RhoA downstream of G(q) and G(12/13) pathways regulates protease-activated receptor-mediated dense granule release in platelets

Abstract

Platelet secretion is an important physiological event in hemostasis. The protease-activated receptors, PAR 1 and PAR 4, and the thromboxane receptor activate the G(12/13) pathways, in addition to the G(q) pathways. Here, we investigated the contribution of G(12/13) pathways to platelet dense granule release. 2MeSADP, which does not activate G(12/13) pathways, does not cause dense granule release in aspirin-treated platelets. However, supplementing 2MeSADP with YFLLRNP (60muM), as selective activator of G(12/13) pathways, resulted in dense granule release. Similarly, supplementing PLC activation with G(12/13) stimulation also leads to dense granule release. These results demonstrate that supplemental signaling from G(12/13) is required for G(q)-mediated dense granule release and that ADP fails to cause dense granule release because the platelet P2Y receptors, although activate PLC, do not activate G(12/13) pathways. When RhoA, downstream signaling molecule in G(12/13) pathways, is blocked, PAR-mediated dense granule release is inhibited. Furthermore, ADP activated RhoA downstream of G(q) and upstream of PLC. Finally, RhoA regulated PKCdelta T505 phosphorylation, suggesting that RhoA pathways contribute to platelet secretion through PKCdelta activation. We conclude that G(12/13) pathways, through RhoA, regulate dense granule release and fibrinogen receptor activation in platelets.

Fig. 1. Effect of G_12/13 activation on 2MeSADP-induced platelet dense granule release in human platelets

Aspirin-treated and washed human platelets were stimulated with 100 nM 2MeSADP, 60 μM YFLLRNP or 60 μM YFLLRNP+100 nM 2MeSADP at 37°C while stirring. Platelet aggregation (A) and secretion (B) were measured in a Lumi aggregometer. The tracings are representative of data from at least three independent experiments.

Fig. 1. Effect of G_12/13 activation on 2MeSADP-induced platelet dense granule release in human platelets

Aspirin-treated and washed human platelets were stimulated with 100 nM 2MeSADP, 60 μM YFLLRNP or 60 μM YFLLRNP+100 nM 2MeSADP at 37°C while stirring. Platelet aggregation (A) and secretion (B) were measured in a Lumi aggregometer. The tracings are representative of data from at least three independent experiments.

Fig. 2. Effect of selective activation of Phospholipase C and G_12/13 pathways on platelet dense granule release in human platelets

Aspirin-treated and washed human platelets were stimulated with 80 μM m-3MFBS, 500 μM AYPGKF or 80 μM m-3MFBS+500 μM AYPGKF at 37°C while stirring. Platelet samples were incubated with 50 nM YM254890 (G_q protein inhibitor) at 37°C for 5 min before the addition of agonists. Platelet aggregation (A) and secretion (B) were measured in a Lumi aggregometer. The tracings are representative of data from at least three independent experiments.

Fig. 2. Effect of selective activation of Phospholipase C and G_12/13 pathways on platelet dense granule release in human platelets

Aspirin-treated and washed human platelets were stimulated with 80 μM m-3MFBS, 500 μM AYPGKF or 80 μM m-3MFBS+500 μM AYPGKF at 37°C while stirring. Platelet samples were incubated with 50 nM YM254890 (G_q protein inhibitor) at 37°C for 5 min before the addition of agonists. Platelet aggregation (A) and secretion (B) were measured in a Lumi aggregometer. The tracings are representative of data from at least three independent experiments.

Fig. 3. Effect of selective activation of Phospholipase C and G_12/13 pathways on platelet dense granule release in Gα_q null mouse platelets

Aspirin-treated and washed Gq−/− mouse platelets were stimulated with 80 μM m-3MFBS, 500 μM AYPGKF or 80 μM m-3MFBS+500 μM AYPGKF (A and B),100 nM 2 MeSADP, 500 μM AYPGKF or 100 nM 2MeSADP + 500 μM AYPGKF (C and D) at 37°C while stirring. Platelet aggregation (A and C) and secretion (B and D) were measured in a Lumi aggregometer. The tracings are representative of data from at least three independent experiments.

Fig. 3. Effect of selective activation of Phospholipase C and G_12/13 pathways on platelet dense granule release in Gα_q null mouse platelets

Aspirin-treated and washed Gq−/− mouse platelets were stimulated with 80 μM m-3MFBS, 500 μM AYPGKF or 80 μM m-3MFBS+500 μM AYPGKF (A and B),100 nM 2 MeSADP, 500 μM AYPGKF or 100 nM 2MeSADP + 500 μM AYPGKF (C and D) at 37°C while stirring. Platelet aggregation (A and C) and secretion (B and D) were measured in a Lumi aggregometer. The tracings are representative of data from at least three independent experiments.

Fig. 3. Effect of selective activation of Phospholipase C and G_12/13 pathways on platelet dense granule release in Gα_q null mouse platelets

Aspirin-treated and washed Gq−/− mouse platelets were stimulated with 80 μM m-3MFBS, 500 μM AYPGKF or 80 μM m-3MFBS+500 μM AYPGKF (A and B),100 nM 2 MeSADP, 500 μM AYPGKF or 100 nM 2MeSADP + 500 μM AYPGKF (C and D) at 37°C while stirring. Platelet aggregation (A and C) and secretion (B and D) were measured in a Lumi aggregometer. The tracings are representative of data from at least three independent experiments.

Fig. 3. Effect of selective activation of Phospholipase C and G_12/13 pathways on platelet dense granule release in Gα_q null mouse platelets

Aspirin-treated and washed Gq−/− mouse platelets were stimulated with 80 μM m-3MFBS, 500 μM AYPGKF or 80 μM m-3MFBS+500 μM AYPGKF (A and B),100 nM 2 MeSADP, 500 μM AYPGKF or 100 nM 2MeSADP + 500 μM AYPGKF (C and D) at 37°C while stirring. Platelet aggregation (A and C) and secretion (B and D) were measured in a Lumi aggregometer. The tracings are representative of data from at least three independent experiments.

Fig. 4. Effect of C3 Transferase on PAR-mediated dense granule secretion in human platelets

Aspirin-treated and washed human platelets were stimulated with 500 μM AYPGKF, 10 μM SFLLRN or 0.2 U/ml Thrombin at 37°C while stirring. Platelet samples were incubated with 20 μg/ml exoenzyme C3 Transferase at 37°C for 4 hrs before the addition of agonists. Platelet shape change (A) aggregation (B) and secretion (C and D) were measured in a Lumi aggregometer. The tracings are representative of data from at least three independent experiments. The secretion data in panel D were normalized to the maximum secretion (taken as 100%).

Fig. 4. Effect of C3 Transferase on PAR-mediated dense granule secretion in human platelets

Aspirin-treated and washed human platelets were stimulated with 500 μM AYPGKF, 10 μM SFLLRN or 0.2 U/ml Thrombin at 37°C while stirring. Platelet samples were incubated with 20 μg/ml exoenzyme C3 Transferase at 37°C for 4 hrs before the addition of agonists. Platelet shape change (A) aggregation (B) and secretion (C and D) were measured in a Lumi aggregometer. The tracings are representative of data from at least three independent experiments. The secretion data in panel D were normalized to the maximum secretion (taken as 100%).

Fig. 4. Effect of C3 Transferase on PAR-mediated dense granule secretion in human platelets

Aspirin-treated and washed human platelets were stimulated with 500 μM AYPGKF, 10 μM SFLLRN or 0.2 U/ml Thrombin at 37°C while stirring. Platelet samples were incubated with 20 μg/ml exoenzyme C3 Transferase at 37°C for 4 hrs before the addition of agonists. Platelet shape change (A) aggregation (B) and secretion (C and D) were measured in a Lumi aggregometer. The tracings are representative of data from at least three independent experiments. The secretion data in panel D were normalized to the maximum secretion (taken as 100%).

Fig. 4. Effect of C3 Transferase on PAR-mediated dense granule secretion in human platelets

Aspirin-treated and washed human platelets were stimulated with 500 μM AYPGKF, 10 μM SFLLRN or 0.2 U/ml Thrombin at 37°C while stirring. Platelet samples were incubated with 20 μg/ml exoenzyme C3 Transferase at 37°C for 4 hrs before the addition of agonists. Platelet shape change (A) aggregation (B) and secretion (C and D) were measured in a Lumi aggregometer. The tracings are representative of data from at least three independent experiments. The secretion data in panel D were normalized to the maximum secretion (taken as 100%).

Fig. 5. Rho A activation downstream of Gq pathways

A) Aspirin-treated and washed human platelets were stimulated with 1 μM 2MeSADP at 37°C for the indicated times with or without 5 minute pre-incubation with the Gq inhibitor YM-254890. Active RhoA was then assayed using agarose coupled GST-Rhotekin-RBD and Western Blotting probing for RhoA. (B) GST-pull down of active RhoA was performed on lysates from unstimulated platelets or platelets pre-incubated with or without the PLC inhibitor U73122 or its control compound (U73343) for 8 minutes prior to activation with 2MeSADP for 10 sec. Active RhoA was detected via Western Blotting with an anti –Rho monoclonal antibody. The blot is representative of data from at least three independent experiments.

Fig. 5. Rho A activation downstream of Gq pathways

A) Aspirin-treated and washed human platelets were stimulated with 1 μM 2MeSADP at 37°C for the indicated times with or without 5 minute pre-incubation with the Gq inhibitor YM-254890. Active RhoA was then assayed using agarose coupled GST-Rhotekin-RBD and Western Blotting probing for RhoA. (B) GST-pull down of active RhoA was performed on lysates from unstimulated platelets or platelets pre-incubated with or without the PLC inhibitor U73122 or its control compound (U73343) for 8 minutes prior to activation with 2MeSADP for 10 sec. Active RhoA was detected via Western Blotting with an anti –Rho monoclonal antibody. The blot is representative of data from at least three independent experiments.

Fig. 6. Effect of C3 Transferase on PAR-mediated activation-dependent PKCδ phosphorylation

Aspirin-treated and washed human platelets were stimulated with 250 μM AYPGKF, 10 μM SFLLRN, or 0.2 U Thrombin at 37°C while stirring. Platelet samples were incubated with 20 μg/ml exoenzyme C3 Transferase at 37°C for 4 hrs before the addition of agonist. PKCδ activation was measured by detecting T-505 phosphorylation by western blotting using phospho-specific antibodies. Total Erk1/2 was used as lane loading control. The blot is representative of data from at least three independent experiments.

Fig. 7. Model depicting RhoA pathways are activated downstream of both G_q and G_12/13 pathways

PAR activation by thrombin results in activation of G_q and G_12/13 pathways. G_12/13 pathways potentitate the G_q-mediated dense granule release and fibrinogen receptor activation. In the absence of G_q pathways these events do not occur. Maximum stimulation of G_q pathways by PARs can circumvent requirement of G_12/13 pathways due to possible direct activation of RhoA pathways.