Sphingosine 1-Phosphate Induces Erythrocytes Sickling in Sickle Cell Disease By Promoting Hemoglobin S Aggregation

Sickle cell disease (SCD) is a genetic hemolytic disease with high morbidity and mortality affecting millions of individuals worldwide. Although SCD was identified a century ago, we still lack effective mechanism-based therapies to treat this disease. Our recent study showed inhibition of sphingosine kinase 1 (Sphk1, the enzyme that produces S1P) significantly decreased S1P level in sickle erythrocytes, attenuated sickling cells. We also found sickling was induced by high concentration (10µM) but not low concentration (0.5 µM) of S1P, which indicates S1P induced sickling is independent of its receptors. However, the mechanism of S1P inducing sickling in SCD is unknown. Because sickling is induced by formation of polymers of deoxygenated sickle hemoglobin, we hypothesized that S1P functions intracellularly to induce sickling by directly promoting polymerization of deoxygenated HbS. To test this possibility, we determined the effects of S1P on deoxy-HbS polymerization using temperature jump method. We found that 5µM S1P began to induce aggregation of deoxy-HbS (0.25mM) with shorter delay time at 17.65 ± 0.57min compared to HbS treated with solvent (methanol) which with delay time at 19.28 ± 0.17 min. Moroever, S1P at 10µM induced deoxy-HbS aggregation with further decreased delay time at 15.71 ± 0.38 min, clearly demonstrating a dose-dependent effect. Thus, this study indicates that S1P at concentrations higher than 5µM dramatically and specifically accelerated HbS aggregation. Next, we found that aggregation amplitude (ΔA) of deoxy-HbS was significantly increased by S1P at a concentration of 5µM and was further elevated at 10mM of S1P. indicating that S1P enhanced polymerization ability of deoxy-HbS in a dosage-dependent manner. Finally, we detected the effect of S1P on deaggregation of aggregated deoxy-HbS. We found that S1P significantly reduced temperature drop-mediated unaggregated HbS and this effect was in a dosage dependent manner. In order to test whether anti-sickling drug can increase delay time of Hb S aggregation, we chose 5-HMF that was reported anti-sickling. We found 5-HMF significantly increased delay time of HbS. Taken together, we demonstrated that S1P induces HbS polymerization via three mechanisms: 1) Accelerating polymerization of HbS by shortening temperature jump-mediated delay time; 2) enhancing the aggregate formation during temperature jumping; and 3) reducing deaggregation ability during temperature dropping. Thus, this study strongly supports our in vivo animal, in vitro cellular and functional evidence that S1P contributes to sickling by promoting polymerization.