Cleavage of purified 5B9 by IdeS. Representative Coomassie blue SDS-PAGE (n = 3 experiments) (A) and percentages of intact 5B9 IgG, single cleaved 5B9 (sc5B9), and F(ab′)₂ 5B9 (mean values, n = 3), as defined by ImageJ, after incubation of 5B9 with IdeS (0.02 U/µg IgG) at 37°C (B). (C) Binding of cleaved 5B9 (1 µg/mL) to PF4/H complexes, as evaluated by ELISA with a secondary Ab specific to murine K chain (mean ± standard error of the mean [SEM], n = 2 experiments). A, absorbance; O/N, overnight. (D) Inhibition of IV.3 binding on platelets with increasing concentrations of 5B9 or sc5B9 (mean ± SEM; n = 3 platelet donors). (E) Representative platelet aggregation curve (n = 2 experiments) with untreated 5B9 (50, 25, 12.5, and 6.25 µg/mL) or sc5B9 (50 µg/mL) added to platelet-rich plasma with UFH (0.5 IU/mL). (F) Representative profile of serotonin release assay (n = 2 experiments) with untreated 5B9 (20 µg/mL) and sc5B9 (20 µg/mL) and different concentrations of UFH. (D-F) Single cleaved 5B9 (sc5B9) is the IgG fraction obtained after incubating 5B9 with IdeS (0.02 U/µg IgG; 6 minutes) at 37°C. *P < .05.