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.
Department of Biochemistry and Molecular Biology, University of Calgary and the Foothills Hospital, Calgary, Alberta, Canada. brain@ucalgary.ca
Michael C
MC
Brain
Bernard
B
Ruether
Karen
K
Valentine
Christopher
C
Brown
Henk
H
ter Keurs
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