Thrombospondin-1 (TSP-1), a Ca2+-binding trimeric glycoprotein secreted by multiple cell types, has been implicated in the pathophysiology of several clinical conditions. Signaling involving TSP-1, through its cognate receptor CD47, orchestrates a wide array of cellular functions including cytoskeletal organization, migration, cell-cell interaction, cell proliferation, autophagy, and apoptosis. In the present study, we investigated the impact of TSP-1/CD47 signaling on Ca2+ dynamics, survival, and deformability of human red blood cells (RBCs). Whole-cell patch-clamp was employed to examine transmembrane cation conductance. RBC intracellular Ca2+ levels and multiple indices of RBC cell death were determined using cytofluorometry analysis. RBC morphology and microvesiculation were examined using imaging flow cytometry. RBC deformability was measured using laser-assisted optical rotational cell analyzer. Exposure of RBCs to recombinant human TSP-1 significantly increased RBC intracellular Ca2+ levels. As judged by electrophysiology experiments, TSP-1 treatment elicited an amiloride-sensitive inward current alluding to a possible Ca2+ influx via non-selective cation channels. Exogenous TSP-1 promoted microparticle shedding as well as enhancing Ca2+- and nitric oxide-mediated RBC cell death. Monoclonal (mouse IgG1) antibody-mediated CD47 ligation using 1F7 recapitulated the cell death-inducing effects of TSP-1. Furthermore, TSP-1 treatment altered RBC cell shape and stiffness (maximum elongation index). Taken together, our data unravel a new role for TSP-1/CD47 signaling in mediating Ca2+ influx into RBCs, a mechanism potentially contributing to their dysfunction in a variety of systemic diseases.

中文翻译：

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Thrombospondin-1/CD47 信号调节人类红细胞的跨膜阳离子电导、存活和变形能力。

血小板反应蛋白-1 (TSP-1) 是一种由多种细胞类型分泌的 Ca2+ 结合三聚体糖蛋白，与多种临床病症的病理生理学有关。涉及 TSP-1 的信号传导，通过其同源受体 CD47，协调广泛的细胞功能，包括细胞骨架组织、迁移、细胞间相互作用、细胞增殖、自噬和细胞凋亡。在本研究中，我们研究了 TSP-1/CD47 信号传导对人红细胞 (RBC) 的 Ca2+ 动力学、存活和变形能力的影响。全细胞膜片钳用于检查跨膜阳离子电导。RBC 细胞内 Ca2+ 水平和 RBC 细胞死亡的多个指标使用细胞荧光分析来确定。使用成像流式细胞术检查红细胞形态和微泡。使用激光辅助光学旋转细胞分析仪测量红细胞变形能力。红细胞暴露于重组人 TSP-1 显着增加红细胞细胞内 Ca2+ 水平。根据电生理学实验判断，TSP-1 治疗引发了阿米洛利敏感的内向电流，暗示可能通过非选择性阳离子通道的 Ca2+ 流入。外源性 TSP-1 促进微粒脱落以及增强 Ca2+-和一氧化氮介导的 RBC 细胞死亡。使用 1F7 的单克隆（小鼠 IgG1）抗体介导的 CD47 连接概括了 TSP-1 的细胞死亡诱导作用。此外，TSP-1 处理改变了 RBC 细胞的形状和刚度（最大伸长指数）。总之，我们的数据揭示了 TSP-1/CD47 信号在介导 Ca2+ 流入红细胞中的新作用，