Gp96 does not interfere with platelet function.
(A) Freshly isolated platelets from 3 healthy donors were preincubated with 2.5 μM ADP, 100 μg/mL Gp96, or ovalbumin at 37°C for 15 minutes. Thereafter, thrombin at different concentrations was added. After an additional 5-minute incubation at 37°C, platelets were fixed and activation was analyzed by flow cytometry after staining with PE-labeled anti-CD40L antibody. Because of varying levels of CD40L expression between different donors, the obtained fluorescence intensities were converted to relative activation values. The median of fluorescence after maximal stimulation with 500 mU/mL thrombin was set as 100% activation (donor 1, 23.71; donor 2, 28.13; donor 3, 18.11). The value for nonstimulated platelets was set as 0% (2.94 ± 0.24). (B) The influence of Gp96 on the aggregation of platelet-rich plasma (2.5 × 105 cells/μL) was measured in an aggregometer. Ten seconds after the start of measurement, Gp96 was added to a final concentration of 50 μg/mL. Control samples were treated with buffer only. After 3 minutes, 2.5 μM or 10 μM ADP, 10 μg/mL collagen, or 50 μM adrenaline were added, and aggregation was followed for an additional 320 seconds. Experiments were performed in triplicate and were repeated for 3 different donors.
