P-selectin and VWF string formation. (A) Wells in an enzyme-linked immunosorbent assay plate were coated with either 20 μg/mL purified P-selectin (R&D Systems) or 1% BSA. U937 cells, which express P-selectin ligand PSGL-1, were resuspended at 10⁶/mL, preincubated with buffer (Ctrl) or a polyclonal antibody against P-selectin (Ab) at 5 μg/mL or 10 μg/mL, and adhesion assays were performed as described in “U937 cell adhesion assay.” Results indicate the mean plus or minus SD for quadruplicate wells. (B) Perfusion assays were performed at a shear stress of 2.5 dyne/cm². Numbers of VWF string formed under control conditions (Ctrl), with P-selectin antibody (Ab), or with purified P-selectin (P-selectin) at the indicated concentrations (μg/mL) are shown. (C) Perfusion assays were conducted in Ca²⁺- and Mg²⁺-free DPBS at a shear stress of 2.5 dyne/cm², and the numbers of VWF strings (■) and platelet-decorated VWF strings (□) are shown as the mean plus or minus SEM from 10 fields per experiment. Cells were treated with no agonist, or with 100 μM of histamine in the absence (Ctrl) or presence of anti–P-selectin antibody (Ab) or soluble P-selectin at the indicated concentrations (μg/mL). Each experiment was performed 3 times.