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

不是所有的细胞都有相同的行为方式，即使它们看起来是同一类型的。在这个项目中，我们将调查血小板异质性的来源。血小板对正常止血是必不可少的，但也在动脉血栓形成和心肌梗死中发挥关键作用，心肌梗死是英国死亡的主要原因。在动脉血栓形成过程中，血小板的一个亚群成为促凝血剂。这些血小板暴露出磷脂酰丝氨酸，加速血栓生成，促进闭塞性血栓的形成。目前，人们对单个血小板为什么变成促凝剂而不是促凝剂知之甚少。通过流式细胞分选(FACS)和定量蛋白质组学比较，将活化的、促凝血的和促聚集的血小板分离出来。已确定的差异将通过生化技术得到证实。随着年龄的增长，血小板也显示出异质性，而且有人认为年轻的血小板最容易血栓形成。血小板是无核的。它们合成新蛋白质的能力仅限于由其巨核母细胞遗传的信使核糖核酸编码的蛋白质。血小板含有活跃的蛋白酶体和溶酶体，因此蛋白质在血小板的生命周期内很可能被降解。年轻的血小板表现出RNA含量的增加，这也可以用核酸染料的FACS来纯化这些血小板，用于定量的蛋白质组学分析，并直接比较年轻和老年血小板的促聚集和促凝血潜力。

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 inplatelets. Platelets are essential to normal haemostasis but also play a critical role in arterial thrombosis and myocardial infarction, a major cause of deathin the UK.During arterial thrombosis a subpopulation of platelets becomes pro-coagulant. These platelets expose phosphatidylserine and accelerate thrombingeneration, promoting occlusive thrombus formation. Currently, very little is known about why an individual platelet becomes pro-coagulant, rather thanpro-aggregatory. Platelets will be activated and procoagulant and pro-aggregatory platelets separated by flow-assisted cell sorting (FACS) and comparedby 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. Theircapacity to synthesise new proteins is limited to those encoded by the mRNA inherited from its megakaryocyte parent. Platelets contain an activeproteasome and lysosomes, so proteins are likely to be degraded during the platelet lifespan. Young platelets show increased RNA content, which canalso be used to purify these platelets by FACS using nucleic acid dyes, for quantitative proteomic analysis, and to directly compare the pro-aggregatoryand pro-coagulant potential of young and older platelets.