Newly formed cell bodies are functional. (A) The bar graph depicts P-selectin and PAC-1 surface expression (percentage of positive cells) as assessed by flow cytometry in freshly isolated (0 hours) or cultured (6 hours) platelets with or without thrombin (Thr) stimulation for 15 minutes. The data are compared with isotype-matched control antibodies (IgG, IgM). (B) Platelets were cultured in suspension for 6 hours and subsequently placed on immobilized fibrinogen. As shown in these sequential images (i-xii), a platelet process adheres, spreads, and forms 2 distinct cell bodies that eventually separate from one another (gray dashed lines). Scale bar represents 5 μm. Panel B is representative of 4 independent experiments. (C-D) Platelets were cultured for 6 hours and subsequently treated with vehicle or thrombin (0.005 U/mL). After 60 seconds, the platelets were fixed in solution, the permeabilization step was skipped, and then the cells were coimmunostained for (C) P-selectin and sialic acids (WGA) or (D) annexin V and actin. Panels C and D are representative of 3 independent experiments. Scale bars represent 10 μm.