Effect of trypsinization of intact, resting platelets on exposure of SNAP-23 and syntaxin-2. (A) To determine whether indomethacin inhibits platelet activation following incubation with trypsin, platelets were incubated in the presence or absence of 20 μM indomethacin, exposed to 5 μg/mL trypsin, and analyzed for platelet activation using a 14C-serotonin release assay. Error bars represent the standard deviation of 3 independent experiments. (B) To assess membrane integrity following exposure to trypsin, platelets were incubated in the presence or absence of 5 μg/mL trypsin, subsequently exposed to either buffer alone or α-toxin to permeabilize platelets, and incubated with sulforhodamine. Error bars represent the standard deviation of 3 independent experiments. (C) Gel-filtered platelets were incubated in the presence (trypsinized) or absence (untreated) of 5 μg/mL trypsin and subsequently stimulated with PMA. Platelets were then analyzed for SNAP-23 and syntaxin-2 surface expression. Values are reported as percent of control compared with signal detected in unstimulated samples exposed to nonimmune antibodies. Error bars represent the standard deviation of 3 to 6 independent experiments. (D) Platelets treated with indomethacin were exposed to buffer alone (Resting) or 100 μM SFLLRN (Activated) and subsequently incubated with buffer alone (No addition) or 5 μg/mL trypsin (Trypsin). Platelets were then stained with anti–SNAP-23 or anti–syntaxin-2 antibodies and evaluated by immunofluorescence microscopy. Controls using nonimmune antibody demonstrated no signal. Differential interference contrast (DIC) imaging confirmed the presence of platelets in all imaged fields. Bars, 5.0 μm. See “Materials and methods, Immunofluorescence microscopy” for image acquisition information.
