Identity of TSP-4 receptors on stimulated PMNs. (A) PMA-stimulated PMNs were pretreated with function-blocking mAbs to various integrin β subunits (20 μg/mL) for 20 minutes at room temperature (RT) and seeded onto polyvinylopyrrolidone (PVP)– or TSP-4–coated plates (80 nM). Alternatively, the pretreated cells were added to the upper chamber of Boyden chambers (B). Adhesion and migration assays were performed as described in Figure 1. (C) PMNs were pretreated with increasing concentrations of NIF (right) or with blocking mAbs (left) to all members of the β₂ integrin family for 20 minutes at RT, and their binding to TSP-4 variants was estimated as described in Figure 1. (D) The adhesion assays of HEK293 cells expressing α_Mβ₂, α_Lβ₂, chimeric α_Lβ₂-containing the I-domain of α_M (α_M-I-α_Lβ₂) and control mock cells to TSP-4–coated plates (80 nM) were performed as described in Figure 1. Cell adhesion to PVP was subtracted as a background. The data are expressed as means ± SEMs of quadruplet wells from 3 blood donors. (E) Microtiter wells were coated with the α_MI-domain (5 μg/well) for 20 hours at 4°C and coated afterward with 3% BSA for 1 hour at 37°C. TSP-4 variants (0-1.5 μM) were incubated for 4 hours at 37°C in the presence of magnesium and calcium. After washing, bound TSP-4 variants were detected by an ELISA using anti–TSP-4 Ab. The data are means ± SEMs (n = 5).