Molecular basis of red cell membrane rheology. Part 1.

The biorheological properties and behavior of red blood cells (RBCs), as other types of cells, have a biochemical and molecular basis. The shape maintenance and deformability of RBCs depend on the structural and functional integrity of the membrane proteins. These proteins are composed of transmembrane proteins inserted in the lipid bilayer, the skeletal proteins forming a network lining the membrane endoface, and the linking proteins which link together the other two types of proteins to form a three-dimensional protein structure to effect the complex and intricate biorheological functions of the RBC. The application of molecular biological techniques has led to the establishment of the molecular structures of all major RBC membrane proteins and generated insights into the nature and energy of protein interactions in the membrane. Abnormalities or deficiencies of these proteins in hereditary disorders in humans and animals have offered opportunities to assess the rheological significance of each of these proteins and their interactions. Parallel molecular biological and biorheological studies on RBC membranes under a variety of conditions can provide the fundamental information required for theoretical modeling of RBC membrane rheology at the molecular level. Such interdisciplinary research will contribute to not only the elucidation of normal rheology of RBCs and other types of cells, but also the understanding of pathorheology of their disorders and the development of new methods of diagnosis and treatment.

Impaired echinocytic transformation of ankyrin- and spectrin-deficient erythrocytes in mice.

The membrane skeleton of the red blood cell plays an important role in the determination of cell deformability and cell shape. Under various in vitro conditions, red blood cells undergo an echinocytic or stomatocytic shape transformation. The mechanism of this fundamental process is not well understood. We have studied the red cell shape transformation in normoblastic anemia mice (nb/nb) and spherocytic anemia mice (sph/sph), which are deficient in ankyrin and spectrin, respectively. We found that both ankyrin-deficient cells (nb/nb) and spectrin-deficient cells (sph/sph) have a reduced capacity to undergo echinocytic transformation with various echinocytogenic treatments, that is, incubation with sodium salicylate (40 and 120 mM), calcium loading (50 microM A23187 + 2.2 mM Ca2+), or metabolic depletion (24 hr at 37 degrees C). These results suggest that the functional integrity of the membrane skeleton is essential for the maintenance and transformation of the red cell shape.

Quantitative relationship between Heinz body formation and red blood cell deformability.

The ultimate cause of destruction of red blood cells (RBCs) after oxidative damage with Heinz body formation is not well understood. We correlated the changes in RBC morphology and membrane protein composition after oxidant treatment with the alterations in deformability of whole cells and cell membranes. The incubation of RBCs with phenylhydrazine concentrations of 0.3 to 100 mg/dL at 37 degrees C for one hour led to a dose-dependent formation of Heinz bodies, ranging from isolated Heinz bodies at 1 mg/dL to a confluent coating of the inner membrane surface at 100 mg/dL phenylhydrazine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a large quantity of hemoglobin bound to the ghost membrane of treated RBCs. Electrophoresis with and without dithiothreitol indicated that disulfide bridges are abundant between hemoglobin molecules and are also present among membrane proteins but are not the major bond between hemoglobin and membrane. Changes of spectrin, ankyrin, band 3, and band 6 and the appearance of a 260,000-dalton complex were also observed. With phenylhydrazine concentrations below 30 mg/dL, even in the presence of multiple Heinz bodies, the RBC deformability measured by filtration through 2.6-, 4.5-, and 6.8-microns pores and the membrane deformability determined by a filter aspiration technique were not altered. With 100 mg/dL phenylhydrazine, when the entire membrane was coated with Heinz bodies, RBC filterability and membrane deformability were drastically reduced. These results indicate that oxidative damage of RBCs with discrete Heinz body formation causes focal membrane rigidification but does not affect the global cellular deformability until the Heinz bodies nearly cover the entire cell endoface.