ICs containing patient-derived anti-PF4/heparin antibodies induce robust, heparin-dependent neutrophil degranulation. (A) ICs containing patient-derived anti-PF4/heparin antibodies cause MMP-9 release. Whole blood was incubated with buffer or KKO (25 µg/mL) along with PF4 (25 µg/mL) and heparin (1 U/mL), with plasma from 2 individual HIT patients (HIT1 and HIT2; 1:10 dilution), or plasma from a normal healthy donor (Control, 1:10 dilution) in the presence of buffer or PF4 with or without heparin. Released MMP-9 was measured by enzyme-linked immunosorbent assay (ELISA). Data shown are representative of whole blood from 3 healthy donors. (B) ICs containing patient-derived anti-PF4/heparin antibodies cause release of all neutrophil granule populations. Plasma from a patient with anti-PF4/heparin antibodies (diluted 1:10) was added to whole blood from a healthy donor, along with PF4 (25 µg/mL) and heparin (1 U/mL). After incubation for varying amounts of time (0-120 minutes), granules were measured in plasma. Data shown are representative of whole blood from 2 healthy donors. (C) ICs containing patient-derived anti-PF4/heparin antibodies induce heparin/LMWH-dependent neutrophil activation. Whole blood from a healthy donor was incubated with plasma from a patient with anti-PF4/heparin antibodies or with plasma from a healthy donor as a control (both at a 1:10 dilution) along with PF4 (25 µg/mL) and varying amounts of UFH or LMWH (0-1000 µg/mL). After 30 minutes, MMP-9 release was measured in plasma. Data shown are representative of whole blood from 3 healthy donors. All data are representative of at least 3 independent experiments. Results are expressed as mean ± standard deviation values for triplicate wells.