The neutrophil response to KKO-PF4/heparin ICs varies among individuals and represents a fixed phenotype. (A) The neutrophil response to anti-PF4/heparin ICs in healthy donors is heterogeneous. Whole blood from healthy donors (n = 71) was incubated with buffer, PF4/heparin (PF4 25 µg/mL; heparin [H] 1 U/mL) alone or in the presence of KKO or isotype control (Iso, 25 µg/mL), and MMP-9 was measured in plasma. Median and interquartile range are plotted, with individuals >Q3 depicted in blue and individuals <Q1 in red. (B) The neutrophil response to anti-PF4/heparin ICs represents a fixed phenotype. Whole blood from 3 healthy donors who had been designated as high responders (blue) and from 3 donors who had been designated as low responders (red) were repeatedly sampled over a course of 358 days. Whole blood from all donors was incubated with KKO-PF4/heparin ICs, and released MMP-9 was measured in plasma. Results are expressed as mean ± standard deviation values for triplicate wells. (C) The neutrophil activation phenotype is consistent among all 3 neutrophil granule populations. Samples from 10 representative high responders (blue) and 10 representative low responders (red) from panel A were tested for MPO (top) or lactoferrin (bottom) release. (D) The neutrophil activation phenotype is preserved with ADA-PRT/heparin ICs. Whole blood from a designated high responder and a low responder was incubated with ADA (50 µg/mL) along with protamine (25 µg/mL) and varying amounts of UFH (0-100 U/mL). After 30 minutes, MMP-9 release was measured in plasma. Data are representative of 3 high-responding and 3 low-responding donors. Results are expressed as mean ± standard deviation values for triplicate wells. (E) The neutrophil activation phenotype is preserved with heat-aggregated (agg) IgG. Whole blood from 3 representative high responders (blue) and 3 representative low responders (red) was incubated with buffer or heat-aggregated IgG (100 µg/mL). After 3 hours of incubation, MMP-9 release was measured in plasma.