Hemoglobin S Polymerization Can be Inhibited By Alterations in Erythrocyte Glycolysis

Sickle cell disease (SCD) is caused by the polymerization of deoxy Hemoglobin S (HbS) resulting in lifelong complications including anemia, infections, stroke, tissue damage, organ failure, intense painful episodes, and premature death. Each year more than 300,000 children are born with SCD, and greater than 95% of these children live in resource-poor countries. Although bone marrow transplantation and/or gene therapy are curative or potentially curative treatments for SCD, these options are not available for the vast majority of people with SCD. The most effective palliative treatment for SCD is hydroxyurea, a drug that was approved by the FDA in 1998. Clearly there is a global need for low cost, portable treatments for SCD.

We have previously shown that erythrocytes sense oxygen through reversible binding of deoxy Hb to the cytoplasmic domain of Band 3 using 3 lines of transgenic mice: 1) a mouse in which the mouse Hb/glycolytic enzyme binding region of Band 3 is replaced with the equivalent human sequences (humanized), 2) a humanized mouse lacking the Band 3 Hb binding site (-Hb) and 3) a humanized mouse with a deletion of glycolytic enzyme binding sites, resulting in increased affinity for deoxy Hb (HI affinity). Complete blood counts of these animals demonstrated only a mild increase in the MCV for the -Hb mice (p<0.03) compared to the humanized control. In the humanized control mouse, under oxygenated conditions glycolytic enzymes associate with Band 3, inhibiting glycolysis. In deoxy conditions, deoxy Hb displaces the glycolytic enzymes, activating glycolysis. In the -Hb mouse, the glycolytic enzymes were permanently bound to Band 3 (inactive glycolysis), while in the HI affinity mouse, the glycolytic enzymes were constitutively in the cytoplasm. (Chu et al. Blood 128:2708-16, 2016, Zhang et al. JBC 284:2519-25, 2019, Zhou et al. Sci Adv. 5:4466, 2019).

To determine whether the alteration in glycolysis had effects on the polymerization of HbS, each of the mutant mouse strains were crossed to the Townes SCD mouse model. The end result was 3 different strains of animals that were 1) homozygous for one of the Band 3 mutations, 2) homozygous for the human alpha globin genes and 3) either HbA/HbA, HbA/HbS or HbS/HbS. Changes in CBC were related to the beta globin genotype. An elevated MCV was observed in the -Hb animals of all 3 genotypes (p<0.02).

We compared the rate of sickling of S/S cells (Li et al. PNAS 114 (5) E689-E696, 2017) and showed that the -Hb S/S mice had a 16% increase in both the absolute number of sickled cells and the rate of appearance of the sickled cells relative to the humanized control (p<0.02). In contrast, sickling of HI affinity S/S cells was inhibited.

We hypothesized that alterations in glycolysis in the -Hb and HI affinity erythrocytes were responsible for the changes in HbS polymerization. To test this hypothesis, we analyzed the metabolites in erythrocytes from our mutant mice, including those with HbA/A, HbA/S and HbS/S. We found that the ADP concentration was 2-fold higher in -Hb S/S cells compared to HI affinity S/S cells (p <0.05). The relative levels of ATP were 1.5 fold lower in -Hb S/S cells compared to HI affinity S/S cells (p <0.05). Pentose phosphate pathway (PPP) intermediates were elevated in HI Affinity S/S cells compared to -Hb S/S cells, indicating that the Hi Affinity S/S mice have recovered PPP activity.

To test whether altering glycolysis chemically could alter sickling, we treated humanized S/S cells with phosphoenolpyruvate (PEP), a membrane-permeable, rate-limiting glycolytic substrate that is rapidly metabolized to lactate and/or processed up the glycolysis pathway to generate 2,3-bisphosphoglycerate, which can initiate the PPP. We found that PEP treatment led to a dose dependent suppression of sickling compared to untreated cells (p<10^-16). At a PEP concentration of 10mM, sickling was reduced by 15% compared to untreated cells with no effects on either membrane stability or MCV (p<10^-15).

In summary, we have shown that alterations in glycolysis can enhance or suppresses the sickling of red blood cells in a mouse model. Our results offer the prospect of a non-invasive, dietary therapy to potentially inhibit sickling and improve the outcome for SCD patients in resource poor areas of the world.