L5-mediated EC dysfunction. (A) HAECs were cultured in the presence of vehicle (endothelial growth media), L1 (500 μg/mL), or L5 (25 μg/mL) for 24 hours. Cells were then incubated with freshly collected whole blood from control subjects for 15 minutes, followed by 5 washes with PBS. Platelets were attached and aggregated on L5-treated HAECs but not L1-treated HAECs. Olympus BX51; bar = 10 µm. (B) Results of parallel plate flow chamber analysis with BAECs are shown. Cells were stained with 1 µM Hoechst (blue) to visualize cell nuclei. Human PRP samples containing 1 part acid sodium citrate (an anticoagulant), 10 µM calcein AM (makes platelets green), and unstimulated (treated with PBS) or L5-stimulated platelets were perfused at a flow rate of 750 S−1, and platelet-EC interactions were analyzed by fluorescence microscopy. (B, upper) Unstimulated (PBS-treated) platelets were attached to L5-treated BAECs. (B, lower) L5-stimulated platelets were slightly attached to PBS- or L1-treated BAECs but attached to L5-treated BAECs. Olympus IX70; bar = 100 µm. (C) Immunofluorescence staining with Alexa Fluor 555 (red) shows the expression of (upper) tissue factor and (lower) P-selectin in BAECs treated with PBS (Ctl), 50 μg/mL L1, or 50 μg/mL L5 for 24 hours. Cell nuclei were stained with DAPI (n = 3). Olympus IX70; bar = 10 µm.
