Effect of neutrophil granule secretion on P-selectin binding.
(A) Specific binding of 125I-labeled P-selectin (1 μg/mL, final concentration) to unstimulated neutrophils (2 × 107/mL, final concentration) at 22°C was measured in the presence of 1 mM Ca++ (NPH) or 1 mM EDTA (EDTA). Neutrophils were stimulated for 15 minutes with 5 μg/mL cytochalasin B (cytB), 30 minutes with 0.1 μM fMLP, or sequentially with 5 μg/mL cytochalasin B then 0.1 μM fMLP (cytB+fMLP), washed, and resuspended at 2 × 107/mL before measuring binding of125I-labeled P-selectin in the presence of 1 mM Ca++. Results shown represent the mean and standard deviation of 3 separate experiments performed with different donors. (B) Neutrophils were treated as in panel A. Some samples were treated with elastase inhibitor (EI) or chymostatin (CS) alone or in combination before the addition of cytochalasin B and fMLP. All samples were lysed in buffer containing Triton X-100. Neutrophil lysates were electrophoresed on SDS–polyacrylamide gels, transferred to nitrocellulose, and probed with either PL-1 (upper panel) or PL-2 (lower panel). Data are representative of at least 3 experiments with different donors.
