PMN-derived MPs interact with platelets in time-dependent and saturable manner involving α_Mβ₂ and PSGL-1 receptors. (A,B) Gel-filtered human platelets were incubated with increasing concentrations (0-50 μg/mL) of Alexa488-labeled MPs derived from resting or PMA-stimulated human PMNs for 0 to 90 minutes at 37°C. To identify platelets the samples in addition to MPs contained PE-labeled anti–human α_IIb mAbs or isotype control mouse antibody (IgG₁). On incubation, MP binding was measured by FACS, and 10 000 of α_IIb-positive events (which accounted for 98% of total events) were analyzed in every sample. (C) Gel-filtered human platelets were preincubated in the absence or presence of function-blocking mAbs to GPIbα, P-selectin, or the β₃ integrin subunit (10 μg/mL), whereas MPs (25 μg/mL final concentration) were pretreated with NIF (20 nM) or anti-α_M mAb (44a; 10 μg/mL) for 20 minutes at 37°C. Next, the cells and MPs were combined and incubated for 45 minutes at 37°C in the presence of anti–α_IIb-PE mAb and analyzed by FACS as described in panels A and B. The data are expressed as MFI (± SEM) from 4 experiments using platelets from 4 different blood donors and MPs from 2 preparations.