miRNA is transferred to endothelial cells by a vesicle-dependent mechanism. (A) The effect of brefeldin A on platelet microparticle release was assessed with flow cytometry. Brefeldin A (10 μg/mL) was added to platelet-rich plasma 30 minutes before the addition of 1 U/mL thrombin. The microparticle population was defined based on size and expression of CD42a. ***P < .001 comparing untreated with thrombin-stimulated, ++P < .01 comparing thrombin vs thrombin + brefeldin A, n = 3. (B) Mean fluorescence in the FL1 channel within the microparticle population was used to assess miR-Scr-FITC content (***P < .001 comparing no thrombin vs thrombin-stimulated; +P < .05 comparing thrombin versus thrombin + brefeldin A, n = 6). (C) Quantification of FITC-labeled miRNA in HMEC-1 cells. Pixels of intensity greater than background staining were pseudocolored white. In each image the percentage of cells positive for white pixels were counted manually (*P < .05 comparing HMEC-1 cells cocultured with platelets in the presence or absence of thrombin; ++P < .01 comparing HMEC-1 cells cocultured with platelets in the presence of thrombin alone or with brefeldin A).