Heparin-containing ICs induce heparin-dependent neutrophil activation. (A) KKO-PF4/heparin ICs cause neutrophil degranulation and MMP-9 release. First, 100 µL of whole blood from a healthy donor was incubated with buffer, PMA (500 nM), KKO (25 µg/mL), or IgG2b isotype control (25 µg/mL) in the presence of the following antigens alone or in combination: PF4 (25 µg/mL) or heparin (Hep; 1 U/mL). After incubation for 30 minutes at 37°C, samples were centrifuged at 300g for 5 minutes. Plasma was removed, and MMP-9 was quantified. (B) KKO-PF4/heparin ICs cause release of all neutrophil granule populations. KKO (25 µg/mL) along with PF4 (25 µg/mL) and heparin (1 U/mL) was added to whole blood. After incubation for varying amounts of time (0-120 minutes), release of MMP-9, lactoferrin, or MPO was measured in plasma. (C) KKO-PF4/heparin IC–induced neutrophil activation is heparin/low molecular weight heparin (LMWH) dependent. Whole blood was incubated with KKO (25 µg/mL) along with PF4 (25 µg/mL) and UFH or LMWH at varying concentrations (ranging from 0 to 1000 µg/mL) or IgG2b isotype control (25 µg/mL) along with PF4 (25 µg/mL) and varying levels of UFH/LMWH. After 30 minutes, release of MMP-9 was measured in the plasma. (D) ADA-PRT/heparin ICs also induce heparin-dependent MMP-9 release. Whole blood was incubated with a monoclonal anti-PRT/heparin antibody (ADA 50 µg/mL) along with PRT (25 µg/mL) and varying amounts of heparin (0-100 U/mL). After 30 minutes, MMP-9 released in plasma was measured. Data are representative of 3 independent experiments and results using whole blood from 3 healthy donors. Results are expressed as mean ± standard deviation values for triplicate wells.