α_M^−/− MPs have an impaired ability to interact and activate platelets. (A) Gel-filtered mouse WT platelets were incubated with increasing concentrations (0-50 μg/mL) of Alexa488-labeled MPs derived from resting or PMA-stimulated WT or α_M^−/− PMNs for 1 hour at 37°C. MPs binding was analyzed by FACS as described in Figure 3A with one exception that PE-labeled rat anti–mouse α_IIb antibody was used to identify platelets. The data are MFI (± SEM) from 3 experiments. (B) Gel-filtered mouse WT platelets (4 × 10⁷ in 200 μL) were incubated in Tyrode buffer containing 1 mM Ca²⁺ in the absence or presence of MPs derived from resting (25 μg/mL each) or PMA-stimulated (5 and 25 μg/mL) mouse WT or α_M^−/− PMNs for 30 minutes at 37°C. After this incubation, platelets were lysed and Akt activation was analyzed as described in Figure 5A. (C,D) Mouse WT platelets were incubated in the absence or presence of WT or α_M^−/− MPs as indicated (at 50 μg/mL each) in Tyrode buffer containing 1 mM Ca²⁺ and FITC-conjugated mAb to mouse P-selectin or its isotype control (IgG₁) for 45 minutes at 37°C. The data represent MFI (± SEM) from 3 independent experiments.