MOLECULAR DEFECTS WITHIN THE SPECTRIN MEMBRANE SKELETON OF SICKLED CELLS

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(Adapted from the Applicant's Abstract) During the current funding cycle, the investigators have demonstrated that a major cause of the slow dissociation of the irreversibly sickled cell (ISC) membrane skeleton, and resultant inability of the ISC to model, is a single post-translational modification in ISC beta-actin. In ISC beta-actin a disulfide bridge is formed between cysteine 284 and cysteine 373 which is not found in reversibly sickled cell (RSC) or control-beta actin. However, spectrin was also demonstrated to contribute to the slow dissociation of the IS core skeleton, although its structural defect remains to be determined. Furthermore, the investigators have demonstrated that the reducing agents dithiothreitol (DTT) and N-acetylcysteine both have the ability to inhibit ISC formation in vitro. In the proposed grant period the investigators will determine any structural and functional modification in the actin binding domain of beta spectrin (Aim 1); utilize a rabbit autoantibody which reacts with control alpha spectrin but not ISC alpha spectrin to define the structural (and functional) modifications that exist in alpha spectrin (Aim 2); and begin human trials to determine whether N- acetylcysteine can reduce the number of dense cells and ISC in patients with sickle cell anemia and whether this reduction will lead to less sickle cell crises per year (Aim 3).