Costimulation with both anti-PF4 mAb and HIT patient IgG results in Mac-1 up-regulation that is blocked by chondroitinase ABC and heparin. The ability of 1 U/mL chondroitinase ABC (ABC), heparin, and 10 μg/mL anti-CD32/FcγRII mAb (IV.3) to block Mac-1 up-regulation following 9-minute costimulation with 2 μM hPF4, and either 10 μg/mL anti-PF4 mAbs (197.3 and 197.2; A,C) or HIT patient IgG (B,D) was examined. Whereas 10 μg/mL nonbinding isotype-matched Abs (IgG_2b and IgG₁) were used in some runs, anti–PSGL-1/CD162 mAb KPL-1 served as an isotype-matched, neutrophil-binding control Ab for studies performed with 197.2. Protamine sulfate (PS; 25 μg/mL) was added in lieu of human PF4 in other controls. (A,B) Mac-1 up-regulation (%). (C,D) Total antibody binding to neutrophils (MFI) determined using Alexa 488 goat anti–mouse F(ab′)₂ IgG (C) and Alexa 488 goat anti–human F(ab′)₂ IgG (D). *P < .05, with respect to all negative controls (no stimulus, hPF4 or 197.3 alone, hPF4 plus IgG₁ or IgG_2b isotype controls, hPF4 plus KPL-1, and PS plus 197.3) and blocker treatments (ABC, ≥ 1U/mL heparin, IV.3). †P < .05, with respect to all other treatments except that HIT + PF4 is not different from HIT + PF4 + IV.3. In panels B and D, whereas blood was drawn in heparin in some runs, EDTA was used as anticoagulant in other runs. Low doses of unfractionated heparin (0.05 U/mL) enhance PF4-HIT complex binding to neutrophils. Error bars represent SEM (n ≥ 3).