Membrane bending and sphingomyelinase-associated, sulfatide-dependent hypoxic adhesion of sickle mature erythrocytes

Abstract

Abnormal erythrocyte adhesion due to polymerization of sickle hemoglobin is central to the pathophysiology of sickle cell disease (SCD). Mature erythrocytes constitute >80% of all erythrocytes in SCD, and the relative contributions made by erythrocytes to acute and chronic vasculopathy in SCD are not well understood. Here, we show that the bending stress exerted on the erythrocyte plasma membrane by the sickle hemoglobin polymerization under hypoxia enhances sulfatide-mediated abnormal mature erythrocyte adhesion. We hypothesized that sphingomyelinase activity that is upregulated by accumulated bending energy leads to elevated membrane sulfatide availability and thus hypoxic mature erythrocyte adhesion. 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 (early erythrocyte) adhesion, high to begin with, does not change (1281 ±299 vs. 1258±328, mean±SEM). We show that greater mean accumulated bending energy of adhered mature erythrocytes is associated with higher acid SMase activity and increased mature erythrocyte adhesion (p=0.022, for the acid SMase activity and p=0.002 for the increase in mature erythrocyte adhesion with hypoxia, N=5). In addition, hypoxia results in sulfatide exposure on the erythrocyte membrane, and sphingomyelinase increases while anti-sulfatide inhibits the enhanced adhesion of erythrocytes. These results suggest that lipid components of the plasma membrane contribute to the complications in SCD. Therefore, sulfatide and the components of its upregulation pathway, particularly sphingomyelinase should be further explored as potential therapeutic targets to inhibit sickle erythrocyte adhesion.

Microfluidic concurrent assessment of red blood cell adhesion and microcapillary occlusion: potential hemorheological biomarkers in sickle cell disease

“…For instance, our recent published study shows that sickle reticulocytes and sickle mature RBCs bear different adhesion characteristics under hypoxia, which is membrane-sulfatide dependent and varies from patient to patient. 94 Third, manual counting of RBC adhesive and occlusive events in the microchannel greatly hinders the OcclusionChip potential for clinical translation. Future work will prospectively focus on demonstrating the clinical utility of the functionalized OcclusionChip assay as an in vitro therapeutic benchmark in a larger patient population with homozygous SCD, under normoxic and hypoxic conditions, and monitoring diverse treatments including conventional, targeted, or curative therapies.…”

Motion Blur Microscopy

“…HbSS whole blood was diluted 1:4 with PBS for the dilution experiment. Hypoxic conditions (SpO2 of approximately 83% in the blood sample), using a micro-tube gas exchanger as we described previously 23 . To prevent non-specific cellular interactions, microchannels were incubated with bovine serum albumin at 4°C overnight, and additionally, 250 μ l of bovine serum albumin was injected into the microchannels at room temperature with a 5 μ l/min flow rate 2 hours prior to the experiments.…”

“…MBM also has the potential of revealing more subtle dynamical relationships that have not been systematically explored, for example how morphological features of cells are correlated with dynamical behaviors at the single-cell level. For example, a longstanding question about sickle red blood cells is whether elongated, irreversibly sickled cells are more adhesive to endothelium than sickle discocytes 23 . In Fig 8 a, we show MBM data for the relationship between average sickle red blood cell eccentricity (calculated over the entire cell trajectory) and adhesion duration to laminin, revealing a significant negative trend ( p < 0.03, linear regression): cells with longer adhesion durations are slightly less elongated on average.…”

Metabolite and protein shifts in mature erythrocyte under hypoxia

“… 53 , 81 , 82 A recent finding indicated that the polymerization of HbS added bending stress on the erythrocyte membrane. 83 Upregulated sphingomyelinase caused erythrocytes to produce higher-than-normal levels of sulfatides, promoting abnormally adherent. Meanwhile, the increase in plasma Hb and free heme during hypoxia activates the vascular endothelium, causing sickle erythrocyte adhesion 84 and increased adhesion to laminin.…”