Characterization of the binding properties of scIV.3. (A) Increasing concentrations of FAM-conjugated scIV.3 (scIV.3-FAM) were incubated with platelets from wild-type mice (hFcγRII^null) or hFcγRIIA transgenic mice (hFcγRIIA^TGN), and the median fluorescence intensity (MFI) of 3 independent experiments was reported. (B) Representative histograms of THP-1 cells stained with select concentrations of scIV.3-FAM. THP-1 cells (green dashed line) or human platelets (black line) were incubated with increasing concentrations of scIV.3-FAM, and data are reported as percentage of maximum MFI for each cell type (n = 3-4). (C) Representative microscopic images (60× objective) of human platelets (CD41⁺) spread on fibrinogen, treated with control or cIV.3 (30 nM), and then stained with scIV.3-FAM (5 nM). (D) Flow cytometry was used to quantify the binding of scIV.3-FAM to human platelets after incubation with or without cIV.3 (data represent n = 4; mean ± standard deviation [SD]). (E) Human whole blood was incubated with increasing concentrations of scIV.3-FAM and normalized to cIV.3 (30 nM). (F) Human platelets were treated with scIV.3 (50 nM) or control and then stimulated with mouse anti-human CD9 antibodies, an FcγRIIA-specific agonist (data represent n = 5; mean ± SD). Two-way statistical analysis of variance with Bonferroni correction was performed. K_d, distribution coefficient. *P < .05, **P < .01, ***P < .001.