Red cell vesiculation - A common membrane physiologic event

Loss of red blood cell membrane material in the form of microvesicles has been noted in sickle cells, in Ca⁺⁺-loaded and adenosine triphosphate (ATP)-depleted normal red blood cells; and during storage of normal red blood cells. To further understand the vesiculation process, we have studied vesicles generated by a variety of perturbations of the red blood cell membrane. Vesicles were isolated by centrifugation at 30,000 x g from plasma of heparinized pathologic blood samples (sickle cell anemia, hemoglobin H disease, hereditary spherocytosis, hereditary elliptocytosis, and protein 4.1 deficiency) incubated overnight at 4° C. Vesicle formation also was induced in normal erythrocytes by ATP depletion, by heating to 49°C, by incubation at pH 5.4, and by incubation in 5 mmol/L diamide. Membrane protein composition was characterized on denaturing polyacrylamide gels and by immunoblot. The vesicles all contained band 3, glycophorin A, and band 4.1. Spectrin was depleted in all vesicles. Thiol disulfide exchange chromatography revealed evidence of oxidative cross-linking of spectrin in pathologic and normal red blood cells that had undergone vesiculation. This suggests that the mechanism of vesiculation may be related to cross-linking of membrane proteins. Membrane phospholipid composition of sickle cell and acid-induced vesicles was similar to that of normal red cells as determined by thin-layer chromatography. Possible pathophysiologic effects of vesiculation were assessed by using a modified Russell's viper venom assay. All vesicles examined shortened Russell's viper venom clotting time by 55% to 70% of control values. In addition, ektacytometer studies reveal that cells remaining after acid-induced vesiculation are rigid. These observations indicate that the vesicles may play a role in the hypercoagulation seen in some hemolytic disorders and that the process of vesiculation itself may contribute to increased rigidity of red cells and their subsequent removal from the circulation.