Platelet-derived TSP-1 modulates intracellular cAMP levels by increasing PDE3 activity. (A) WT (red) and TSP-1^−/− (blue) platelets (2 × 10⁸ platelets/mL) were treated with PGI₂ (0-50 nM) for 1 minute and lysed before intracellular cAMP concentrations were measured. Mean ± standard error of the mean (SEM) (N = 5). (B) TSP-1^−/− (red) platelets (2 × 10⁸ platelets/mL) were treated with PGI₂ (100 nM) for 1 minute in the presence or absence of human TSP-1 (hTSP-1; 10 μg/mL) and lysed before intracellular cAMP concentrations were measured. Mean ± SEM (N = 5, *P < .05, Mann-Whitney U test₂). (C) WT (red bars) and TSP-1^−/− (blue bars) platelets (2 × 10⁸ platelets/mL) incubated with apyrase were treated with PGI₂ (10 nM) alone for 2 minutes or stimulated with collagen (10 μg/mL) 1 minute after PGI₂ treatment. Reactions were stopped with lysis buffer, and intracellular cAMP levels were measured by using enzyme-linked immunosorbent assay. Intracellular cAMP levels are presented as mean ± SEM (N = 6; *P < .05, Mann-Whitney U test₂). (D) WT (red bars) and TSP-1^−/− (blue bars) platelets (5 × 10⁸ platelets/mL) incubated with apyrase were stimulated with collagen (20 μg/mL) for 1 minute before stopping the reaction with lysis buffer. PDE3A was immunoprecipitated, and enzyme activity was measured. Data are presented as percent activity above basal and given as mean ± SEM (N = 6; P < .05). (E) Whole blood from WT (red bars) and TSP-1^−/− (blue bars) mice were incubated with apyrase and treated with PGI₂ (10 nM) alone for 2 minutes or stimulated with CRP-XL (10 μg/mL) for 1 minute after PGI₂ treatment. Blood was fixed, permeabilized, and incubated with anti-VASP^Ser157 followed by secondary fluorescent-conjugate (Alexa 647) and analyzed by flow cytometry. Data are presented as mean ± SEM fold increase in phosph-VASP^Ser157 over basal (N = 4; P < .03, *P < .05, Mann-Whitney U test).