GPVI shedding in Adam10^−/−/Adam17^ex/ex platelets in vitro. Platelet count (A) and size (B) in Adam10^−/−/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. *P < .05; ***P < .001). (D) Washed platelets were treated with the indicated reagents (W7: 150μM, 1 hour at 37°C; CCCP: 100μM, 1 hour at 37°C), stained with a fluorophore-labeled antibody recognizing the indicated glycoprotein, and analyzed in a flow cytometer. (E) Washed platelets were incubated with the biotinylated anti-GPVI antibody, JAQ1, and then treated with CCCP (100μM) or W7 (150μM) for 1 hour at 37°C. Supernatant was collected and applied on a JAQ3-coated ELISA plate and incubated with HRP-conjugated streptavidin. (F) Washed platelets were treated with CCCP (100μM) or W7 (150μM) for 1 hour at 37°C and immediately lysed with 1% Nonidet P-40. The lysates were separated on a SDS-PAGE and incubated with an HRP-labeled anti-GPVI antibody. GPIIIa was used as a loading control. Results of experiments (A-E) are mean ± SD (n = 4 mice per group, representative for 3 individual experiments).