NADPH oxidase 2 inhibitor GSK2795039 exerts antiplatelet and antithrombotic activity

Figures

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  Fig. 1. GSK2795039 attenuates collagen-stimulated platelet aggregation and ROS production. (A) Washed human platelets (5 × 108/mL) were pretreated with vehicle (0.5% DMSO) or various concentrations of GSK2795039 for 5 min before stimulation with collagen (10 µg/mL). Platelet aggregation was monitored by measuring changes in light transmission, with 100% transmission defined as the buffer control. Data represent the mean ± standard deviation from three independent donors. (B–D) For analyzing the levels of intracellular ROS (B), extracellular ROS (C), and extracellular superoxide anion (D), following CM-H2DCFDA loading (B); in the presence of DCFH2 (C) or L-012 (D), washed human platelets were treated as in A. (B, C) Fluorescence change was measured in a fluorometer cuvette under constant stirring conditions. Upper panels show representative traces. (D) Chemiluminescence was measured in a luminometer. (E) NOX activity assay. Washed human platelets were treated as in A. Cell lysates were prepared and then incubated with NADPH. Superoxide anion production in the lysates was measured by a chemiluminescence assay using L-012. (F) To quantify extracellular H2O2, the supernatant from the…

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  Fig. 2. GSK2795039 inhibits protein tyrosine phosphorylation in collagen-stimulated platelets. Washed platelets were treated with vehicle or GSK2795039 at the indicated concentrations for 5 min and stimulated with collagen (10 µg/mL) for 2 min. (A) Total protein tyrosine phosphorylation levels were analyzed by immunoblotting, with β-actin serving as a loading control. (B) After treatment with iodoacetamide and N-ethylmaleimide in a hypoxic chamber, platelet lysates were further incubated with dithiotreitol. The activity of total protein tyrosine phosphatases (PTPs), which were recovered from oxidation, was determined using tyrosine phosphopeptide as the substrate. (C) The phosphorylation of Syk, LAT, Vav1, and Btk at specific tyrosine residues was assessed by immunoblotting with phospho-specific antibodies. Representative immunoblots are shown alongside quantitative analysis of band intensity. Data represent the mean ± standard deviation. Statistical significance: * p < 0.05, ** p < 0.01 and *** p < 0.001.

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  Fig. 3. GSK2795039 suppresses PLCγ2/PKC/Ca2+ pathway in collagen-stimulated platelets. (A and B) Washed human platelets were treated with vehicle or GSK2795039 for 5 min and stimulated with collagen (10 µg/mL) for 2 min. Phosphorylation of PLCγ2 at Tyr753 (A) and PKC substrates (B) were analyzed by immunoblotting, respectively. Representative immunoblots are shown alongside quantitative analysis of band intensity. (C) Intracellular calcium mobilization was measured in Fluo-3-AM-loaded washed human platelets stimulated with collagen for 2 min in the presence of either 0.5 mM EGTA (left) or 1 mM extracellular CaCl2 (right). Fluorescence intensity expressed in arbitrary units (AU) was recorded, and maximum intensity values are presented. All data represent the mean ± standard deviation. Statistical significance: * p < 0.05, ** p < 0.01 and *** p < 0.001.

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  Fig. 4. GSK2795039 inhibits collagen-stimulated granule release in platelets. Washed human platelets were treated with vehicle or GSK2795039 for 5 min and stimulated with collagen (10 µg/mL) for 5 min. (A) Platelet dense granule secretion was assessed by measuring ATP release using a luciferin-luciferase assay. Data are presented as representative tracings and maximum luminescence (relative luminescence units, RLU) generated by ATP. (B) Surface expression of P-selectin (CD62P) as a marker of α-granule secretion was measured by flow cytometry after staining with CD62P-PE. Representative histograms and mean fluorescence intensity (MFI) values are shown. All data represent the mean ± standard deviation, with statistical significance indicated: ***p < 0.001.

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  Fig. 5. GSK2795039 suppresses the p38 MAPK/cPLA2/TXA2 signaling pathway in collagen-stimulated platelets. (A, B) Washed human platelets were pretreated with vehicle or GSK2795039 and stimulated with collagen (10 µg/mL) for 2 min. Phosphorylation of ASK1, MKK3/6, p38 MAPK (A), and cPLA2 (B) was analyzed by immunoblotting. Representative immunoblots and quantitative data are presented. (C) Thromboxane B2 (TXB2), a stable metabolite of TXA2, was measured in the supernatant of collagen-stimulated platelets using an ELISA. All data represent the mean ± standard deviation. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 6. GSK2795039 reduces ERK5 activation and PS exposure in collagen-stimulated platelets. (A) Washed human platelets were treated with GSK2795039 or vehicle and stimulated with collagen (10 µg/mL) for 2 min. Phosphorylation of ERK5 at Thr218/Tyr220 was analyzed by immunoblotting. Representative immunoblots and quantitative data are presented. (B) Washed human platelets surface exposure of phosphatidylserine was assessed by annexin V-FITC staining and analyzed using flow cytometry. All data represent the mean ± standard deviation. Statistical significance: ***p < 0.001.

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  Fig. 7. GSK2795039 modulates the cGMP/PKG/VASP/integrin-αIIbβ3 signaling pathway in collagen-stimulated platelets. Washed human platelets were treated with GSK2795039 (10 or 30 µM) or vehicle for 5 min, then stimulated with collagen (10 µg/mL). (A) Activation of integrin-αIIbβ3 was assessed by PAC1-FITC binding using flow cytometry after 5 min of collagen stimulation. Representative histograms and mean fluorescence intensity (MFI) are shown. (B) Phosphorylation of VASP at Ser239 was analyzed by immunoblotting after 2 min of collagen stimulation. Representative blots and densitometric quantification are presented. (C) Intraplatelet cGMP levels were quantified by ELISA after 2 min of collagen stimulation. All data represent the mean ± standard deviation. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001 and ns, p > 0.05 (not significant).

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  Fig. 8. GSK2795039 inhibits thrombus formation under flow and reduces thrombosis in vivo without impairing hemostasis. (A) Washed human platelets labeled with DiOC6 were perfused over a collagen-coated surface at a shear rate, and platelet adhesion and thrombus formation were visualized. Representative images and quantification of surface coverage are presented. Scale bar represents 100 μm. (B–D) Mice were pretreated with vehicle or GSK2795039 (50 or 100 mg/kg). (B) Mice were subjected to ferric chloride-induced carotid artery injury. Occlusion times were recorded using a flowmeter. The left panel shows representative tracings of blood flow. (C) Pulmonary thromboembolism was induced by retro-orbital injection of collagen and epinephrine. Time for thromboembolic death was measured and plotted. (D) Bleeding time upon tail-tip transection is shown. All data represent the mean ± standard deviation. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001 and ns, p > 0.05 (not significant).