Platelet-derived TSP-1 modulates PDE3 activity in a CD36-dependent manner. (A) Platelet-rich plasma (PRP) from WT and CD36^−/− mice (treated with apyrase) were stimulated with collagen (10 μg/mL), in the presence or absence of PGI₂ (5 nM), and aggregation was measured. Mean ± standard error of the mean (SEM) (N = 5, **P < .01, Mann-Whitney U test). (B) PRP from WT and CD36^−/− mice (treated with apyrase) were treated with PGI₂ (10 nM) for 1 minute before stimulation with CRP-XL (5 μg/mL) for 20 minutes, and JonA binding was measured by using flow cytometry (N = 7; P < .05). (C) As in (B), except TLT-1 surface expression was measured (N = 7; P < .05). (D) PRP from TSP-1^−/− (treated with apyrase) was stimulated with collagen (10 μg/mL) in the presence and absence of PGI₂ (5 nM) for 1 minute and aggregation measured. In some cases, platelets were pretreated with CD36-derived releasates and the CD36 receptor blocking antibody FA6.152 (2 μg/mL) or CD36-derived releasates and immunoglobulin G (IgG) (2 μg/mL). Percentage aggregation is presented as mean ± SEM (N = 5; **P < .01 compared with platelets treated with thrombin and PGI₂, Mann-Whitney U test). (E) PRP from CD36^−/− mice (treated with apyrase) was stimulated with collagen (10 μg/mL) or treated with PGI₂ (5 nM) for 1 minute before stimulation in the presence or absence of CD36-derived releasates. Platelet aggregation is presented as mean ± SEM (N = 6). (F) As in (E), except platelets were treated with human TSP-1 (hTSP-1) (10 µg/mL). (G) WT and CD36^−/− platelets (5 × 10⁸ platelets/mL; incubated with apyrase) were stimulated with collagen (10 μg/mL) for 1 minute before stopping the reaction with lysis buffer. PDE3A was immunoprecipitated, and its activity was measured. Data are presented as percent activity above basal activity and presented as mean ± SEM (N = 3; P < .05). (H) Whole blood from WT and CD36^−/− mice (incubated with apyrase) was treated with PGI₂ (50 nM) alone, CRP-XL (10 μg/mL) alone, or PGI₂ followed by CRP-XL. Blood was fixed, permeabilized, and stained for VASP (Ser157) phosphorylation. Quantification is presented as fold increase in median fluorescence intensity over basal (N = 4; *P < .05, Mann-Whitney U test). NS, not significant.