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

Background: Thrombospondin-1 (TSP-1), a Ca-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 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 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 levels. As judged by electrophysiology experiments, TSP-1 treatment elicited an amiloride-sensitive inward current alluding to a possible Ca influx via non-selective cation channels. Exogenous TSP-1 promoted microparticle shedding as well as enhancing Ca- 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 influx into RBCs, a mechanism potentially contributing to their dysfunction in a variety of systemic diseases. Video abstract.