivo. Transgenic lines enable visualisation of leukocyte migration, whilst inoculation with pathogens enables modelling of resistance to infection. Temporary gene knockdown is achieved by injection of morpholino oligonucleotides and recent advances have enabled targeted mutation of specific gene loci. This thesis describes my investigations into the role of P2Y12 in thrombosis, inflammation and infection in these zebrafish models. I found that p2y12 knockdown significantly reduced thrombus area in response to vessel injury. I also investigated the effect of knockdown of several platelet microRNAs on thrombosis. I found that knockdown of miR-223 significantly increased thrombus area after vessel injury, a novel finding which indicates a previously unsuspected role of miR-223 in thrombus formation. Leukocyte migration to sites of inflammation was examined in both control and p2y12 morphants however I found no significant difference, suggesting P2Y12 does not play a role in leukocyte migration in this model. I investigated resistance to S. aureus infection and found a statistically significant reduction in survival of p2y12 morphants. I generated a p2y12 mutant which results in a frame shift proximal to the N-terminus, however I observed no effect on thrombosis or resistance to infection. This requires further investigation. My data shows that the function of P2Y12 in thrombosis is conserved in the zebrafish and that P2Y12 may play a role in resistance to S. aureus infection.