Disruption of the cytoskeleton primes platelets for SNARE protein-mediated α-granule secretion. (A) Platelets were incubated in the presence of vehicle (no addition), 2 μM latrunculin A (LAT A), or 5 mM MgATP (ATP) and permeabilized with SL-O. Following a 15-minute incubation, platelets were exposed to buffer (□) or 10 μM Ca²⁺ (▪) and assayed for P-selectin expression by flow cytometry. P-selectin expression after exposure to MgATP alone followed by Ca²⁺ represents 100%. (B) Platelets were incubated in the presence of vehicle (no addition), 2 μM latrunculin A (LAT A), or 5 mM MgATP (ATP) and permeabilized with SL-O. Following a 15-minute incubation, platelets were exposed to buffer (□) or 33 μM GTP-γ-S (▪) and assayed for P-selectin expression by flow cytometry. P-selectin expression after exposure to MgATP alone followed by Ca²⁺ represents 100%. (C) Platelets were incubated with either vehicle (▪), 8 μM jasplakinolide (▨), or 100 μM phalloidin oleate (□) for 20 minutes. Platelets were then permeabilized in the presence of vehicle (no addition) or 2 μM latrunculin A (LAT A). Following a 15-minute incubation, platelets were exposed to 10 μM Ca²⁺ and assayed for P-selectin expression. P-selectin expression after exposure to latrunculin A alone followed by Ca²⁺ represents 100%. (D) Platelets were incubated with either buffer (lane 1), 50μg/mL nonimmune IgG (lane 2), or 50 μg/mL anti-VAMP antibody (lane 3). Samples were permeabilized in the presence of latrunculin A for 15 minutes, exposed to 10 μM Ca²⁺ (▪) or 33 μM GTP-γ-S (□), and then assayed for P-selectin expression. P-selectin expression after exposure to latrunculin A alone followed by agonist represents 100%.