Ligation of Glycophorin A Generates Reactive Oxygen Species Leading to Decreased Red Blood Cell Function

Acute, inflammatory conditions associated with dysregulated complement activation are characterized by significant increases in blood concentration of reactive oxygen species (ROS) and ATP. The mechanisms by which these molecules arise are not fully understood. In this study, using luminometric- and fluorescence-based methods, we show that ligation of glycophorin A (GPA) on human red blood cells (RBCs) results in a 2.1-fold, NADPH-oxidase-dependent increase in intracellular ROS that, in turn, trigger multiple downstream cascades leading to caspase-3 activation, ATP release, and increased band 3 phosphorylation. Functionally, using 2D microchannels to assess membrane deformability, GPS-ligated RBCs travel 33% slower than control RBCs, and lipid mobility was hindered by 10% using fluorescence recovery after photobleaching (FRAP). These outcomes were preventable by pretreating RBCs with cell-permeable ROS scavenger glutathione monoethyl ester (GSH-ME). Our results obtained in vitro using anti-GPA antibodies were validated using complement-altered RBCs isolated from control and septic patients. Our results suggest that during inflammatory conditions, circulating RBCs significantly contribute to capillary flow dysfunctions, and constitute an important but overlooked source of intravascular ROS and ATP, both critical mediators responsible for endothelial cell activation, microcirculation impairment, platelet activation, as well as long-term dysregulated adaptive and innate immune responses.伴随补体激活失调的急性炎症性疾病，其典型特征为血液中活性氧（reactive oxygen species, ROS）与三磷酸腺苷（ATP）浓度显著升高。目前学界尚未完全阐明这类分子的产生机制。本研究采用发光法与荧光法开展实验，结果显示：人红细胞（red blood cells, RBCs）表面的糖蛋白A（glycophorin A, GPA）发生交联后，细胞内活性氧水平可升高2.1倍，该过程依赖于烟酰胺腺嘌呤二核苷酸磷酸氧化酶（NADPH-oxidase）；活性氧进一步激活多条下游信号级联反应，最终介导半胱天冬氨酸蛋白酶3（caspase-3）活化、三磷酸腺苷释放以及带3蛋白（band 3）磷酸化水平上调。功能验证实验中，本研究通过二维微通道检测红细胞膜变形能力，发现经GPA交联的红细胞迁移速度较对照组红细胞降低33%；同时利用荧光漂白恢复技术（fluorescence recovery after photobleaching, FRAP）检测发现，红细胞的脂质流动性下降10%。若预先使用可穿透细胞膜的活性氧清除剂单乙基谷胱甘肽（glutathione monoethyl ester, GSH-ME）处理红细胞，则可完全阻断上述实验效应。本研究通过抗GPA抗体在体外获得的实验结果，经分离自健康对照者与脓毒症患者的补体失调红细胞验证后，得到了确认。本研究结果提示：在炎症状态下，循环红细胞会显著加剧毛细血管血流功能障碍，同时也是血管内活性氧与三磷酸腺苷的重要但此前被忽视的来源；这两类分子均为关键介质，分别参与内皮细胞活化、微循环障碍、血小板活化，以及长期适应性与固有免疫应答失调过程。