Wang L, Ostberg O, Wihlborg A-K, Brogren H, Jern S, Erlinge D
Department of Cardiology, Lund University Hospital, Lund, Sweden.
J Thromb Haemost. 2003 Feb;1(2):330-6. doi: 10.1046/j.1538-7836.2003.00070.x.
The mechanism of ADP-mediated platelet activation has been difficult to unravel due to the large number of receptors for extracellular nucleotides (P2 receptors). mRNA levels in circulating platelets are very low, but have been shown to be translationally active. By optimizing mRNA extraction and using real time (RT)-PCR we were able to establish a protocol for highly sensitive platelet mRNA quantification in human regular blood samples. In platelets from healthy volunteers, only P2X1, P2Y1 and P2Y12 were found in significant levels, with the following order of expression: P2Y12 >> P2X1 > P2Y1. Other P2 receptors (P2Y2, P2Y4, P2Y6, P2Y11, P2Y13, P2X4, P2X7) had very low expression. As a control measurement to exclude contamination, P2 receptors in buffy coat were quantified but had a completely different profile. Incubation in vitro revealed a more rapid degradation rate for P2X1 receptor mRNA than for P2Y1 and P2Y12, indicating that the level of P2X1 may be relatively higher in newly released platelets and in megacaryocytes. In conclusion, we have developed the first protocol for quantifying mRNA expression in human platelets limiting the P2 receptor drug development targets to P2Y12, P2Y1 and P2X1. Furthermore, the method could be used to study platelet expression for any gene in human materials.
由于细胞外核苷酸的受体（P2受体）数量众多，ADP介导的血小板激活机制一直难以阐明。循环血小板中的mRNA水平非常低，但已被证明具有翻译活性。通过优化mRNA提取方法并使用实时（RT）-PCR，我们能够建立一种在人类常规血液样本中对血小板mRNA进行高灵敏度定量的方案。在健康志愿者的血小板中，仅发现P2X1、P2Y1和P2Y12的表达水平较高，其表达顺序如下：P2Y12 >> P2X1 > P2Y1。其他P2受体（P2Y2、P2Y4、P2Y6、P2Y11、P2Y13、P2X4、P2X7）的表达水平非常低。作为排除污染的对照测量，对血沉棕黄层中的P2受体进行了定量，但结果显示出完全不同的表达模式。体外孵育显示，P2X1受体mRNA的降解速度比P2Y1和P2Y12更快，这表明在新释放的血小板和巨核细胞中，P2X1的水平可能相对较高。总之，我们开发了首个用于定量人类血小板中mRNA表达的方案，将P2受体药物开发靶点限定为P2Y12、P2Y1和P2X1。此外，该方法可用于研究人类材料中任何基因的血小板表达情况。
