Reducing animal use in thrombosis research with an ex vivo injury model

Not all cells behave in the same way, even when they are seemingly of the same type. In this project we will investigate sources of heterogeneity in
platelets. Platelets are essential to normal haemostasis but also play a critical role in arterial thrombosis and myocardial infarction, a major cause of death
in the UK.
During arterial thrombosis a subpopulation of platelets becomes pro-coagulant. These platelets expose phosphatidylserine and accelerate thrombin
generation, promoting occlusive thrombus formation. Currently, very little is known about why an individual platelet becomes pro-coagulant, rather than
pro-aggregatory. Platelets will be activated and procoagulant and pro-aggregatory platelets separated by flow-assisted cell sorting (FACS) and compared
by quantitative proteomics. Identified differences will be confirmed by biochemistry techniques.
Platelets also show heterogeneity with age, and it has been suggested that younger platelets are the most thrombotic. Platelets are anucleate. Their
capacity to synthesise new proteins is limited to those encoded by the mRNA inherited from its megakaryocyte parent. Platelets contain an active
proteasome and lysosomes, so proteins are likely to be degraded during the platelet lifespan. Young platelets show increased RNA content, which can
also be used to purify these platelets by FACS using nucleic acid dyes, for quantitative proteomic analysis, and to directly compare the pro-aggregatory
and pro-coagulant potential of young and older platelets.