Measurement of membrane phospholipid asymmetry in normal and sickle-cell erythrocytes by means of annexin V binding.
We investigated the use of annexin V (placental anticoagulant protein I), a calcium-dependent protein that binds tightly to phosphatidylserine-containing membranes, as a means to measure membrane phospholipid asymmetry in human erythrocytes. Iodine 125-labeled annexin V bound to erythrocytes in a specific, reversible, calcium-dependent reaction (dissociation constant = 25 +/- 4 nmol/L at 37 degrees C and 2.5 mmol/L calcium). Lysed erythrocytes contained 1.2 x 10(6) binding sites for annexin V. Treatment of erythrocytes with 1 mumol/L A23187 in the presence of 2.5 mmol/L calcium at 37 degrees C caused a gradual but marked increase in annexin V binding sites, reaching a level of approximately 300,000 sites per cell after 8 hours of incubation. We also noted a very gradual spontaneous exposure of annexin V binding sites during storage of purified erythrocytes, reaching a level of approximately 20,000 sites per cell after 30 days. Measurements could also be made directly on diluted whole blood specimens. In samples freshly drawn from 35 normal donors, a mean number of 276 sites per cell were present; this increased to 858 sites per cell after storage of specimens at 4 degrees C for 24 hours. Measurement of annexin V binding to samples from patients with sickle-cell anemia revealed a marked increase in binding (mean of 12,430 sites per cell for all samples); serial measurements in a patient hospitalized with sickle-cell crisis showed a progressive decline in annexin V binding over a period of 6 days.(ABSTRACT TRUNCATED AT 250 WORDS)
Tait
J F
JF
Department of Laboratory Medicine, University of Washington, Seattle 98195.
Gibson
D
D
D017304:Annexin A5 / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D008563:Membrane Lipids / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D012016:Reference Values; D012805:Sickle Cell Trait / Q000097:blood*
D017304:Annexin A5; D008563:Membrane Lipids; D010718:Phosphatidylserines; D010743:Phospholipids; D000001:Calcimycin; D002118:Calcium