Small molecule targeting the Rac1-NOX2 interaction prevents collagen-related peptide and thrombin-induced reactive oxygen species generation and platelet activation

Abstract

Essentials Reactive oxygen species (ROS) generation by NOX2 plays a critical role in platelet activation. Rac1 regulation of NOX2 is important for ROS generation. Small molecule inhibitor of the Rac1-p67^phox interaction prevents platelet activation. Pharmacologic targeting of Rac1-NOX2 axis can be a viable approach for antithrombotic therapy.

Summary: Background Platelets from patients with X-linked chronic granulomatous disease or mice deficient in nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase isoform NOX2 exhibit diminished reactive oxygen species (ROS) generation and platelet activation. Binding of Rac1 GTPase to p67^phox plays a critical role in NOX2 activation by facilitating the assembly of the NOX2 enzyme complex. Objective We tested the hypothesis that Phox-I, a rationally designed small molecule inhibitor of Rac-p67^phox interaction, may serve as an antithrombosis agent by suppressing ROS production and platelet activation. Results Collagen-related peptide (CRP) induced ROS generation in a time-dependent manner. Platelets from Rac1^-/- mice or human platelets treated with NSC23766, a specific Rac inhibitor, produced significantly less ROS in response to CRP. Treatment of platelets with Phox-I inhibited diverse CRP-induced responses, including: (i) ROS generation; (ii) release of P-selectin; (iii) secretion of ATP; (iv) platelet aggregation; and (v) phosphorylation of Akt. Similarly, incubation of platelets with Phox-I inhibited thrombin-induced: (i) secretion of ATP; (ii) platelet aggregation; (iii) rise in cytosolic calcium; and (iv) phosphorylation of Akt. In mouse models, intraperitoneal administration of Phox-I inhibited: (i) collagen-induced platelet aggregation without affecting the tail bleeding time and (ii) in vivo platelet adhesion/accumulation at the laser injury sites on the saphenous vein without affecting the time for complete cessation of blood loss. Conclusions Small molecule targeting of the Rac1-p67^phox interaction may present an antithrombosis regimen by preventing GPVI- and non-GPVI-mediated NOX2 activation, ROS generation and platelet function without affecting the bleeding time.

Keywords: NADPH oxidase; Rac1 GTP-binding protein; platelet activation; reactive oxygen species; thrombosis.

Fig. 1.

Gene targeting of Rac1 GTPase or small molecule inhibition of Rac1 diminished CRP-induced ROS generation in platelets. (A) CRP (0.5 μg mL⁻¹) was added to washed human platelets and ROS was measured at the indicated time-points. CRP increased ROS generation in a time-dependent manner. (B) NSC23766, a Rac GTPase inhibitor [23], was added to platelets 2 min before stimulation with CRP (0.5 μg mL⁻¹) and ROS was recorded at 10 min. (C) CRP (0.5 μg mL⁻¹) was added to platelets from conditional Rac1 knockout mice [23] and ROS was measured at 10 min. Generation of ROS in dcf-da loaded platelets was monitored by flow cytometry as detailed in the Methods section. The data are mean ± SE, n = 4. *P < 0.0001, **P < 0.001. CRP, collagen-related peptide; MFI, mean fluorescence intensity; ROS, reactive oxygen species; SE, standard error.

Fig. 2.

Phox-I inhibited CRP-induced ROS generation, release of p-selectin, secretion of ATP and platelet aggregation. (A) Phox-I, a Rac1-p67^phox inhibitor [22], was added to washed human platelets 2 min before addition of CRP and ROS generation was measured at 10 min. Phox-I inhibited ROS generation in a concentration-dependent manner (n = 4, mean ± SE *P < 0.0001). (B) Phox-I was added to washed human platelets 2 min prior to addition of CRP and the release of p-selectin from platelet α-granules was quantified by flow cytometry in aspirin-treated (1 mM) washed platelets, containing 0.2% bovine serum albumin and apyrase (0.4 U mL⁻¹) as detailed in the Methods section. Phox-I inhibited the release of p-selectin in a concentration-dependent manner (mean ± SE, n = 4, *P < 0.0001). (C) CRP was added to washed human platelets 2 min after addition of Phox-I and ATP secretion was recorded using the Chrono-Log Lumi-Aggregometer (Havertown, PA). Phox-I inhibited ATP secretion (mean ± SE, n = 4, *P < 0.0001). (D) CRP was added to washed human platelets 2 min after addition of Phox-I and platelet aggregation was recorded using the Chrono-Log Lumi-Aggregometer. Addition of Phox-I to platelets inhibited aggregation in a concentration-dependent manner. (E) Platelet aggregation was analyzed by quantifying percent transmittance (mean ± SE, n = 4, *P < 0.0001, **P < 0.001). CRP, collagen-related peptide; ROS, reactive oxygen species; SE, standard error. [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 3.

Phox-I blocked thrombin-induced ATP secretion, platelet aggregation and rise in platelet cytosolic calcium. (A–C) Addition of thrombin to washed human platelets induced ATP secretion and platelet aggregation. A 2 min pre-incubation with Phox-I inhibited ATP secretion (mean ± SE, n = 4, *P < 0.0001) and platelet aggregation (n = 4, *P < 0.0001, **P < 0.001, ***P < 0.05) in a concentration-dependent manner. A Lumi-Aggregometer from Chrono-Log-Corporation was used to monitor platelet ATP secretion and aggregation. (D) Addition of thrombin to washed human platelets increased the cytosolic calcium level. Treatment of platelets with Phox-I 2 min before addition of thrombin inhibited the rise in calcium in a concentration-dependent manner. Changes in calcium levels were quantified in Fura2/AM loaded platelets as detailed in the methods section. [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 4.

Phox-I inhibition of platelet aggregation is surmountable. Phox-I was added to platelets 2 min prior to addition of CRP (left panels) or thrombin (right panels) and aggregation was recorded using a Chrono-Log Aggregometer. Phox-I inhibited aggregation induced by CRP or thrombin. Increase in CRP or thrombin concentrations gradually reversed the inhibitory effect of Phox-I. The aggregation tracings are representative of three experiments. CRP, collagen-related peptide. [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 5.

Phox-I diminished platelet spreading on immobilized fibrinogen. Washed human platelets in the absence (A) or presence of (B) Phox-I (10 μM) were layered over fibrinogen (5 μg mL⁻¹) coated cover slips in the presence of apyrase (3 U mL⁻¹) for 10 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 Phox-I (B), as compared with DMSO (A), exhibited diminished spreading on immobilized fibrinogen. (C) The bar graph shows that spreading of Phox-I, as compared with DMSO, is diminished (***P < 0.05). Spreading of washed platelets on fibrinogen was quantified using Image J software (http://rsbweb.nih.gov/ij). DMSO, dimethylsulfoxide. [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 6.

Phox-I inhibited CRP or thrombin-induced phosphorylation of Akt. Phox-I was added to washed human platelets 2 min prior to addition of CRP or thrombin. The reactions were terminated at 5 min by adding 4× sample buffer and processed for western blotting and probed for Akt, p-Akt and β-tubulin. Phosphorylation was quantified by densitometry. The data in the bar graphs are mean ± SE from three experiments (*P < 0.0001, **P < 0.001, ***P < 0.05). CRP, collagen-related peptide; SE, standard error

Fig. 7.

Administration of Phox-I to wild-type mice blocked ex vivo platelet aggregation without altering the tail bleeding times. (A–D) Collagen-induced platelet aggregation in citrated platelet-rich plasma from mice administered Phox-I is completely blocked at the lower collagen concentrations and is partially recovered at the higher collagen concentrations. (E) Platelet aggregation was analyzed by quantifying percent transmittance (*P < 0.0001, **P < 0.001). (F) Tail bleeding times were assessed by cutting 5 mm off the tails of mice after 20 min of intraperitoneal administration of Phox-I. The dot plot shows that Phox-I administration did not affect the tail bleeding time. [Color figure can be viewed at wileyonlinelibrary.com]

Fig. 8.

Administration of Phox-I to wild-type mice diminished accumulation of platelets at the laser injury sites at the saphenous vein without affecting the bleeding time. Mice were administered Phox-I (12 mg kg⁻¹) or DMSO vehicle control. (A) Representative images taken 1 min after laser injury. (B) Sum platelet intensity over time recorded at the injury sites is diminished in the Phox-I-treated mice. (C) Sum platelet intensity at 1 min after laser injury is significantly more diminished in the Phox-I-treated than control mice (**P < 0.001). (D) Time to complete stoppage of blood loss (in vivo bleeding time) after laser injury is essentially unaltered. [Color figure can be viewed at wileyonlinelibrary.com]