scIV.3-IdeS bound to platelet FcγRIIA cleaves antiplatelet antibodies and prevents in vitro phagocytosis. (A) Platelets treated with scIV.3 (5 nM), IdeS, and scIV.3-IdeS (5 nM) were incubated with rabbit anti-human CD41 or CD42b antibodies, and the amount of undigested antibody bound to platelets was quantified by flow cytometry with an anti-rabbit heavy chain antibody. Data normalized to control median fluorescence intensity (MFI). (B) Platelets coated in rabbit anti-human CD41 polyclonal antibodies were treated with scIV.3-IdeS (5 nM) or control. The amount of undigested antibody bound to platelets was quantified by flow cytometry using an antibody specific for the heavy chain of rabbit IgG. (C) Human IgG western blot, representative of 3 independent experiments, of platelet-poor plasma (PPP) incubated with increasing concentrations of scIV.3-IdeS–coated platelets (18.7-150 × 10⁶/mL) for 1 hour. PPP was also incubated with control, untreated platelets, or IdeS (1 μM). IdeS-treated samples lacked the full-length IgG heavy chain (arrow; upper band) and had an Fc cleavage product (arrow; lower band). (D) CFSE-stained platelets treated with scIV.3 or scIV.3-IdeS in the presence of rabbit anti-human (CD41 and CD42b) antibodies were added to THP-1 cells. After 1 hour, THP-1 cells were stained with a platelet-specific (PE-conjugated CD42a) antibody to distinguish THP-1 cells that had adhered (CFSE⁺/CD42a⁺) or internalized (CFSE⁺/CD42a⁻) platelets (data represent mean ± standard deviation; 1-way analysis of variance; n = 4). *P < .05, ***P < .001.