Abstract
BackgroundThrombospondin-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).MethodsWhole-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.ResultsExposure 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).ConclusionsTaken 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.FKY5xz95WsHn6abiyZ6BGLVideo abstract
Highlights
Thrombospondin-1 (TSP-1), a Ca2+-binding trimeric glycoprotein secreted by multiple cell types, has been implicated in the pathophysiology of several clinical conditions
In the present study, using cytofluorometric and electrophysiological approaches we examined the influence of CD47-dependent signaling, evoked by exogenous TSP-1 or antibody-mediated CD47 ligation, on Ca2+ dynamics in human Red blood cell (RBC)
RBCs were incubated with recombinant human thrombospondin-1 (1–50 μg/mL; R&D Systems, Minneapolis, MN, USA) or with anti-human CD47 mAb 1F7, which was purified from hybridoma supernatants [29,30,31]
Summary
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. Thrombospondin-1 (TSP-1), a multimodular Ca2+-binding trimeric matricellular glycoprotein, is secreted by a wide array of cells such as platelets, fibroblasts, macrophages, dendritic cells, vascular smooth muscle cells, keratinocytes, epithelial cells, endothelial cells, and several cancer cells [1,2,3]. TSP-1 possesses interacting domains for a variety of proteins, which relay signals regulating a diverse range of cellular functions such as cytoskeletal organization, migration, cell-cell interaction, cell proliferation, autophagy, and apoptosis [2, 7, 8]. TSP-1 signaling via its cognate receptor CD47 has further been implicated in orchestrating cytoplasmic Ca2+ dynamics and, influencing various physiological functions [12]
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