Abstract LBA-3
Sickle cell disease (SCD) is a debilitating hemolytic disorder with high morbidity and mortality affecting millions of individuals worldwide. Although SCD was first identified a century ago, we still lack effective mechanism-based safe therapies to treat this disease. Thus, identification of specific molecules triggering sickling, the central pathogenic process of the disease, is extremely important to advance our understanding of the molecular basis for the pathogenesis of SCD and to develop novel therapeutics.
Using non-biased metabolomic screening, we found that sphingosine-1-phosphate (S1P) is significantly elevated in the blood of SCD mice. Further analysis revealed that the activity of sphingosine kinase 1 (Sphk1, the enzyme that produces S1P) is significantly elevated in erythrocytes of SCD mice. Chronic treatment of SCD mice with a SphK1 inhibitor significantly attenuated sickling, hemolysis, inflammation and multiple tissue damage by reducing erythrocyte and plasma S1P levels. Erythrocyte S1P levels were further elevated following hypoxia/reoxygenation-induced acute sickle crisis (ASC) in SCD mice and blocking its elevation by a Sphk1 specific inhibitor significantly reduced hallmark features associated with ASC. As with SCD mice, we found that erythrocyte Sphk1 activity and erythrocyte and plasma S1P levels were significantly elevated in humans with SCD compared to normal individuals. Inhibition of SphK1 in cultured primary human erythrocytes isolated from SCD patients inhibited hypoxia-induced elevation of erythrocyte S1P levels and reduced sickling. Thus, we have revealed for the first time that SphK1-mediated S1P elevation in SCD erythrocytes is a key contributor to sickling in SCD and that Sphk1 inhibition can attenuate both acute and chronic sickling events and disease progression.
S1P is an important signaling molecule regulating diverse biological processes. Although S1P is predominantly produced and stored in RBCs, nothing was known about the physiological role of S1P in normal RBCs or the pathophysiological role of S1P in SCD until we conducted a metabolomic screen. In an effort to determine the molecular mechanism underlying S1P-induced sickling, we unexpectedly found that S1P directly binds with Hb and results in a reduced Hb-O2 affinity. This finding led us to further discover that 2,3-diphosphoglycerate, another erythrocyte specific allosteric modulator, is required for S1P-mediated allosteric modulation and that these two endogenous heterotropic modulators work cooperatively to induce a substantial reduction in Hb-O2 affinity. Supporting the biochemical and functional findings, molecular modeling predicts that S1P binds near the water filled central cavity of HbA at a site that is different from the Hb-2,3-DPG binding site. Thus, our discovery adds a significant new chapter to erythrocyte physiology by revealing S1P is a novel allosteric modulator of Hb-O2 affinity and also providing a mechanism underlying S1P-mediated sickling by promoting the formation of deoxyHbS. Thus, the work reported here could be the foundation leading to future human trials and a possible therapy for SCD, a life-threatening hemolytic disorder for which the current treatment is extremely limited.
The significance of our findings extends well beyond SCD. Our findings reveal a previously unrecognized important role for S1P in erythrocyte physiology and indicate a new concept for the regulation of O2 release from Hb under normal and sickle cell disease conditions. For SCD, elevated S1P is detrimental because reduced Hb-O2 affinity leads to more deoxygenation of HbS, increased sickling and subsequent multiple life-threatening complications. However, for normal erythrocytes, elevated S1P is likely beneficial by decreasing Hb-O2 affinity allowing for more O2 release to hypoxic tissues. Thus, for humans with normal Hb, if elevated S1P can induce O2 release to hypoxic tissues it may be a novel therapeutic target for a range of disorders, from chronic heart failure to diabetic retinopathy, traumatic blood loss, pulmonary disease and even cancer. In this way our findings reveal important novel opportunities to treat and prevent not only SCD but also multiple cardiovascular and pulmonary diseases associated with hypoxia. Thus, the impact of our novel finding is significant and enormous.
Disclosures:
No relevant conflicts of interest to declare.
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