PGE₁ modulates thrombin-induced shape change and MLC phosphorylation. (A) Washed platelets (3 × 10⁸ platelets per milliliter) were preincubated with apyrase (2 U/mL), indomethacin (10 µM), and EGTA (1 mM) followed by stimulation with thrombin (0.05 U/mL) in the presence and absence of PGE₁ (0.2 to 100 nM), and traces were recorded for 2 minutes. Shown are representative traces of 3 separate experiments. (B) Washed platelets (3 × 10⁸ platelets per milliliter) were stimulated with thrombin (0.05 U/mL) for the indicated time points and the levels of MLC^ser19 were assessed by immunoblotting. (i) Representative immunoblots (IB) from 3 independent experiments. (ii) Densitometric analysis of MLC^ser19 phosphorylation of 3 different experiments expressed as arbitrary units (AU). **P < .01 compared with basal levels. (C) Washed platelets (3 × 10⁸ platelets per milliliter) were stimulated with thrombin (0.05 U/mL) for 1 minute in the presence or absence of PGE₁ (0.2 to 100 nM) and phospho-MLC^ser19 was assessed by immunoblotting. (i) Representative immunoblots from 4 independent experiments. (ii) Densitometry of MLC^ser19 phosphorylation from 4 different experiments. **P < .01 compared with thrombin alone. (D) Washed platelets (2 × 10⁸ platelets per milliliter) were treated with PGE₁ (100 nM) in the presence and absence of RO1138452 (1 µM) or Rp-8-CPT-cAMPS (RP; 500 µM)/KT-5720 (20 µM), and cAMP levels were measured. Data are mean ± standard error of the mean of 3 experiments and are expressed as fmol cAMP/1 × 10⁷ platelets. **P < .01 compared with basal levels. (E) Same as in (D), except platelets were lysed and immunoblotted for phospho-VASP-ser¹⁵⁷. Blot is representative of 5 independent experiments. (F) Same as in (A), except that experiments were performed in the presence of RO1138452 (1 µM) or Rp-8-CPT-cAMPS (500 µM)/KT-5720 (20 µM). Shown are representative traces of 3 separate experiments.