Effect of APT1 and botulinum toxin C light chain on extracellular SNAP-23 and syntaxin-2. (A) To assess membrane integrity following exposure to 10 μg/mL APT1 and/or 5 μg/mL botulinum toxin C light chain, platelets were incubated in the presence or absence of the indicated enzymes, 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. (B) Gel-filtered platelets were incubated with 20 μM indomethacin and subsequently exposed to either buffer alone, 10 μg/mL APT1, or 5 μg/mL botulinum toxin C light chain as indicated. Platelets were subsequently pelleted at 100 000g for 2 hours, and supernatants were evaluated for SNAP-23 or syntaxin-2 by immunoblot analysis. (C) Gel-filtered platelets were incubated with both APT1 and botulinum toxin C light chain and then pelleted. SNAP-23 and syntaxin-2 were immunoprecipitated from supernatants. Immunoprecipitates were then analyzed for SNAP-23 and syntaxin-2 by immunoblot analysis.