Modulation of Erythrocyte Adhesion by Changes in Cellular Tonicity and Volume.
Authors: Wandersee Nancy J. , Punzalan Rowena C. , Sillah Nyama M. , Rettig Michael P. , Kennedy Michael D. , Scott J. Paul , Low Philip S. , Hillery Cheryl A. 
Published: October 2019

The sickle red blood cell (SS-RBC) membrane manifests many abnormal properties, including oxidative damage of membrane proteins and lipids, disruption of the lipid bilayer, abnormal clustering of surface proteins, dehydration, and increased adhesive properties. Conditions that alter the RBC membrane may modify or abnormally expose its native components and contribute to the adhesive properties of the RBC. We have previously reported that sickle RBCs have enhanced adhesion to the plasma and subendothelial matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. Since a significant proportion of sickle RBCs are inherently dehydrated, in part due to intracellular K+ loss via the Gardos and K-Cl co-transport channels, we test the hypothesis that dehydration-induced alterations in membrane organization or composition contribute to sickle cell adhesion in sickle cell disease (SCD). To examine the role of RBC hydration in adhesion to the subendothelial matrix protein thrombospondin-1 (TSP), normal and sickle RBCs were incubated in buffers of varying tonicity and tested for adhesion to immobilized TSP under flow conditions. Expanding on our previous preliminary results, we found that sickle RBCs exhibited a decrease in TSP binding with increasing cell hydration (p<0.005), suggesting that cellular dehydration may contribute to its adhesive phenotype. Consistent with this hypothesis, normal RBCs showed an increase in TSP adhesion with increasing dehydration (p<0.01). Furthermore, TSP adhesion of both sickle RBCs and dehydrated normal RBCs was inhibited by the anionic polysaccharides chondroitin sulfate A and high molecular weight dextran sulfate, but not by the anionic polysaccharide chondroitin sulfate C. TSP adhesion of sickle and dehydrated normal RBCs in our in vitro system was also not inhibited by competitors of CD47-, band 3-, or RBC phosphatidylserine-meditaed adhesion. Increased TSP adhesion of normal RBCs could also be induced by isotonic dehydration using nystatin-sucrose buffers; this adhesion was also inhibited by chondroitin sulfate A and high molecular weight dextran sulfate but not by chondroitin sulfate C. More importantly, we found increased adhesion of nystatin-sucrose dehydrated normal mouse RBCs, as compared to untreated normal murine RBCs, to kidney capillaries following reinfusion in vivo. In summary, these findings demonstrate that changes in hydration can significantly impact adhesion, and suggest that changes in volume are more important than changes in intracellular tonicity in the adhesion of RBCs to TSP. There are striking similarities in the adhesive properties of sickle and dehydrated normal RBCs, supporting the theory that adhesion of sickle RBCs arises as the result of exposure or modification of normal erythroid membrane components. Characterization of the membrane epitope(s) responsible for adhesion of dehydrated normal erythrocytes to TSP may thus provide a parallel avenue to identify reagents that can inhibit RBC adhesion and resultant vasoocclusion in SCD.