Rheologic impairment of sickle RBCs induced by repetitive cycles of deoxygenation-reoxygenation

The transformation of less-dense, discoidal homozygous sickle cells (HbSS) RBCs into dehydrated, rheologically impaired cells is believed to be an important factor in the pathophysiology of sickle cell disease. We investigated this process by subjecting the less-dense fraction of HbSS RBCs, which contains a low percentage of irreversibly sickled cells (ISCs), to cyclic deoxygenation-reoxygenation for 15 hours at 37 degrees C. This incubation procedure caused cell shrinkage, shifts in intracellular Na and K content, and formation of ISCs that closely resembled endogenous ISCs found in sickle blood. The viscoelasticity of the treated cells was tested using micropipette techniques to measure the membrane shear elastic modulus (mu) and the time constant for extensional shape recovery (tc); mu represents the "static rigidity" of the cells, and the product mu.tc was taken as a measure of their "dynamic rigidity". Density separation of the incubated cells showed that their rheologic impairment (ie, elevation of both static and dynamic rigidities) paralleled cellular dehydration and that the newly formed dense cells had viscoelastic characteristics very similar to those of endogenous dense cells. Rehydration by osmotic swelling tended to normalize the dynamic rigidities of dense cells but had no significant effect on their static rigidities. Thus, cellular dehydration contributes to the observed changes of viscoelasticity, although an irreversible alteration of membrane structure also appears to be involved. Dense ISCs could be formed without added calcium, implying that entry of external calcium is not an essential requirement for cellular dehydration; ISCs formed without calcium tended to be less rigid (ie, to have lower static and dynamic rigidities) than those formed with calcium. Our results indicate that the cyclic incubation procedure closely mimics RBC rheologic deterioration in vivo and thus suggest its usefulness as a model for investigating this phenomenon.