Detection of Sema3A binding to platelets and expression of Sema3A receptors in platelets. (A) Silver stain of purified Sema3A fusion proteins; 0.25 μgof Sema3A/Fc (∼ 125 kDa, lane c) and Sema3A/AP (∼ 150 kDa, lane d) were loaded on a 7.5% SDS-PAGE gel under reducing conditions and silver staining was performed. Sema3A/Fc and Sema3A/AP samples contain BSA as a carrier protein. In lane c, only BSA was loaded. Molecular weight marker was loaded in lane a. (B) Binding of Sema3A/Fc or Sema3A/AP to platelets. Washed platelets (5 × 10⁵) were incubated with Sema3A/Fc or hIgG, followed by incubation with FITC anti–human Fc. Sema3A/Fc binding was detected by flow cytometry, and mean fluorescence intensity (MFI) was plotted in the top panel. Washed platelets (5 × 10⁶) were incubated with Sema3A/AP or AP, and after washing, AP activity was measured using disodium phenylphosphate as a substrate. Change in optical density (ΔOD) was plotted on the bottom panel. Shown are representative results of 3 independent experiments. (C) Inhibition of Sema3A/AP binding by Sema3A/Fc. Washed platelets were first incubated with 125 μg/mL hIgG or Sema3A/Fc. Then, platelets were incubated with 10 μg/mL Sema3A/AP, and AP activity was measured. Shown is mean and SE of relative binding to hIgG-incubated sample of 3 independent experiments. (D) Expression of plexin-A1, -A2, or -A3 in platelets was detected by RT-PCR assay (left). Human umbilical vein endothelial cell (HUVEC) was used as a positive control. Expression of neuropilin-1 in platelets was detected by Western blotting and flow cytometric analysis (right). In Western blotting, neuropilin-1 expression was detected by anti–neuropilin-1 antibody, followed by incubation with HRP anti–mouse IgG. MDA-MB231 was used as a positive control. In flow cytometry, platelets were incubated with mouse monoclonal anti–neuropilin-1 antibody (filled curve) or control antibody (MOPC21; open curve), followed by incubation with Alexa488-conjugated anti–mouse IgG. MWM indicates molecular weight marker.