Abstract/Summary

Background: Reactive oxygen species (ROS) are key signalling components in virtually all cells, acting either as second messengers or oxidants to target proteins. It has been reported that NADPH oxidase 1 (Nox-1) and the thiol isomerase protein disulphide isomerase (PDI) may interact physically and functionally to fine-tune ROS production in vascular cells. Both Nox-1 and PDI are able to individually regulate platelets, which are circulating cells of importance for thrombosis and haemostasis. However, little is known on how Nox-1 and PDI may collaborate to fine-tune platelet activity. Likewise, there is a current need to uncover novel inhibitors of these proteins and understand how ROS in platelets are implicated in metabolic diseases. Aims: The overarching aim of this thesis is to deepen our understanding of how redox processes regulate platelets in health and disease. Redox processes include the consequences of ROS production, as well as interest in specific redox proteins, namely PDI and Nox-1. Results: First, the flavonoid myricetin was studied and shown to be a novel inhibitor of PDI. Myricetin was able to reduce platelet aggregation, fibrinogen binding, granule exposure and in vitro thrombus formation, with no effect on tail bleeding in vivo. It is proposed that this flavonoid binds non-covalently near the active site of PDI and ERp5, which is also a thiol isomerase. Since PDI has been shown to associate with Nox-1 in vascular cells, the functional association of PDI and Nox-1 was studied. Through the use of selective inhibitors and Nox-1 deficient murine platelets, it was evident that PDI and Nox-1 collaborated to GPVI-mediated signalling in a process that involved phosphorylation of p38 mitogen-activated kinase and p47phox, which is a cytosol organizer of Nox-1. Moreover, in a healthy population study (n=137) PDI was positively correlated with waist/hip ratio and insulinaemia, whereas Nox-1 was correlated with body mass index (BMI) and systolic blood pressure. In line with the relevance of redox proteins PDI and Nox-1 to the regulation of platelet function, the redox regulation of platelets was also shown in a murine obesity model, in which it is shown for the first time that maternal high-fat diet intake is able to programme platelets from male offspring in a process associated with higher oxidative stress. Of importance, it is described a novel ‘double-hit’ effect in which maternal and offspring high-fat diet ingestion results in platelet hyperactivity. Conclusions: Altogether, data herein presented advance our understanding of how platelets are fine-tuned by redox processes. Myricetin is proposed as a novel inhibitor of thiol isomerases, while a novel mechanistic association between PDI and Nox-1 is also shown. Furthermore, it is suggested for the first time that platelets can be programmed by maternal insults. Therefore, platelets are highly regulated by redox processes and the study of these processes will contribute to improve the development of more effective antiplatelet therapy.