TSP-1 inhibits activation of PKA independently of changes in cAMP concentrations. (A) Platelets (3 × 10⁸/mL) were preincubated with TSP-1 (10 μg/mL) for 15 minutes, followed by addition of 8-bromo-cAMP (500μM) for 15 minutes, and then stimulated with collagen (5 μg/mL) for 4 minutes under stirring conditions. Representative traces of 3 independent experiments. (B) Platelets (3 × 10⁸/mL) were treated with 8-bromo-cAMP (500μM) alone for 15 minutes or TSP-1 (10 μg/mL) for 15 minutes followed by 8-bromo-cAMP for 15 minutes in the presence and absence of FA6-152 (1 μg/mL) or control IgG. Platelets were lysed, separated by SDS-PAGE and blotted for phosphoVASP-ser¹⁵⁷, followed by reprobing for β-tubulin. Immunoblots are representative of 3 independent experiments. (C) Platelets (2 × 10⁸/mL) were preincubated with PGE₁ (0.1μM) alone for 30 seconds or with TSP-1 (10 mg/mL) for 15 minutes before PGE₁, in the presence or absence KT-5720 (5μM) and Rp-cAMPS (0.5mM). cAMP levels were measured as per manufacturer's instructions and are expressed as fmol cAMP/1 × 10⁷ platelets. Data are presented as mean ± SEM of 3 individual experiments. §P < .05, PGE₁-stimulated cAMP compared with basal; *P < .05, PGE₁-stimulated cAMP compared with the absence of TSP-1. Platelets (3 × 10⁸/mL) were treated with PGE₁ (0.1μM) for 30 seconds in the presence or absence KT-5720 (5μM) and Rp-cAMPS (0.5mM), and then immunoblotted for phosphoVASP-ser¹⁵⁷ with a representative blot presented.