CD148-deficient megakaryocytes do not spread or migrate. (Ai) Cultured bone marrow–derived megakaryocytes from wild-type (WT) and CD148 transmembrane-knockout (KO) mice were plated on fibrinogen-, collagen-, and fibronectin-coated surfaces. Adherent megakaryocytes were fixed, permeablized, and actin fibers stained with rhodamine-phalloidin. Representative images from 3 separate experiments (20 μm scale bar). (Aii) The surface area of spread megakaryocytes was quantified and presented as mean ± SEM. (Bi) WT and KO megakaryocytes were placed on a fibronectin-coated surface and allowed to migrate toward a SDF-1α gradient in a Dunn chamber. Images were captured at the indicated times by differential interference contrast microscopy (20 μm scale bar). (Bii) The direction and distance traveled by individual megakaryocytes was tracked in real time and plotted as shown. Each trace was generated by a single megakaryocyte over a 4-hour period. Representative images from 3 separate experiments. See also Figure S6 Videos S3,S4. (C) Whole-cell lysates (WCLs) prepared of nonadherent WT, and KO megakaryocytes were Western blotted with an anti-Src family kinase (SFK) activation p-Tyr antibody. Membranes were stripped and reblotted with an anti–Src-pan antibody (Src). Representative images from 2 experiments. (D) Delayed thrombopoiesis in CD48 TM-KO mice after complement-mediated immune thrombocytopenia. Thrombocytopenia was induced in WT and KO mice by an intraperitoneal injection of anti–mouse GPIbα antibody (2 μg/g of mouse). Peripheral platelet counts were measured 7 days before injection (time = 0) and at 3, 48, 72, 96, 120, 144, and 172 hours after injection (n = 3-4 mice per time point). Mean platelet count (± SEM) was plotted as a function of time (**P < .01).