Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance, survival, and deformability of human red blood cells

Bissinger, Rosi; Petkova-Kirova, Polina; Mykhailova, Olga; Oldenborg, Per-Arne; Novikova, Elena; Donkor, David A.; Dietz, Thomas; Bhuyan, Abdulla Al Mamun; Sheffield, William P.; Grau, Marijke; Artunc, Ferruh; Kaestner, Lars; Acker, Jason P.; Qadri, Syed M.

BACKGROUND: Thrombospondin-1 (TSP-1), a Ca(2+)-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 Ca(2+) dynamics, survival, and deformability of human red blood cells (RBCs). METHODS: Whole-cell patch-clamp was employed to examine transmembrane cation conductance. RBC intracellular Ca(2+) 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. RESULTS: Exposure of RBCs to recombinant human TSP-1 significantly increased RBC intracellular Ca(2+) levels. As judged by electrophysiology experiments, TSP-1 treatment elicited an amiloride-sensitive inward current alluding to a possible Ca(2+) influx via non-selective cation channels. Exogenous TSP-1 promoted microparticle shedding as well as enhancing Ca(2+)- 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). CONCLUSIONS: Taken together, our data unravel a new role for TSP-1/CD47 signaling in mediating Ca(2+) influx into RBCs, a mechanism potentially contributing to their dysfunction in a variety of systemic diseases.