Recombinant human PF4 and 197.3 costimulation induce neutrophil Mac-1 up-regulation. (A) In costimulation studies, 197.3 dose was varied from 0.5 to 30 μg/mL in the presence of 1 μM wild-type (WT) recombinant PF4 (rPF4). Mac-1 up-regulation (left axis) and 197.3 binding to neutrophil (right axis) at 9 minutes after stimulation were measured. Mac-1 expression increased with 197.3 binding to cells. (B) In costimulation studies where rPF4 dose was varied in the presence of constant levels of 197.3 (30 μg/mL), Mac-1 up-regulation was maximum at 1 μM. For studies in panels C-E, 5 PF4 mutants were generated based on the primary sequence of rat-PF4. Costimulation experiments were performed with 1 μM PF4 (recombinant or rat source) and 30 μg/mL 197.3 for 9 minutes. Fixed samples were used to quantify (C) PF4 binding to neutrophils using polyclonal rabbit anti–human PF4 Ab followed by Alexa 488 goat anti–rabbit IgG, (D) 197.3 binding to neutrophil using Alexa 488 goat anti–mouse F(ab′)2 IgG, and (E) Mac-1 up-regulation using PE-labeled anti–Mac-1 mAb. rPF4 binding to neutrophils was abrogated by R49S mutation. Binding of 197.3 was partially dependent on residues 37 to 39. *Significantly different from WT rPF4 run (P < .05). Error bars represent SEM (n = 3).
