FcγRIIa (CD32a) mediated neutrophil Mac-1 up-regulation. The ability of 10 μg/mL anti-CD32/FcγRII (IV.3), anti-CD16 (3G8 F(ab′)₂), and isotype control for IV.3 (IgG_2b) to block Mac-1 up-regulation induced by 2 μM hPF4 and 10 μg/mL 197.3 was measured. Blocking reagents were added 6 minutes prior to stimulation. (A) Mac-1 expression was measured 9 minutes after stimulation in fixed samples. (B) Total mouse antibody bound to cells was measured using Alexa 488–conjugated goat anti–mouse F(ab′)₂ IgG. IV.3 significantly reduced Mac-1 up-regulation without dramatically altering the binding of PF4 to cells. *P < .05, with respect to positive control with hPF4 plus 197.3. (C-D) Confocal microscopy images showed clustering and colocalization of FcγRIIa and anti-PF4 antibody. Two Alexa 647–conjugated anti-CD32 monoclonal antibodies were used for these runs, IV.3 (Ci-Civ) and CIKm5 (Di-Div). In panels Ci and Di, CD32 distribution on unstimulated neutrophils determined using IV.3–Alexa 647 and CIKm5–Alexa 647, respectively, was seen to be uniform. Cells were stimulated with hPF4 and Alexa 488–conjugated 197.2. After fixation, these cells were labeled with either IV.3–Alexa 647 or CIKm5–Alexa 647. Panels Cii and Dii show signal from 197.2–Alexa 488 channel, panels Ciii and Diii show data from IV.3–Alexa 647 and CIKm5–Alexa 647, respectively, and panels Civ and Div show merged fluorescence images along with differential interference contrast (DIC) image. Error bars represent SEM (n ≥ 3).