PGE₁ modulates the formation of a RhoA/ROCK/MYPT1 signaling complex. (A) Washed platelets (5 × 10⁸ platelets per milliliter) were stimulated with thrombin (0.05 U/mL) for 1 minute in the presence or absence of Y27632 (10 µM) and BAPTA-AM (20 µM); phospho-MYPT1-thr⁸⁵³ was then assessed by immunoblotting. (i) Representative immunoblots from 3 independent experiments. (ii) Densitometry of phospho-MYPT1-thr⁸⁵³ from 3 different experiments. **P < .01 thrombin compared with basal levels; §P < .05 thrombin alone compared with Y27632. (B) Same as in (A), except platelets were treated with PGE₁ (100 nM) or GGTI-298 (50 µM) prior to stimulation with thrombin, and phospho-MYPT1-thr⁸⁵³ was assessed by immunoblotting. (i) Representative immunoblots from 3 independent experiments. (ii) Densitometry of MYPT1-thr⁸⁵³ phosphorylation of 3 different experiments. **P < .01 thrombin alone compared with thrombin/PGE₁ and GGTI-298. (C) Washed platelets (5 × 10⁸ platelets per milliliter) were stimulated with thrombin (0.05 U/mL) for 1 minute in the presence or absence of PGE₁ (100 nM), and phospho-MYPT1-thr⁸⁵³ was assessed by immunoblotting. In some cases, platelets were also treated with Rp-8-CPT-cAMPS (500 µM)/KT-5720 (20 µM). (i) Representative immunoblots from 3 independent experiments. (ii) Densitometric analysis of phospho-MYPT1^thr853 of 3 different experiments. *P < .05 compared with thrombin only. (D) Same as in (B), except platelets were treated with Rho Activator I (30 µM) for 2 minutes instead of thrombin. (E) Same as in (B), except platelets were treated with oxLDL (50 μg/mL) for 15 seconds instead of thrombin. (F) Washed platelets (8 × 10⁸ platelets per milliliter) were stimulated with thrombin (0.05 U/mL) for 1 minute in the presence and absence of PGE₁ (100 nM). Reaction was stopped with lysis buffer and ROCK1 was immunoprecipitated. The immunoprecipitates were then immunoblotted for the presence of ROCK2, MYPT1, and RhoA. (i) Representative immunoblots of 4 independent experiments. (ii) Densitometric analysis of amount of RhoA present in the immunoprecipitates. *P < .05 thrombin compared with thrombin alone. (G) Same as in (F), except PP1δ was immunoprecipitated and was incubated for 1 hour at room temperature with 5 mM para-nitrophenyl phosphate, and absorbance was measured at 405 nm. Graph is representative of 5 different experiments. **P < .01 thrombin compared with basal activity; *P < .05 thrombin alone compared with thrombin with PGE₁. (H) Samples from (G) were immunoblotted for PP1δ and MYPT1. (i) Representative immunoblots of 4 independent experiments. (ii) Densitometric analysis of total MYPT1 of 4 different experiments. *P < .05 thrombin compared with basal levels; §P < .05 thrombin alone compared with thrombin with PGE₁.