Inhibition of metalloproteinase activity prevents cleavage of GPIbα on damaged platelets. (A) Surface expression of GPIbα and GPIbβ as determined by flow cytometry. Upper panel: washed platelets treated for 60 minutes with DMSO, CCCP, or GM6001/CCCP. Middle panel: PRP aged for 16 hours at 37°C in the presence (GM6001/16 h) or absence (16 h) of GM6001. Lower panel: washed platelets treated for 60 minutes with calpeptin (25 μM), CCCP, or calpeptin/CCCP. Results are representative of 5 experiments. (B) Washed platelets treated for 60 minutes with DMSO, CCCP, or GM6001/CCCP were surface labeled by 10 nm anti-GPIbα immunogold. As previously reported, anti-GPIbα immunogold is found in linear arrays on the surface of the resting mouse platelet.48 This topology is preserved when platelets are treated with CCCP in the presence of GM6001. Anti-GPIbα immunogold does not label the surface of CCCP-treated platelets. Shown are representative electron micrographs; the bar is 100 nm; arrows point to gold particles bound to surface-expressed GPIbα.
