INU1 induces the loss of CLEC-2 in circulating platelets in vivo. (A) Mice received the indicated amounts of purified IgG or Fab fragments of INU1 intravenously in 200 μL sterile PBS, and platelet counts were determined by flow cytometry at the indicated time points. Results are expressed as the mean platelet count ± SD for 4 mice per group and are representative of 3 individual experiments. Parallel analyses on an automated cell analyser (Sysmex) yielded similar results (not shown). (B) Two-color flow cytometric analysis of integrin αIIbβ3 activation (JON/A-PE) and P-selectin exposure (FITC) in platelets from mice 1 or 3 days after INU1-IgG or INU1-Fab injection. Diluted whole blood was stimulated with 1 μg/mL rhodocytin (RC). The data are representative of 8 mice per group. (C) Mice were injected with the indicated amounts INU1-IgG or INU1-Fab, and surface-bound IgG or Fab was detected by flow cytometry using anti–rat IgG-FITC. Maximal binding was determined by incubating platelets from untreated mice with INU1-IgG or INU1-Fab (10 μg/mL) in vitro. (D) INU1-FITC binding to platelets from the indicated mice. Results are expressed as mean fluorescence intensity (MFI) ± SD (C-D). (E) Immunoprecipitation of CLEC-2 and GPVI from surface-biotinylated platelets from control and INU1 (8 μg/g)–treated mice on day 5 under reducing conditions.