CCCP-induced GPVI shedding is abolished in Adam17^ex/ex platelets. Platelet count (A) and size (B) in Adam17^ex/ex BMc and control mice. (C) Surface protein expression was determined by flow cytometry. Platelets were stained with the indicated fluorophore-labeled antibodies for 15 minutes and analyzed directly. (D) Glycocalicin levels in plasma were determined by Western blot. (E) Washed platelets were treated with the indicated reagents (W7: 150μM, 1 hour at 37°C; CCCP: 100μM, 1 hour at 37°C), stained with the indicated fluorophore-labeled antibody, and analyzed on a FACSCalibur. (F) Washed platelets were incubated with biotinylated JAQ1 and then treated with CCCP (100μM) or W7 (150μM) for 1 hour at 37°C. Supernatants were applied on a JAQ3-coated ELISA plate, and GPVI-JAQ1^biotin complexes were detected with HRP-conjugated streptavidin. (G) Western blot detection of intact GPVI (JAQ1-HRP) in CCCP (100μM), W7 (150μM), or vehicle-treated platelets. GPIIIa was used as a loading control. Results of experiments (A-C and E-F) are mean ± SD (n = 4 mice per group, representative of 3 individual experiments).