Src kinases mediate TSP-1 inhibition of AC and PKA activity. (A) Platelets (2 × 10⁸/mL), incubated with either PP1 or PP3 (20μM), were preincubated with TSP-1 (10 μg/mL) or CD36 peptide (10μM) for 15 minutes followed by treatment with PGE₁ (0.1μM) for 30 seconds. 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 4 individual experiments. §P < .05 for cAMP levels induced by PGE₁ alone compared with the presence of TSP-1 or CD36 peptide. *P < .05 for cAMP levels induced by PGE₁ in the presence TSP-1 or CD36 peptide, where platelets had been pretreated with PP1. (B) Platelets (3 × 10⁸/mL), incubated with either PP1 (20μM), PP3 (20μM), JNK peptide inhibitor 1 (10μM), or SB202190 (10μM), were preincubated with TSP-1 (10 μg/mL) for 15 minutes followed by treatment with PGE₁ (0.1μM) for 1 minute. Samples were then lysed and phosphorVASPser¹⁵⁷ was analyzed. (C) As in panel B, but the CD36 stimulatory peptide (10μM) was used in the place of TSP-1. All immunoblots are representative of 4 independent experiments.