The phosphorylation of platelet RhoA in response to PGE₁ is cAMP-PKA–dependent. (A) Washed platelets (3 × 10⁸ platelets per milliliter) were incubated with PGE₁ (0 to 100 nM) for 1 minute, and the phosphorylation of RhoA-ser¹⁸⁸ and VASP-ser¹⁵⁷ were determined by immunoblotting. (i) Representative immunoblots from 3 independent experiments. (ii) Densitometry for phospho-RhoA-ser¹⁸⁸ from 3 different experiments. *P < .05 for untreated compared with PGE₁ (100 nM). (B) Washed platelets (3 × 10⁸ platelets per milliliter) were treated with PGE₁ (100 nM) for 1 minute or 8-CPT-6-Phe-cAMP (50 µM) for 2 minutes in the presence or absence of Rp-8-CPT-cAMPS (500 µM)/KT-5720 (20 µM). RhoA-ser¹⁸⁸ and VASP-ser¹⁵⁷ phosphorylation were assessed by immunoblotting. (i) Representative immunoblots from 3 independent experiments. (ii) Densitometry for phospho-RhoA-ser¹⁸⁸ (black bars) and VASP-ser¹⁵⁷ (white bars) from 3 different experiments. *P < .05 PGE₁ only compared with PGE₁ with PKA inhibitors. §P < .05 8-CPT-6-Phe-cAMP compared with 8-CPT-6-Phe-cAMP with PKA inhibitors. (C) Washed platelets (8 × 10⁸ platelets per milliliter) were lysed, and RhoA was immunoprecipitated from the lysate overnight. RhoA and PKAc in the immunoprecipitates (IP) were detected by immunoblotting. Representative blots for 3 experiments. (D) In lanes 1 to 4, recombinant active PKAc_β (0.1 to 0.5 µg) was incubated with recombinant His-tagged RhoA (55 ng) in the presence of adenosine triphosphate (ATP; 400 µM) for 15 minutes at 37°C. Reactions were stopped with 2× Laemmli buffer, and RhoA^ser188 phosphorylation was assessed by immunoblotting. In lane 4, ATP was omitted from the reaction mixture. In lane 5, whole-cell lysates from PGE₁ (100 nM) –treated platelets were immunoblotted for phospho-RhoA-ser¹⁸⁸. Shown are representative immunoblots from 3 independent experiments.