MPs derived from PMA-stimulated PMNs activate platelets. (A,B) Gel-filtered human platelets (6 × 10⁶ in 150 μL) were incubated with increasing concentrations (0-100 μg/mL) of MPs as indicated in Tyrode buffer containing 1 mM Ca²⁺ and PE-conjugated mAb to human P-selectin or its isotype control (IgG₁-PE) for 1 hour at 37°C. After incubation, the platelets were fixed and analyzed by FACS. (C) The experiments were performed as described in panels A and B, but in addition to resting or PMA MPs, platelets were stimulated with human thrombin (1 U/mL), ADP (10 μM) for 0 to 60 minutes at 37°C. (D) Human platelets were incubated with MPs (50 μg/mL) and PAC-1-FITC mAb (filled histograms) or FITC-labeled mouse isotype control (IgM; open histogram) as described above. PAC-1 binding was analyzed by FACS. (E) Gel-filtered human platelets were preincubated in the absence or presence of function-blocking mAbs to GPIbα or P-selectin or both or normal mouse IgG (10 μg/mL), whereas MPs (50 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 in the presence of PE-conjugated mAb to human P-selectin or its isotype control and incubated for 30 minutes at 37°C. The data are expressed as MFI (± SEM) from 3 separate experiments using platelets from 4 different blood donors and MPs from 3 preparations.