Expression of activated α2 integrins does not alter the location of megakaryocytes in the bone marrow in vivo or migration through collagen ex vivo. (A) Immunostaining for VWF, GFP, and Flk-1 in the bone marrow of mice expressing wild-type and activated α2 integrins. Femurs from mice expressing wild-type and activated α2 integrins were sectioned longitudinally and stained with anti-VWF antibody to identify megakaryocytes, with anti-GFP antibody to identify cells derived from hematopoietic stem cells that were transfected with retroviruses, and with anti–Flk-1 antibody to identify sinusoidal endothelial cells. White arrowheads indicate the location of 5 representative megakaryocytes that express the indicated α2 integrins in each panel. (B) Migration of bone marrow cells expressing wild-type or activated α2 integrins across a collagen-coated filter ex vivo. Freshly harvested cells were cultured ex vivo in the presence of thrombopoietin to induce megakaryocyte maturation and allowed to migrate across transwell filters coated or uncoated with collagen for 24 hours in response to an SDF-1 gradient. The chemotactic index is defined as the number of cells that migrated across the transwell filter divided by the total number of cell input. The error bars indicate SD of the chemotactic index from 3 independent experiments. No statistically significant difference was observed between groups (P > .05). (C) Migration across collagen is associated with loss of surface expression of activated but not wild-type α2 integrins. Surface expressions of α2 integrins on CD41⁺ cells that migrated through a collagen-coated filter are shown in red and those that migrated across a non–collagen-coated filter are shown in blue. The gray shadowed area represents the α2 levels of nontransfected cells.