THE INSTABILITY OF THE MEMBRANE SKELETON IN THALASSEMIC RED-BLOOD-CELLS

The thalassemias are a heterogeneous group of disorders characterized by accumulation either of unmatched alpha or beta globin chains. These in turn cause the intramedullary and peripheral hemolysis that leads to varying anemia. A partial explanation for the hemolysis came out of our studies on material properties that showed that beta-thalassemia (beta-thal) intermedia ghosts were very rigid but unstable. A clue to this instability came from the observation that the spectrin/band 3 ra tio was low in red blood cells (RBCs) of splenectomized beta-thal inte rmedia patients, The possible explanations for the apparent decrease i n spectrin content included deficient or defective spectrin synthesis in thalassemic erythroid precursors or globin chain-induced membrane c hanges that lead to spectrin dissociation from the membrane during gho st preparation. To explore the latter alternative, samples from differ ent thalassemic variants were obtained, ie, beta-thal intermedia, HbE/ beta-thal, HbH (alpha-thal-1/alpha-thal-2), HbH/Constant Spring (CS), and homozygous HbCS/CS. We searched for the presence of spectrin in th e first lysate of the standard ghost preparation. Normal individuals a nd patients with autoimmune hemolytic anemia, sickle cell anemia, and anemia due to chemotherapy served as controls. Using gradient sodium d odecyl sulfate-polyacrylamide gel electrophoresis analysis, no spectri n was detected in identical aliquots of the supernatants of normals an d these control samples. Varying amounts of spectrin were detected in the first lysate supernatants of almost all thalassemic patients. The identification of spectrin was confirmed by Western blotting using an affinity-purified, monospecific, rabbit polyclonal antispectrin antibo dy. Relative amounts of spectrin detected were as follows in decreasin g order: splenectomized beta-thal intermedia including HbE/beta-thal; HbCS/CS; nonsplenectomized beta-thal intermedia, HbH/CS; and, lastly, HbH. These findings were generally confirmed when we used an enzyme-li nked immunosorbent assay technique to measure spectrin in the first ly sate. Subsequent analyses showed that small amounts of actin and band 4.1 also appeared in lysates of thalassemic RBCs. Therefore, the three major membrane skeletal proteins are, to a varying degree, unstably a ttached in severe thalassemia. From these studies we would postulate t hat membrane association of abnormal or partially oxidized alpha-globi n chains has a more deleterious effect on the membrane skeleton than d o beta-globin chains. (C) 1995 by The American Society of Hematology.