Platelets express truncated TrkB receptors. (A) Immunoblotting of TrkB from human platelet lysates (100 µg for Sino Biological clone 7H6E7B3 and 25 µg for Abnova clone 3D12) obtained from 4 different healthy volunteers. TrkB-Fc fusion protein (5 ng; expected molecular weight, 120 kDa) and human cortex whole cell lysate (5 µg; expected molecular weight of truncated TrkB and the full-length TrkB receptor, 95- and 140-kDa, respectively) were used as positive controls. β-Actin was used a loading control. Two different antibodies (clone 7H6E7B3 and clone 3D12) targeting the extracellular domain of TrkB were used. Blots are representative of ≥3 independent experiments. (B) Confocal fluorescence microscopy of platelets expressing TrkB. Washed platelets, PBMCs, U251-MG cells, and HEPG2 cells were labeled with anti-TrkB primary antibodies and Alexa Fluor 488–conjugated secondary antibodies. Nuclei of PBMCs, U251-MG cells, and HEPG2 cells were stained with 4′,6-diamidino-2-phenylindole, and IgG_2B/IgG₁ was used as an isotype control. Images were visualized at room temperature with Zeiss LSM510 using a 100× objective lens for platelets and PBMCs, a 63× objective lens for HEPG2 cells, and a 20× objective lens for U251-MG cells as well as 3× magnification. Scale bar represents 5 µm for PBMCs and platelets, 10 µm for HEPG2 cells, and 200 µm for U251-MG cells. Images are representative of 3 independent experiments. (C) Flow cytometry of surface and intracellular TrkB on washed human platelets; platelets expressed TrkB on both their surface (TrkB⁺: 27% ± 11%, n = 8) and their intracellular compartment (TrkB⁺: 82% ± 9%, n = 6). PBMCs were used as positive controls for TrkB labeling on both the surface (TrkB⁺: 45% ± 15%, n = 4) and the intracellular compartment (TrkB⁺: 88% ± 6%, n = 4). U251-MG cells were used as positive controls for TrkB labeling on both the surface (TrkB⁺: 30% ± 2%, n = 3) and the intracellular compartment (TrkB⁺: 94% ± 2%, n = 3). IgG_1/2B was used as isotype control. HEPG2 cells were used as TrkB-low controls for TrkB labeling on both the surface (TrkB⁺: 6% ± 0.12%, n = 2) and the intracellular compartment (TrkB⁺: 17% ± 1%, n = 2). FITC, fluorescein isothiocyanate.