Microvesicles fuse with activated platelets. (A) Fluorescence microscopy (NBD channel) of THP-1 cells labeled with NBD-PE and Rh-PE (left panel, top) and of their shed microvesicles (left panel, bottom). Original magnifications, × 40 and × 400 on a Nikon Eclipse EP00 upright microscope equipped with 4 ×/0.13 NA and 40 ×/0.75 NA objective lenses (Nikon, Melville, NY), respectively. Images were captured using a Photometrics CoolSnap CS digital camera (Photometrics, Tucson, AZ). Solubilization of the labeled microvesicles with 1% SDS diluted the probes, resulted in an increase in NBD fluorescence, and a decrease in Rh fluorescence (right panel). (B) Labeled microvesicles were incubated with either: (x) unstimulated platelets; (▪) activated platelets; (♦) activated platelets + annexin V (100 μg/mL); (▴) activated platelets + anti–PSGL-1 antibody, KPL-1 (5 μg/mL). (C) Transfer of PSGL-1 and TF to activated platelets by monocyte microvesicles, as assessed by flow cytometry. Annexin V (100 μg/mL) did not affect the number of platelets that acquire these proteins. Background fluorescence was determined using fluorescent nonspecific mouse IgG. n = 3. (D) Membrane fusion between activated platelets and THP-1 microvesicles at 37°C (▪), and at 4°C (▪). (E) Fluorescence microscopy in rhodamine channel (×40) showing the transfer of the fluorescent lipid R18 from labeled microvesicles to platelets adhered on immobilized fibrinogen. Transfer of the lipid resulted in the spread platelets becoming fluorescent, an effect that was blocked by annexin V. The small fluorescent spots in the annexin V–treated sample correspond to the labeled microvesicles. These images are representative of 4 experiments. Act indicates activated; Plts, platelets; Unstim, unstimulated; An V, annexin V. Error bars indicate ± standard deviation (SD) in panels B-D.