Yu Lu, Yu Shifang, He Yunlei, Deng Gang, Li Qiang
The Ningbo Central Blood Station, Ningbo, China.
The Department of Transfusion Medicine, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China.
Indian J Hematol Blood Transfus. 2023 Oct;39(4):670-678. doi: 10.1007/s12288-023-01638-1. Epub 2023 Mar 8.
Platelets undergo remarkable morphological changes during storage. Platelets change into different sizes and densities and differ in their biochemistry and functions. However, the correlation between structural heterogeneity and platelet autophagy is largely unknown. The aim of this study was to investigate the autophagy process in vitro, such as routine storage of platelets, and explore the role of reactive oxygen species (ROS) involved in the regulation of platelet autophagy. The ROS and autophagy levels of platelet concentrates from apheresis platelets were evaluated through flow cytometry. The expression levels of autophagy-associated proteins (LC3I, LC3II, Beclin1, Parkin, and PINK1) were measured via Western blot. All biomarkers were dynamically monitored for seven days. Moreover, the morphological characteristics of platelet morphology during storage were analyzed through transmission electron microscopy (TEM). Flow cytometry showed that the levels of total cell ROS and mitochondria ROS increased in the stored platelets. Together with the increase in mitochondrial ROS, the autophagy signal LC3 in the platelets was strongly amplified. The number of swollen platelets (large platelets) considerably increased, and that of autophagy signal LC3 was remarkably higher than that of the normal platelets. Western blot revealed that the expression levels of Beclin1 and LC3 II/LC3 I ratio were enhanced, whereas those of Parkin and PINK1 almost did not change during the seven days of storage. The existence of autophagosomes or autophagolysosomes in the platelets at the middle stage of platelet storage was observed via TEM. Our data demonstrated that the subpopulation of large (swollen) platelets exhibited different autophagy patterns. Furthermore, increased platelet autophagy was associated with mitochondrial ROS. These preliminary results suggest that swelling platelets have a higher autophagy pattern than normal platelets during storage.
血小板在储存过程中会发生显著的形态变化。血小板会变成不同的大小和密度，其生物化学和功能也有所不同。然而，结构异质性与血小板自噬之间的相关性在很大程度上尚不清楚。本研究的目的是在体外研究自噬过程，如血小板的常规储存，并探讨活性氧（ROS）在血小板自噬调节中的作用。通过流式细胞术评估单采血小板制备的血小板浓缩物中的ROS和自噬水平。通过蛋白质免疫印迹法检测自噬相关蛋白（LC3I、LC3II、Beclin1、Parkin和PINK1）的表达水平。所有生物标志物均动态监测7天。此外，通过透射电子显微镜（TEM）分析储存期间血小板形态的形态学特征。流式细胞术显示，储存的血小板中总细胞ROS和线粒体ROS水平升高。随着线粒体ROS的增加，血小板中的自噬信号LC3被强烈放大。肿胀血小板（大血小板）的数量显著增加，自噬信号LC3的数量明显高于正常血小板。蛋白质免疫印迹法显示，在储存的7天内，Beclin1的表达水平和LC3 II/LC3 I比值升高，而Parkin和PINK1的表达水平几乎没有变化。通过TEM观察到血小板储存中期血小板中存在自噬体或自噬溶酶体。我们的数据表明，大（肿胀）血小板亚群表现出不同的自噬模式。此外，血小板自噬增加与线粒体ROS有关。这些初步结果表明，储存期间肿胀血小板的自噬模式高于正常血小板。
