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

并不是所有的细胞都有相同的行为方式，即使它们看起来是相同的类型。在这个项目中，我们将研究血小板异质性的来源。血小板对正常止血至关重要，但在动脉血栓形成和心肌梗死中也起着关键作用，这是英国死亡的主要原因。在动脉血栓形成时，血小板亚群变成促凝剂。这些血小板暴露磷脂酰丝氨酸，加速凝血生成，促进闭塞性血栓形成。目前，对于为什么单个血小板变成促凝剂，而不是促聚集，所知甚少。血小板将被激活，通过流动辅助细胞分选（FACS）分离促凝血小板和促聚集血小板，并通过定量蛋白质组学进行比较。确定的差异将通过生物化学技术加以证实。血小板也表现出年龄的异质性，有研究表明，年轻的血小板最容易形成血栓。血小板是无核的。它们合成新蛋白质的能力仅限于由巨核细胞亲本遗传的mRNA编码的蛋白质。血小板含有活性蛋白酶体和溶酶体，因此在血小板的生命周期中，蛋白质很可能被降解。年轻血小板显示出增加的RNA含量，这也可以用FACS用核酸染料纯化这些血小板，进行定量蛋白质组学分析，并直接比较年轻血小板和老年血小板的促聚集和促凝潜能。

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.