Effect of eicosanoids on annexin-V binding to erythrocytes.
WB was diluted (1:40; vol/vol) with HEPES buffer and then CaCl2 was added (final Ca++ concentration, 1.5 mM) (“Materials and methods”). Different concentrations of either AA, U46 619, 12-HETE, PGI2, or appropriate solvent controls were added. Ca++-ionophore A23 187 and ionomycin were used as positive controls. Samples were kept without stirring (5 minutes, 22°C) and then FITC–annexin-V or PE–antiglycophorin-A antibodies were added. The samples were kept undisturbed (10 minutes, 4°C, dark) (“Materials and methods”). Samples were quench-diluted with ice-cold HEPES (with 1.5 mM Ca++) at 4°C and examined immediately by flow cytometry (“Materials and methods”). The TXA2-analog U46 619 and free AA dose dependently increased annexin-V binding to erythrocytes (n = 12). In the same time frame, Ca++-ionophore A23 187 (1 μM) induced annexin binding in 8.4% ± 1.03 erythrocytes (n = 17). Ionomycin 1 μM yielded PS exposure in 1.91% ± 0.19% of erythrocytes (n = 10). PGI2 (0.5-1 μM) or 12-HETE (0.1-1 μM) did not induce annexin-V binding (not shown). Results are expressed as mean ± SEM (%) of erythrocytes that bind annexin-V; n = number of different donors studied. *P < .05; **P < .001. Significance was determined by Studentt test.
