Role of Band 3 Tyrosine Phosphorylation in Causing the Vaso-Occlusive Events in Sickle Cell Disease

Background: Sickle cell disease (SCD) derives from a mutation in sickle hemoglobin (HbS) that leads to vaso-occlusive events that eventually cause organ failure. Based on observations from multiple labs, we hypothesize that the events linking HbS denaturation to vaso-occlusion are: HbS denaturation→oxidative stress→inhibition of erythrocyte tyrosine phosphatases→enhanced tyrosine phosphorylation of band 3→dissociation of the spectrin/actin cytoskeleton from the membrane→release of membrane-derived microparticles (MPs) and discharge of cell-free HbS→induction of microthromboses and activation of the vascular endothelium→vascular occlusion. In the studies below, we document each step of this pathway and demonstrate that inhibitors of Band 3 tyrosine phosphorylation block all downstream events. Methods: Band 3 tyrosine phosphorylation, plasma MP counts, plasma cell-free Hb concentration, sickle cell deformability, sickle cell flow through micro-capillaries, and adhesion of sickle erythrocytes to activated endothelial cells are quantitated using fresh sickle blood both before and after incubation with inhibitors of Band 3 tyrosine phosphorylation. Findings: Imatinib and other Syk kinase inhibitors block Band 3 tyrosine phosphorylation, suppress release of membrane MPs, reduce discharge of cell-free HbS, improve sickle cell deformability, enhance flow of sickle cells through microcapillaries, and reduce adherence of sickle cells to heme-activated endothelium. Interpretation: Inhibition of Band 3 Tyrosine phosphorylation restores membrane integrity and thereby suppresses the downstream sequelae that lead to vaso-occlusive events in SCD. Inhibitors of Band 3 tyrosine phosphorylation may therefore constitute a novel effective therapy for SCD. Funding Statement: NIH grants R01GM24417-40 (P.S.L.), 1RF1 NS110049-01 (J.W.), R01HL133574 (U.A.G. and J.A.L.), T32HL134622 (R.A.), and NSF #1552782 (U.A.G.). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All sickle cell blood samples were obtained following informed consent using procedures approved by the local IRBs.