TSP-1 blunts PGE₁-mediated cAMP production and PKA signaling. (A) Platelets (2 × 10⁸/mL) treated with or without milrinone were preincubated with TSP-1 (10 μg/mL) for 15 minutes, followed by treatment with PGE₁ (0.1μM) for 30 seconds. In some cases platelets were treated with fibrinogen (10 μg/mL) instead of TSP-1, before addition of PGE₁. cAMP levels were measured per manufacturer's instructions and are expressed as fmol cAMP/1 × 10⁷ platelets. Data are presented as mean ± SEM of 10 individual experiments. §P < .05 PGE₁ treatment compared with basal cAMP; *P < .05 PGE₁ stimulated cAMP compared with the presence of TSP-1. **P < .01 PGE₁ treatment compared with basal cAMP. (B) Platelets (3 × 10⁸/mL) were preincubated with TSP-1 (0-10 μg/mL) for 1-15 minutes before treatment with PGE₁ (0.1μM) for 1 minute. Platelets were lysed, separated by SDS-PAGE and immunoblotted for phosphoVASP-ser¹⁵⁷, followed by reprobing for β-tubulin. (C) As in panel B, except platelets were incubated with TSP-1 (10 μg/mL) for up to 60 minutes before addition of PGE₁ for 1 minute. (D) As in panel B, except platelets were incubated with fibrinogen (0.001-10 μg/mL) before addition of PGE₁. Immunoblots for panels A-D are representative of 4 independent experiments. (E) Platelets (3 × 10⁸/mL) were preincubated with TSP-1 (10 μg/mL) for 15 minutes, followed by the addition of PGE₁ (0.1μM) for 1 minute. Platelets were lysed, separated by SDS-PAGE, and immunoblotted for phospho-PKA substrates, followed by reprobing for β-tubulin. Shown is a representative blot of 3 independent experiments.