Thrombospondin-1 Stimulates Calcium Influx and Echinocytosis in Sickle Cell-Derived Red Blood Cells

Abstract

Thrombospondin-1 (TSP1) is a secreted matricellular protein found preformed in platelet α-granules and upregulated in the plasma of patients with sickle cell disease (SCD). In preclinical models TSP1 enhances sickle red blood cell (RBC) binding to endothelial cells, while in murine sickle mice TSP1 promotes RBC echinocytosis and vaso-occlusion. Recently we reported elevated plasma TSP1 was associated with vaso-occlusive complications in SCD patients. Herein we tested the hypothesis that TSP1 compromises RBC membrane integrity via enhanced calcium influx leading to morphological changes, such as echinocytosis. RBC were collected via centrifugation of heparinized blood from human HbAA donors (n=3), SCD patients with homozygous HbSS SCD (n=4), and wild type C57BL/6J mice (n=2). Cells were then re-suspended in glucose and calcium-supplemented PBS, and treated with several concentrations of exogenous TSP1 (2.75 nM, 11 nM, 22 nM and 44 nM) for 2 hours at 37°C. RBC morphology was assessed by both light and electron microscopy. TSP1 modulation of RBC calcium influx was studied using the calcium fluorophore Fluo-3, AM. TSP1 treatment, in a dose-dependent manner, stimulated echinocytosis in human SCD and non-SCD RBC and in murine cells. In some instances electron microscopy showed echinocyte membrane projections with a string-of-beads appearance, suggestive of imminent microparticle shedding. TSP1 treatment, again in a dose-dependent manner, also significantly increased calcium influx in SCD (Figure, right panel) and non SCD RBC (Figure, left panel) and murine RBC (not shown). Interestingly, SCD RBC displayed more sensitivity to TSP1-mediated increases in cellular calcium as compared to normal cells. Our results show for the first time that TSP1, at doses found in patient plasma, stimulates significant echinocytosis and calcium influx in both normal and SCD RBC. These data identify a novel role for plasma TSP1 in promoting RBC pathology. Future studies are underway to elucidate this mechanism and its pathogenicity in humans with and without SCD. [Display omitted] DisclosuresIsenberg:Vasculox: Membership on an entity’s Board of Directors or advisory committees; Radiation Control Technologies: Membership on an entity’s Board of Directors or advisory committees.