Membrane Bending Enhances Sulfatide Mediated Sickle Mature Erythrocyte Adhesion

Introduction: Abnormal red blood cell (RBC) adhesion due to polymerization of sickle hemoglobin (HbS) is central to the pathophysiology of SCD. Early erythrocytes (reticulocytes), expanded in number due to sustained stress erythropoiesis stimulated by anemia and hypoxia, are more adhesive to vascular endothelial proteins and surfaces than are mature erythrocytes. However, mature erythrocytes typically constitute >80% of all RBCs in SCD, and the relative contribution made by erythrocytes vs. reticulocytes to acute and chronic vasculopathy is not well understood. We hypothesized that sphingomyelinase (SMase) activity that is upregulated by accumulated bending energy leads to elevated membrane sulfatide availability and thus hypoxic mature erythrocyte adhesion under hypoxia.

Methods: We probed the adhesion of RBCs obtained from SCD patients (#NCT02824471) stained with acridine orange under various physiological conditions in microchannels that were coated with laminin. Then we analyzed the morphology of single sickle reticulocytes and mature erythrocytes to estimate the membrane bending energy.

Results and Discussion: We found that mature erythrocyte adhesion to laminin in controlled microfluidic experiments is significantly greater under hypoxia than under normoxia (1856±481 vs. 78±23, mean±SEM), while sickle reticulocyte adhesion, high to begin with, does not change (1281 ±299 vs. 1258±328, mean±SEM). Furthermore, we show that among the adherent RBCs, mature erythrocytes have greater elongation and accumulated membrane bending energy compared to reticulocytes under hypoxia (Figure 1A). Accumulated membrane bending is known to elicit SMase, and laminin establishes highly specific bonds with sulfatides which are downstream of the SMase. These results suggest that SMase is likely elicited particularly in sickle mature erythrocytes, which become highly adhesive with deoxygenation. This is likely due to loss of membrane surface area with RBC maturation, which may be excessive in SCD due to membrane vesiculation and microparticle release. To elucidate the roles of SMase and sulfatide in the adhesion of mature erythrocytes, we repeated the adhesion experiments with SMase and anti-sulfatide incubation. We found that SMase increases the hypoxia-enhanced adhesion of mature erythrocytes and the anti-sulfatide antibody inhibits the enhanced adhesion of mature erythrocytes seen under hypoxia (Figure 1B). Collectively, these data demonstrate the effect of accumulated membrane damage on the abnormal adhesion of sickle mature erythrocytes.

Conclusions: Patient-to-patient variability in hypoxia enhanced mature erythrocyte adhesion suggests a role in modulating clinical comorbidities and or genetic polymorphisms relevant to red cell health and hypoxic regulation. These results also implicate the lipid components of the plasma membrane in the pathophysiology of RBC dysfunction in SCD. Therefore, sulfatide and the components of its pathway, such as SMase, could be explored as potential therapeutic targets to inhibit sickle erythrocyte adhesion in SCD.