American Society of Hematology

Figure 5

$Figure 5. Inhibition of thrombin-stimulated membrane localization of RhoA by PGE1. (A) Washed platelets (5 × 108 platelets per milliliter) were stimulated with thrombin for 15 to 60 seconds, and reactions were terminated by the addition of fractionation buffer and snap frozen. Samples were separated into soluble (S) and particulate (P) fractions by ultracentrifugation and were analyzed for the presence of RhoA, PLCγ2, and integrin β3 by immunoblotting. (i) Shown are representative blots of 5 independent experiments. (ii) Densitometry for RhoA in the particulate fraction from 5 different experiments. *P < .05 unstimulated compared with thrombin (15 seconds). (B) Washed platelets (5 × 108 platelets per milliliter) were stimulated with thrombin for 15 and 30 seconds in the presence and absence of PGE1 (100 nM) or GGTI-298 (50 µM). Soluble and particulate fractions were analyzed for the presence of RhoA, RhoA-ser188, PLCγ2, and integrin β3 by immunoblotting. (i) Representative blots of 4 independent experiments. (ii) Densitometry of the presence of RhoA in the particulate fractions at 15 seconds. *P < .05 thrombin compared with thrombin/PGE1 or thrombin/GGTI-298 (15 seconds). (iii) Densitometry of the presence of phospho-RhoAser188 in the soluble fraction at 15 seconds compared with untreated cells. **P < .01 thrombin compared with thrombin/PGE1. (C) Washed platelets (5 × 108 platelets per milliliter) were stimulated with thrombin (0.05 U/mL) for 1 minute in the presence and absence of PGE1 (100 nM) or GGTI-298 (50 µM), and reaction was stopped with lysis buffer. Lysates were incubated with Rhotekin-RBD beads (25 ng) for 90 minutes at 4°C. After washing the beads, the level of activated RhoA (GTP-RhoA) was assessed by immunoblotting. Lysates remaining after pull-down assay were used to examine phospho-RhoA-ser188, VASP-ser157, and MLC-ser19. (i) Representative immunoblots from 3 independent experiments. (ii) Densitometry of RhoA-GTP from 3 different experiments. *P < .05 thrombin alone compared with thrombin/PGE1 and GGTI-298. (D) 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 GGTI-298 (50 μM), and traces were recorded for 2 minutes. Shown are representative traces of 3 separate experiments. (E) Washed platelets (5 × 108 platelets per milliliter) was treated with PGE1 (100 nM) for 1 minute in the presence (white bars) and absence (black bars) of GGTI-298 (50 µM), and the phosphorylation of RhoA and VASP was assessed by immunoblotting. (i) Representative immunoblots of phospho-RhoA-ser188 and phospho-VASP-ser157 from 3 independent experiments. (ii) Densitometry of phosphorylation of RhoA from 3 different experiments. NS, not significant.$

Inhibition of thrombin-stimulated membrane localization of RhoA by PGE₁. (A) Washed platelets (5 × 10⁸ platelets per milliliter) were stimulated with thrombin for 15 to 60 seconds, and reactions were terminated by the addition of fractionation buffer and snap frozen. Samples were separated into soluble (S) and particulate (P) fractions by ultracentrifugation and were analyzed for the presence of RhoA, PLCγ₂, and integrin β₃ by immunoblotting. (i) Shown are representative blots of 5 independent experiments. (ii) Densitometry for RhoA in the particulate fraction from 5 different experiments. *P < .05 unstimulated compared with thrombin (15 seconds). (B) Washed platelets (5 × 10⁸ platelets per milliliter) were stimulated with thrombin for 15 and 30 seconds in the presence and absence of PGE₁ (100 nM) or GGTI-298 (50 µM). Soluble and particulate fractions were analyzed for the presence of RhoA, RhoA-ser¹⁸⁸, PLCγ₂, and integrin β₃ by immunoblotting. (i) Representative blots of 4 independent experiments. (ii) Densitometry of the presence of RhoA in the particulate fractions at 15 seconds. *P < .05 thrombin compared with thrombin/PGE₁ or thrombin/GGTI-298 (15 seconds). (iii) Densitometry of the presence of phospho-RhoA^ser188 in the soluble fraction at 15 seconds compared with untreated cells. **P < .01 thrombin compared with thrombin/PGE₁. (C) Washed platelets (5 × 10⁸ platelets per milliliter) were stimulated with thrombin (0.05 U/mL) for 1 minute in the presence and absence of PGE₁ (100 nM) or GGTI-298 (50 µM), and reaction was stopped with lysis buffer. Lysates were incubated with Rhotekin-RBD beads (25 ng) for 90 minutes at 4°C. After washing the beads, the level of activated RhoA (GTP-RhoA) was assessed by immunoblotting. Lysates remaining after pull-down assay were used to examine phospho-RhoA-ser¹⁸⁸, VASP-ser¹⁵⁷, and MLC-ser¹⁹. (i) Representative immunoblots from 3 independent experiments. (ii) Densitometry of RhoA-GTP from 3 different experiments. *P < .05 thrombin alone compared with thrombin/PGE₁ and GGTI-298. (D) 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 GGTI-298 (50 μM), and traces were recorded for 2 minutes. Shown are representative traces of 3 separate experiments. (E) Washed platelets (5 × 10⁸ platelets per milliliter) was treated with PGE₁ (100 nM) for 1 minute in the presence (white bars) and absence (black bars) of GGTI-298 (50 µM), and the phosphorylation of RhoA and VASP was assessed by immunoblotting. (i) Representative immunoblots of phospho-RhoA-ser¹⁸⁸ and phospho-VASP-ser¹⁵⁷ from 3 independent experiments. (ii) Densitometry of phosphorylation of RhoA from 3 different experiments. NS, not significant.

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