RhoA and Rac1 GTPases Differentially Regulate Agonist-Receptor Mediated Reactive Oxygen Species Generation in Platelets

Abstract

Agonist induced generation of reactive oxygen species (ROS) by NADPH oxidases (NOX) enhances platelet aggregation and hence the risk of thrombosis. RhoA and Rac1 GTPases are involved in ROS generation by NOX in a variety of cells, but their roles in platelet ROS production remain unclear. In this study we used platelets from RhoA and Rac1 conditional knockout mice as well as human platelets treated with Rhosin and NSC23767, rationally designed small molecule inhibitors of RhoA and Rac GTPases, respectively, to better define the contributions of RhoA and Rac1 signaling to ROS generation and platelet activation. Treatment of platelets with Rhosin inhibited: (a) U46619 induced activation of RhoA; (b) phosphorylation of p47phox, a critical component of NOX; (c) U46619 or thrombin induced ROS generation; (d) phosphorylation of myosin light chain (MLC); (e) platelet shape change; (f) platelet spreading on immobilized fibrinogen; and (g) release of P-selectin, secretion of ATP and aggregation. Conditional deletion of RhoA or Rac1 gene inhibited thrombin induced ROS generation in platelets. Addition of Y27632, a RhoA inhibitor, NSC23766 or Phox-I, an inhibitor of Rac1-p67phox interaction, to human platelets blocked thrombin induced ROS generation. These data suggest that: (a) RhoA/ROCK/p47phox signaling axis promotes ROS production that, at least in part, contributes to platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC; and (b) RhoA and Rac1 differentially regulate ROS generation by inhibiting phosphorylation of p47phox and Rac1-p67phox interaction, respectively.

Fig 1. Rhosin inhibited RhoA GTPase activation and gene targeting of RhoA deleted expression of RhoA in platelets.

(A-D) Washed human platelets were incubated with U46619 (0.01 μM) for one minute. The reactions were terminated by adding ice-cold HEPES-buffered Tyrode’s solution containing protease inhibitors cocktail. GTP loading of RhoA, Rac1 and Cdc42 was analyzed as described in the methods section. A two minute pre-incubation of platelets with Rhosin inhibited U46619 induced RhoA-GTP formation in a concentration-dependent manner. Rhosin minimally inhibited Cdc42-GTP and Rac1-GTP formation. (E-F) Conditional RhoA knockout mice were generated as described previously [29]. The Western blots and the bar graph show that gene targeting of RhoA completely deleted RhoA expression and partially decreased Cdc42 or Rac1 expression in platelets. Total RhoA, Rac1, Cdc42 and GADPH are shown as loading controls. The data in bar graphs are mean ± SE from three experiments (*p<0.05).

Fig 2. Rhosin and Y27632 but not Phox-I inhibited phosphorylation of p47^phox.

(A-B) A two minute incubation of washed human platelets with U46619 (0.5 μM) induced phosphorylation of p47^phox. Addition of Rhosin (10, 30 μM) or Y27632 (30 M), a known inhibitor of RhoA, two minutes prior to stimulation with U46619 or thrombin inhibited phosphorylation of p47^phox. (C-D) A two minute pre-incubation of platelets with Phox-I (10 μM), an inhibitor of Rac1-p67^phox interaction, did not inhibit phosphorylation of p47^phox. Phosphorylation of p47^phox was quantified by densitometry. Data in the bar graphs are mean ± SE from three experiments (*p<0.01, **p<0.001).

Fig 3. Inhibition of RhoA by Rhosin blocked ROS generation.

(A-B) Incubation of washed human platelets with Rhosin inhibited U46619 (0.1 μM) or thrombin (0.1 U/ml) induced ROS generation in a concentration-dependent manner. (C) Incubation of washed human platelets with Y27632 inhibited thrombin (0.1 U/ml) induced ROS generation. (D) Thrombin (0.1 U/ml) induced ROS generation is diminished in RhoA^-/-, as compared to RhoA^+/+, platelets. Generation of reactive oxygen species in dcf-da loaded washed platelets was monitored by flow cytometry as detailed in the Methods section. (The data are mean ± SE, n = 4. *p<0.01, **p<0.001).

Fig 4. Inhibition of RhoA by Rhosin blocked phosphorylation of myosin light chain and platelet shape change.

(A-B) Addition of Rhosin (30 μM) to aspirin (1 mM) treated washed human platelets containing apyrase (3 U/ml) two minutes prior to stimulation withU46619 (0.01 μM) or thrombin (0.01 U/ml) blocked phosphorylation of myosin light chain. The reactions were terminated at 30 seconds by adding 5x sample buffer and samples were processed for Western blotting and probed for MLC, p-MLC. Phosphorylation of myosin light chain was quantified by densitometry. Data in the bar graphs are mean ± SE from three experiments (*p<0.001). (C-D) Aspirin (1 mM) treated washed human platelets containing apyrase (3 U/ml) were incubated with U46619 (0.01 μM) or thrombin (0.005 U/ml) and platelet shape change was recorded as a decrease in light transmittance using a Lumi-Aggregometer. A two minute pre-incubation with Rhosin blocked platelet shape change by U46619 or thrombin. The shape change racings are representative of four independent experiments.

Fig 5. Inhibition of RhoA by Rhosin or gene targeting of RhoA diminished platelet spreading on immobilized fibrinogen.

(A-C) RhoA^+/+ platelets (A), RhoA^+/+ platelets with Rhosin (30 μM, B) or RhoA^-/- platelets (C) were layered over fibrinogen (3 μg/ml) coated cover slips in the presence of apyrase (3 U/ml) for five min. The cover slips were washed and adherent platelets were processed for immuno-fluorescence confocal microscopy as detailed in the methods section. Platelets treated with Rhosin (B) and RhoA^-/- platelets (C), as compared to DMSO (A) exhibited diminished spreading and filopodia formation on immobilized fibrinogen. (D-E) The bar graphs show that spreading of Rhosin treated (n = 23) or RhoA-deficient (n = 23, S4 Fig), as compared to the matching RhoA^+/+ platelets (n = 28) was diminished significantly (**p<0.001). The Rhosin treated or RhoA-deficient platelets exhibited a significant decrease in the number of filopodia (*p<0.01). Spreading of washed platelets on fibrinogen was quantified using Image J software (http://rsbweb.nih.gov/ij).

Fig 6. Inhibition of RhoA by Rhosin blocked U46619 or thrombin induced release of P-selectin, secretion of ATP and platelet aggregation.

(A-B) Incubation of washed human platelets with U46619 or thrombin induced release of p-selectin from platelet α-granules. Addition of Rhosin to platelets two minutes prior to stimulation with U46619 or thrombin inhibited expression of p-selectin in a concentration dependent manner. P-selectin was quantified by flow cytometry in aspirin treated (1 mM) washed platelets, containing 0.2% bovine serum albumin and apyrase (0.4 U/ml) as detailed in the methods section. Results are reported as means ± SD (n = 4, *p<0.01, **p<0.001). (C-D) Addition of U46619 or thrombin to washed human platelets induced ATP secretion and (E-F) platelet aggregation. A two minute pre-incubation with Rhosin inhibited ATP secretion and platelet aggregation in a concentration dependent manner. A Lumi-Aggregometer from Chrono-Log-Corporation (Havertown, PA) was used to monitor platelet ATP secretion and aggregation. The secretion and aggregation tracings are representative of 3 independent experiments.

Fig 7. Deficiency or inhibition of Rac1 GTPase or inhibition of Rac1-P67^phox interaction prevents ROS generation.

(A) Incubation of washed human platelets with NSC23766 or Phox-I inhibited thrombin (0.1 U/ml, mean ± SE, n = 4, *p < 0.001) induced ROS generation. Diphenyleneiodonium (DPI), a non-selective inhibitor of ROS generation also inhibited thrombin induced ROS generation. (B) Conditional Rac1 knockout mice were generated as described previously [30]. Addition of thrombin (0.1 U/ml) induced significantly less ROS generation in Rac1^-/-, as compared to Rac1^+/+, platelets (mean ± SE, n = 4, *p < 0.001). Generation of reactive oxygen species in dcf-da loaded washed platelets was monitored by flow cytometry as detailed in the Methods section.