Evidence for a third GPVI-cleaving protease in platelets that is insensitive to GM6001 inhibition. (A) Washed platelets were treated with NEM (2mM, 20 minutes, 37°C) in the presence or absence of GM6001 (100μM, 30 minutes, 37°C), stained with a fluorophore-labeled antibody recognizing the indicated glycoprotein, and analyzed in a flow cytometer. (B) Washed platelets were incubated with the biotinylated anti-GPVI antibody, JAQ1, and then treated with NEM (2mM, 20 minutes, 37°C) in the presence or absence of GM6001 (preincubation: 100μM, 30 minutes, 37°C). Supernatant was collected and applied on a JAQ3-coated ELISA plate and incubated with HRP-conjugated streptavidin. Results of experiments are mean ± SD (n = 4 mice per group, representative for 3 individual experiments).