FIGURE 2.

Inhibition of platelet activity by prostacyclin (PGI₂) requires PKA-I/AKAP interactions. (A) Washed platelets (2.5 × 10⁸/ml) were left untreated or pre-incubated with RIAD-Arg₁₁ (2 μM) or scrRIAD-Arg₁₁ (2 μM) for 1 h followed by addition of PGI₂ (50 nM) for 1 mine and then stimulated with collagen (5 μg/ml) for 4 min under stirring conditions. (Ai) representative aggregation traces. (Aii) Collated data of four independent experiments expressed as mean ± SEM. ± p < 0.01 compared to absence of PGI₂; *p < 0.05 compared to PGI₂ alone. (B) As in (A) except platelet ATP secretion was measured by the signal released by a Luciferin-Luciferase reaction using a Chrono-log Lumi-aggregometer. (Bi) representative ATP secretion traces. (Bii) Collated data of four independent experiments expressed as mean ± SEM. ±p < 0.01 compared to absence of PGI₂; *p < 0.05 compared to PGI₂ alone. (C) Washed platelets (5 × 10⁷/ml) were placed on human serum (i) or collagen-coated coverslips (50 μg/ml) (ii–iv) for 60 min at 37°C in the absence (ii) or presence (ii) PGI₂ (100 nM). In (iv) washed platelets were first pre-treated with RIAD-Arg₁₁ (2 μM) for 1 h followed by addition of PGI₂ (100 nM). At the end of the incubation period, non-adherent platelets were removed by washing with PBS. The remaining adherent platelets were fixed, permeabilized with 0.1% Triton, and stained for F-action with TRITC-conjugated phalloidin then visualized with fluorescence microscope. Scale bar is 5 μm.