Archil Shartava

Irreversibly sickled cell β‐actin: Defective filament formation

It has been demonstrated that cysteine modification in irreversibly sickled cell β‐actin slows down the remodeling of membrane skeletons [Shartava et al.: J Cell Biol 128:805‐812, 1995]. This slow remodeling can be due to alterations in spectrin‐actin binding and/or actin‐actin interactions in irreversibly sickled cell (ISC) membrane skeletons. In these studies we demonstrate that ISC actin binds spectrin normally. However, ISC β‐actin polymerizes and depolymerizes more slowly than control β‐actin, and forms unusual aggregates when placed under polymerizing conditions. Electron microscopic analysis of actin polymers indicated that ISC actin generates a large amount of aggregates which we conclude are due to the structural modification caused by the disulfide bridge between cysteine264 and cysteine373 in β‐actin. Am. J. Hematol. 55:97‐103, 1997.

The Gardos Channel Is Responsible for CDNB-Induced Dense Sickle Cell Formation

The red blood cells (RBCs) derived from blood taken from homozygous sickle cell (SS) patients demonstrate densities that are inversely proportional to the intracellular reduced glutathione (GSH) content. Addition of 1 mM 1‐chloro‐2,4‐dinitrobenzene (CDNB) to low‐density sickle cells (LDSS), at 4°C, results in a shift of LDSS erythrocytes to high‐density sickle cells (HDSS), with corresponding decreases in GSH. We have previously demonstrated that this CDNB effect was due to increased K+ leakage and that dense cell formation could be inhibited by clotrimazole (specific for the Gardos channel) but not DIOA (specific for the K+–Cl− co‐transport system) at pH 7.4 (Shartava et al. Am. J. Hematol. 1999;62:19–24). Here we demonstrate that clotrimazole (10 μM) inhibits dense cell formation at pH 7.1 and 6.8, while DIOA (1 mM) has no effect. As pH 6.8 is the optimal pH for the K+–Cl− co‐transport system, we can now reasonably conclude that damage to the Gardos channel is responsible for CDNB‐induced dense cell formation. Am. J. Hematol. 64:184–189, 2000.

Preliminary characterization of a structural defect in homozygous sickled cell alpha spectrin demonstrated by a rabbit autoantibody

We have identified a rabbit autoantibody that strongly reacts with the core membrane skeleton of control red blood cells, and does not react with low‐ or high‐density sickle cell core skeletons upon indirect immunofluorescence. Western blot analysis of red blood cell membrane proteins, utilizing this autoantibody, indicated no reactivity to any protein when SDS‐PAGE was conducted in the presence of the reducing agent, dithiothreitol. However when SDS‐PAGE was performed on control red blood cell membrane proteins separated in the absence of dithiothreitol, the autoantibody specifically reacted with a high molecular weight polypeptide (apparent Mr ≊ 310 kD) representing a DTT sensitive form of control α spectrin, which we refer to as α/ spectrin. There was no staining of high density or low density sickle cell α or α/ spectrin. This autoantibody should be an excellent tool for the fine mapping of structural change(s) in control vs. sickle cell α spectrin, and determination of whether the structural alteration effects spectrin dimer‐tetramer interconversion and/or the spectrin‐actin interaction. The modification in α spectrin, detected by this antibody, is very specific for homozygous SS α spectrin because sickle cell β+ thalassemic α spectrin and sickle cell trait α spectrin react intensely with the autoantibody. Am. J. Hematol. 58:200–205, 1998.