Arf6-GTP decreases upon GPVI activation. (A) Washed platelets were incubated in the presence or absence of 2 mM aspirin with stirring for 30 minutes at 37°C, or in the presence or absence of 0.37 units/mL apyrase for 1 minute prior to the stimulation with 0.1 μg/mL convulxin. Platelets were lysed at the designated time points, and the clarified supernatants were used to pull down Arf6-GTP. Arf6 was detected by Western blot. (B-C) Washed platelets were incubated in the presence or absence of 500 μM RGDS peptide for 1 minute at 37°C, and stimulated with 0.1 μg/mL convulxin. Platelets were lysed at the designated time points and the clarified supernatants were used to pull down Arf6-GTP, followed by Western blot using an anti-Arf6 antibody. (B) Aggregation traces are shown with arrows where time points were taken. (D) Washed platelets were stimulated with the following agonists for 3 minutes and extracts were used to analyze Arf6-GTP levels: thrombin (0.3 U/mL), TRAP (20 μM), U46619 (1 μM), A23187 (1 μM), convulxin (0.1 μg/mL), ADP (10 μM).