Life-threatening hemolytic anemia due to an autoanti-Pr cold agglutinin: evidence that glycophorin A antibodies may induce lipid bilayer exposure and cation permeability independent of agglutination.
BACKGROUND
The hemoglobin of a 29-year-old man fell below 35 g/L over 5 days, despite 14 units of red blood cells (RBCs), due to an anti-Pr cold agglutinin (CA). His hemolytic anemia necessitated respiratory support in intensive care for 4 weeks.


METHODS
The hemolysis was investigated by the effects on blood group-compatible RBCs of this anti-Pr and an anti-I CA and of a rabbit anti-human glycophorin A (GPA) immunoglobulin G (IgG) antibody on Ca(2+) permeability and of phosphatidylethanolamine (PE) exposure. 1) The anti-Pr CA (in a plasmapheresis product from the patient) was absorbed and eluted from RBC ghosts and its immunophenotype was determined by agarose electrophoresis and immunofixation. 2) Ca(2+) permeability was measured by the response of Fluo-3-labeled RBCs to addition of external Ca(2+). 3) Exposed PE was measured with streptavidin-labeled biotinylated peptide Ro 09-0198 (cinnamycin).


RESULTS
1) The patient's anti-Pr CA was a polyclonal IgG. 2) The anti-Pr and the rabbit anti-human glycophorin IgG, but not an anti-I CA, rapidly increased Ca(2+)-dependent fluorescence upon addition of external Ca(2+) in a fraction (15%-25%) of RBCs that also became positive for cinnamycin. 3) Trypsin treatment of RBCs reduced the Ca(2+) influx due to the anti-Pr IgG, but neither trypsin nor neuraminidase changed the responses to the rabbit anti-human GPA IgG.


CONCLUSIONS
The anti-Pr CA and rabbit anti-human GPA increased exposure of PE and increased membrane Ca(2+) permeability that may have caused hemolysis. The difference in the responses to these antibodies to enzyme treatment of RBCs suggests that they react with different epitopes on GPA.
Brain
Michael C
MC
Department of Biochemistry and Molecular Biology, University of Calgary and the Foothills Hospital, Calgary, Alberta, Canada. brain@ucalgary.ca
Ruether
Bernard
B
Valentine
Karen
K
Brown
Christopher
C
ter Keurs
Henk
H
D000328:Adult; D000744:Anemia, Hemolytic, Autoimmune / Q000097:blood / Q000276:immunology* / Q000628:therapy; D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D058846:Antibodies, Monoclonal, Murine-Derived; D001430:Bacteriocins / Q000493:pharmacokinetics; D001789:Blood Group Antigens / Q000276:immunology*; D001803:Blood Transfusion; D002118:Calcium / Q000097:blood*; D002463:Cell Membrane Permeability / Q000276:immunology*; D048430:Cell Shape; D003131:Combined Modality Therapy; D003450:Cryoglobulins / Q000276:immunology; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000276:immunology* / Q000648:ultrastructure; D006021:Glycophorins / Q000276:immunology*; D006461:Hemolysis / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D007075:Immunoglobulin M / Q000276:immunology; D016756:Immunoglobulins, Intravenous / Q000627:therapeutic use; D007166:Immunosuppressive Agents / Q000627:therapeutic use; D008297:Male; D008775:Methylprednisolone / Q000627:therapeutic use; D018448:Models, Immunological; D009103:Multiple Sclerosis / Q000150:complications / Q000276:immunology; D010456:Peptides, Cyclic / Q000493:pharmacokinetics; D010714:Phosphatidylethanolamines / Q000276:immunology; D010956:Plasmapheresis; D000069283:Rituximab
D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D001430:Bacteriocins; D001789:Blood Group Antigens; D003450:Cryoglobulins; D006021:Glycophorins; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D016756:Immunoglobulins, Intravenous; D007166:Immunosuppressive Agents; D010456:Peptides, Cyclic; D010714:Phosphatidylethanolamines; C493795:Pr antigen, human; C029443:cold agglutinins; C000485:cinnamycin; D000069283:Rituximab; D002118:Calcium; D008775:Methylprednisolone