Xiong Guohua, Aras Omer, Shet Arun, Key Nigel S, Arriaga Edgar A
Department of Chemistry, Institute of Technology, University of Minnesota, Minneapolis, MN 55455, USA.
Analyst. 2003 Jun;128(6):581-8. doi: 10.1039/b301035j.
Platelet-derived microparticles (PMPs) formed by vesiculation during platelet activation seem to play a role in blood coagulation and in pathological disease states. Flow cytometry is currently the gold standard to characterize platelets and PMPs. Using this technique we distinguished between platelets and PMPs based on size and the presence of phosphatidyl serine (PS); PMPs were arbitrarily defined to be smaller than one micrometer and capable of forming a stable complex with fluorescently-labeled Annexin V, a protein that forms a calcium-dependent complex with PS. Further confirmation of PMP and platelet identity was done by use of fluorescently-labeled antibodies against CD41a, a glycoprotein found on the surface of both platelets and PMPs. In this report we also introduce the use of capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) for the analysis of fluorescently labeled platelets and PMPs. While both flow cytometry and CE-LIF can measure individual fluorescent events, only CE-LIF allowed us to calculate individual electrophoretic mobilities of activated platelets and PMPs that were then represented as distributions. A comparison between distributions suggests that PMPs have less negative mobilities. The fact that activated platelet preparations include PMPs partially obscure the interpretation of the data. While PMP and platelet number ml(-1) determined by flow cytometry is lower than the same parameter determined by CE-LIF, signal-to-noise ratio was 20 fold better for flow cytometry than for CE-LIF. This is the first time that a direct comparison between these two techniques is reported.
血小板活化过程中通过囊泡化形成的血小板衍生微粒（PMPs）似乎在血液凝固和病理疾病状态中发挥作用。流式细胞术是目前表征血小板和PMPs的金标准。使用该技术，我们根据大小和磷脂酰丝氨酸（PS）的存在来区分血小板和PMPs；PMPs被随意定义为小于1微米且能够与荧光标记的膜联蛋白V形成稳定复合物，膜联蛋白V是一种与PS形成钙依赖性复合物的蛋白质。通过使用针对CD41a的荧光标记抗体进一步确认PMP和血小板的身份，CD41a是一种在血小板和PMPs表面均发现的糖蛋白。在本报告中，我们还介绍了使用柱后激光诱导荧光检测的毛细管电泳（CE-LIF）来分析荧光标记的血小板和PMPs。虽然流式细胞术和CE-LIF都可以测量单个荧光事件，但只有CE-LIF使我们能够计算活化血小板和PMPs的单个电泳迁移率，然后将其表示为分布。分布之间的比较表明PMPs的负迁移率较小。活化血小板制剂中包含PMPs这一事实部分掩盖了数据的解释。虽然通过流式细胞术测定的PMP和血小板数量ml(-1)低于通过CE-LIF测定的相同参数，但流式细胞术的信噪比比CE-LIF好20倍。这是首次报道这两种技术之间的直接比较。
