Crosslinking of the nearest membrane protein neighbors in ATP depleted, calcium enriched and irreversibly sickled red cells.

ATP depleted or Ca2+ (0.1 mM) enriched normal red cells (greater than 80% echinocytes III) subjected to crosslinking by catalytic oxidation contain a greater than 1,000,000 daltons spectrin rich polymer cleavable by dithiothreitol (DTT) reduction. Similar complex is seen after glutaraldehyde crosslinking suggesting spectrin rearrangement into closer contacts or aggregation. In addition, a non-reducible greater than 1,000,000 daltons polymer is produced in fresh rbc or ghosts by high (greater than 0.5 mM) Ca2+ conc. and ionophore A23187. This complex is attributed to endogenous membrane protein crosslinking, catalyzed by a Ca2+ stimulated rbc transglutamidase. ISCs exhibiting a 4 fold increase in Ca2+ and a decrease in ATP do not exhibit these polymers. However, ISCs have an increased propensity to form the spectrin rich polymer during a subsequent ATP depletion and this is associated with a transformation of greater than 60% ISCs into spheroechinocytes. Similar cells are occasionally noted (greater than 4%) in the densest ISC rich fractions separated from fresh blood. We conclude that neither Ca2+, ATP dependent spectrin aggregation nor a spontaneous irreversible crosslinking underlie the membrane lesion of ISCs. Accelerated calcium gain and ATP depletion in ISCs leads to spectrin rearrangement and transformation of ISCs into spheroechinocytes which may represent an end stage ISC lesion resulting in an ISC removal from circulation.