Data from: 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.

与补体激活失调相关的急性炎症性疾病，以血液中活性氧（reactive oxygen species, ROS）与三磷酸腺苷（ATP）浓度显著升高为典型特征。此类分子的产生机制尚未完全阐明。本研究采用发光法与荧光法开展实验，结果显示，靶向结合人类红细胞（red blood cells, RBCs）表面的血型糖蛋白A（glycophorin A, GPA），可使细胞内活性氧水平升高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）；活性氧进而触发多条下游级联反应，最终介导半胱天冬酶-3（caspase-3）激活、三磷酸腺苷释放以及带3蛋白磷酸化水平升高。功能层面，本研究借助二维微通道评估红细胞膜变形能力，发现经抗GPA抗体处理的红细胞，其迁移速度较对照组红细胞慢33%；通过荧光光漂白后恢复（fluorescence recovery after photobleaching, FRAP）实验检测发现，红细胞膜脂流动性降低10%。上述效应可通过使用可透过细胞膜的活性氧清除剂单乙酯谷胱甘肽（glutathione monoethyl ester, GSH-ME）预处理红细胞予以阻断。本研究采用抗GPA抗体开展的体外实验结果，通过分析从健康对照与脓毒症患者体内分离的补体失调红细胞得到了验证。研究结果表明，在炎症状态下，循环红细胞可显著加剧毛细血管血流功能障碍，同时作为一种重要却长期被忽视的血管内活性氧与三磷酸腺苷来源，二者均为介导内皮细胞激活、微循环障碍、血小板激活以及长期适应性与固有免疫应答失调的关键介质。