The relationship between in vivo generated hemoglobin skeletal protein complex and increased red cell membrane rigidity

Abstract

In vitro induced oxidative damage to normal human RBCs has previously been shown to result in increased membrane rigidity as a consequence of the generation of a protein complex between hemoglobin and spectrin. In order to determine if in vivo generated hemoglobin-spectrin complexes may play a role in increased membrane rigidity of certain pathologic red cells, we measured both these parameters in membranes prepared from hereditary xerocytosis (Hx), sickle cell disease (Sc), and red cells from thalassemia minor (beta thal). Membranes were prepared from density-fractionated red cells, and membrane deformability was measured using an ektacytometer. Hemoglobin-spectrin complex was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis, as well as by Western blot analysis using a monoclonal antibody against the beta- subunit of hemoglobin. For these three types of pathologic red cells, progressive cellular dehydration was associated with increased membrane rigidity and increased content of hemoglobin-spectrin complex. Moreover, the increase in membrane rigidity appeared to be directly related to the quantity of hemoglobin-spectrin complex associated with the membrane. Our findings imply that hemoglobin-spectrin complex is generated in vivo, and this in turn results in increased membrane rigidity of certain pathologic red cells. The data further suggest that oxidative crosslinking may play an important role in the pathophysiology of certain red cell disorders.

‘Gardos Channelopathy’: a variant of hereditary Stomatocytosis with complex molecular regulation

“…The history of the patients reported here confirms that congenital hemolytic anemia due to abnormal cation permeability may represent a diagnostic challenge that is now overcome by means of the new Next Generation Sequencing technology approaches. Patient II.4 received a diagnosis of hereditary spherocytosis in infancy and was splenectomized without clinical improvement; the finding with SDS-PAGE of a spectrin deficiency may be considered a secondary effect of a membrane perturbation 30 , 31 disclosed by splenectomy 32 , since it is absent in the daughter, and no mutations in the spectrin or other related genes were detected by whole exome sequencing. The phenotypic variability, the possible high frequency of de novo variants, and the absence of specific biological tests make the diagnosis of this variant of hereditary stomatocytosis particularly difficult.…”

Mechanisms tagging senescent red blood cells for clearance in healthy humans

“…This suggests that the levels of deoxyhemoglobin at the membrane surface exceed those in the cytosol. Furthermore, aging of RBC is associated with an increase in spectrin-hemoglobin complexes (Snyder et al, 1983 ) which contribute to an increased rigidigty of senescent RBC in dense fractions (Fortier et al, 1988 ). Accumulation of hemoglobin at the membrane surface and its auto-oxidation results in production of superoxide anion.…”

Proteome of Stored RBC Membrane and Vesicles from Heterozygous Beta Thalassemia Donors

“…The genetic regulation of redox balance in βThal + subjects consists of interesting modifications in the transcript levels of several redox regulators that could be associated with changes in the erythrocyte proteome [ 6 ]. It has been shown that an excess of Hb chains can bind to spectrin, generating a spectrin-globin complex that increases the rigidity of the RBC membrane [ 7 ]. Moreover, several structural and functional alterations have been observed in the most abundant membrane protein with a critical role in gas transport and RBC structure and metabolism, band 3: extensive phosphorylation [ 8 ], cleavage by caspase-3 [ 9 ], and increased anion exchange [ 10 ].…”