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A B S T R A C T Since unstable hemoglobins have been considered a source of reactive oxygen radicals, and oxidative membrane damage a prehemolytic event,we examined the erythrocyte membranes of six patients (three splenectomized) with hemoglobin Koln disease. In the hydrogen peroxide stress test, the patients' erythrocytes generated more than twice the malonyldialdehyde (a lipid peroxidative product) than control erythrocytes. Fluorescence spectra of lipid extracts of the patients' erythrocytes showed an excitation maximum at 400 nm and an emission maximum of 460 nm, characteristic of malonyldialdehyde lipid adducts. Two types of membrane polypeptide aggregates were found in the erythrocytes of the splenectomized patients. The first, which were dissociable by treatment with mercaptoethanol, contained disulfidelinked spectrin, band 3 and globin. The second, not dissociable by mercaptoethanol, had an amino acid composition similar to that of erythrocyte membranes and spectrin (unlike globin) and like that of aggregates produced by the action of malonyldialdehyde on normal erythrocyte membranes. Atomic absorption spectroscopy of hemoglobin Koln erythrocytes showed no increase in calcium content implying that these cross-links were not due to calcium-stimulated trans aggregates from splenectomized patients were less deformable while aggregate-free erythrocytes from nonsplenectomized patients had normal deformability. We conclude that the erythrocyte membranes in hemoglobin Koln disease show evidence of lipid peroxidation with production of malonyldialdehyde, and that the nondissociable membrane aggregates formed in this disease are likely cross-linked by malonyldialdehyde. Because the erythrocytes containing membrane aggregates from splenectomized patients with unstable hemoglobin disease show decreased membrane deformability, we hypothesize that this abnormality results in premature erythrocyte destruction in vivo.

A case of thrombotic thrombocytopenic purpura (TTP) in a human immunodeficiency virus (HIV)-seropositive homosexual man is reported. The patient improved after corticosteroid and plasma exchange therapy was instituted. The case demonstrated most of the classic features of TTP, including histologic evidence of fibrin thrombi in small blood vessels. Atypical features included reduced number of megakaryocytes in the bone marrow and only partial resolution of thrombocytopenia after therapy was begun. An autoimmune thrombocytopenia has been well-characterized in HIV-antibody-seropositive homosexual men. Physicians caring for HIV-seropositive persons should also be aware of a possible association with TTP.

A B S T R A C T Erythrocytes from patients with chronic hemolytic variants of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency have structural membrane protein abnormalities accompanied by decreased cell membrane deformability which we postulate represent the consequences of oxidant-induced membrane injury. To evaluate the pathophysiologic significance of oxidant-induced membrane injury, we studied the in vitro and in vivo effects of the thiol-oxidizing agent, diamide, on dog erythrocytes. In vitro incubation ofdog erythrocytes with 0.4 mM diamide in Tris-buffered saline for 90 min at 370C resulted in depletion of GSH, formation of membrane polypeptide aggregates (440,000 and >50,000,000 daltons) and decreased cell micropipette deformability, abnormalities similar to those observed in the erythrocytes of patients with chronic hemolytic variants of G-6-PD deficiency. In addition, diamide-incubated cells had increased viscosity and increased membrane specific gravity, but no change in ATP. Reinjection of 51Cr-labeled, diamideincubated cells was followed by markedly shortened in vivo survival and splenic sequestration. Further incubation of diamide-incubated cells in 4 mM dithiothreitol reversed the membrane polypeptide aggregates, normalized micropipette deformability, decreased cell viscosity, prolonged in vivo survival, and decreased splenic sequestration.These studies demonstrate that diamide induces a partially reversible erythrocyte lesion which is a useful model of oxidant-induced membrane injury. They sug-

To investigate the membrane abnormalities that may play a pathophysiologic role in several hemolytic anemias we determined the density distribution on sucrose density gradients of human red blood cell (RBC) membranes from patients with these disorders, from normal controls, and from incubated normal RBC. We analyzed the fractions for membrane-adsorbed hemoglobin (Hb), globin, and nonglobin cytoplasmic proteins. The relationship between the cytoplasmic proteins adsorbed on the membranes and the specific gravity (SG) of the membranes was linear. An increase in SG of the entire membrane population was seen in Hb C disease due to adsorbed Hb. Subpopulations of membranes with increased SG due to adsorption of nonglobin protein were evident in the membranes from two splenectomized patients with hemolytic glucose-6- phosphate dehydrogenase (G6PD) variants. Dense membrane subpopulations found in RBC membranes from three splenectomized patients with Hb Koln were associated with adsorbed globin, while similar subpopulations in RBC membranes from three splenectomized patients with hereditary spherocytosis demonstrated increased SG due to adsorbed Hb. Splenectomized normals had no such abnormality in membrane density. Sucrose density gradients demonstrate that membrane bound cytoplasmic protein is characteristic of the RBC membranes in several hemolytic disorders. Additionally, gradients are useful for the isolation and further analysis of those subpopulations of RBC membranes with abnormal SG and exaggerated membrane protein abnormalities.