Generation and characterization of recombinant scIV.3-IdeS. (A) Increasing concentrations of His-tagged scIV.3-IdeS or scIV.3 were incubated with human platelets and then stained with a fluorescently labeled anti-His Tag antibody and analyzed by flow cytometry (n = 4). (B) Microscopic images (60× objective) of platelets spread on fibrinogen treated with control or cIV.3 and costained with scIV.3-IdeS (5 nM) and an anti-CD41 antibody. (C) Surface-bound scIV.3-IdeS was quantified by flow cytometry every 30 minutes for 90 minutes. (D) Representative Coomassie-stained gel used to evaluate IgG cleavage after the incubation of IgG with predetermined concentrations of scIV.3, Ides, or scIV.3-IdeS. IdeS cleaves IgG in a 2-step process, first generating a single-cleaved IgG (scIgG) and then a double-cleaved F(ab′)2. The cleaved Fc fragment was quantified as a measure of IdeS activity. The relative fluorescence units of the Fc fragment were reported for 4 independent experiments (data given as mean ± standard deviation). *P < .05. RFLU, relative fluorescence units.