PSGL-1–dependent adhesion to VWF. (A) CHO cells transfected to express functionally active PSGL-1 (1 × 105 cells/mL) were added to wells coated with VWF or BSA and incubated for 60 minutes at 37°C. After washing, adherent cells were visualized by light microscopy (original magnification, 400 ×). (B) CHO cells expressing PSGL-1 were incubated in the absence or presence of Nk-protease (5 μg/mL) for 20 minutes at room temperature and subsequently were added to immobilized VWF. After 60 minutes, wells were gently washed, bound cells were lysed with 1% Triton-X100/50 mM acetic acid (pH 5.0), and endogenous alkaline phosphatase activity was determined with PNP as a substrate. Relative adhesion was plotted with adhesion to VWF as a reference (100%, representing 15% of the total added cells). Data are corrected for adhesion to uncoated wells (less than 15% of VWF-coated wells) and represent the mean ± SEM of 3 experiments performed in duplicate. (C) Freshly isolated, nonstimulated PMNs were incubated in the absence or presence of Nk-protease (5 μg/mL) or antibody (10 μg/mL) for 20 minutes at room temperature. Cells (5 × 105 cells/mL) were then perfused over VWF-coated coverslips at a shear rate of 50 s–1. The amount of transient (less than 3 seconds) contact was determined by videoimaging analysis. Bars represent the relative numbers of transient contact (cumulative after 6 minutes) using non–Nk-treated PMNs as a reference (100%). Data are corrected for adhesion to PVP-coated coverslips (less than 2 cells/mm2 after 6 minutes) and represent the mean ± SEM of 5 experiments performed in duplicate.
