Figure 4.
Platelets in bone marrow (BM) and platelet lifespan. (A) BM sections were stained using a primary rabbit-anti-human β1-tubulin antibody and a secondary biotinylated goat anti-rabbit IgG thus highlighting megakaryocytes and platelets; specimens were counterstained with Hematoxylin & Eosin (H&E). Scale bars=40 μm. Pictures were analyzed using Image J software and platelet number in BM was expressed as the number of platelets per 1000 μm2 of cell-covered slide surface (*P<0.05 vs. control). Ten different human BM sections were analyzed from the platelet-type von Willebrand disease (PT-vWD) patient, and ten each from three immune thrombocytopenia (ITP) patients and from three healthy controls. (B) Murine BM biopsies of TgWT and TgG233V mice were stained using a rabbit-anti-mouse CD41 antibody and a secondary biotinylated goat anti-rabbit IgG; specimens were counterstained with hematoxylin & eosin. Scale bars=40 μm. Pictures were analyzed using Image J software and platelet number was expressed as the number of platelets per 1000 μm2 of cells (right) (*P<0.05 vs. TgWT). Ten different BM sections were analyzed from each of three TgWT and three TgG233V mice. (C) TgWT (white circles) and TgG233V mice (gray circles) were injected with a DyLight 488-conjugated anti-GPIX mAb which confers to platelets a green fluorescence, and residual green fluorescent platelets were quantified by flow cytometry at different intervals from DyLight 488-conjugated anti-GPIX mAb injection. Data are expressed as the percentage of GPIX-positive platelets relative to the total CD41/61-PE positive platelet population with time=0 being arbitrarily set at 100%. Data represent mean±Standard Error of Mean (SEM) (n=4; *P<0.05 vs. TgWT). (D) von Willebrand factor (vWF) binding to human (a, c, e) and murine (b, d, f) platelets from EDTA-anticoagulated blood smears as assessed by fluorescence microscopy. vWF was not detected on the platelet surface of healthy controls (a) and TgWT mice (b), while it was bound to the platelet surface of the PT-vWD patient (60-80% of platelets) in the TgG233V mouse (70-90% of platelets) (c) and TgG233V mice (d). Platelet clumps linked by vWF were present in the circulation of the platelet-vWD (PT-vWD) patient (e) and TgG233V mice (f) but not of healthy controls and TgWT mice. vWF is stained red (Alexa Fluor® 568 Goat Anti-Rabbit IgG), CD42b is stained green (Alexa Fluor® 488 Goat Anti-rat IgG). Samples were mounted using the ProLong Antifade medium (Molecular Probes) and analyzed at room temperature using a Carl Zeiss Axio Observer.A1 fluorescence microscope (Carl Zeiss Inc., Oberkochen, Germany). Representative merging images are presented. Scale bars=5 μm, C: healthy control, P: PT-vWD. Data refer to blood smears from five healthy controls, five different blood samples from the PT-vWD patient and from five TgWT and five TgG233V mice.