NADPH Oxidases Are Required for Full Platelet Activation In Vitro and Thrombosis In Vivo but Dispensable for Plasma Coagulation and Hemostasis

Abstract

Objective: Using 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1^-/-/NOX2^-/-/NOX4^-/-), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP-a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride-driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal.

Conclusions: This study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.

Keywords: NADPH oxidases; blood platelets; chlorides; oxidation-reduction; thrombosis.

Figure 1.

Triple NOX (NADPH oxidase) 1, 2, and 4 deficiency attenuates superoxide radical generation (A and B) and platelet aggregation (C and D) in response to collagen or thrombin. Superoxide radicals in response to 3 µg/mL fibrillar Horm collagen (A) or 0.1 unit/mL human thrombin (B) were measured by electron paramagnetic resonance spectroscopy (EPR). Representative EPR spectrograms (top) and quantitative analysis (bottom) are shown. Statistical analysis was tested by analyzed by 1-way ANOVA with Bonferroni post test. In parallel, aggregation in response to 3 µg/mL fibrillar Horm collagen (C) or 0.1 unit/mL human thrombin (D) was measured by turbidimetry. Representative aggregation traces (top) and quantitative analysis (bottom) are shown. Statistical analysis was tested by unpaired Student t test. 3KO indicates triple NOX knockout; CMH, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine; Coll., collagen; ESR, electro spin resonance; ns, nonsignificant; Rest., resting; Throm., thrombin; and WT, wild type. ***P<0.05, **P<0.01, ***P<0.001.

Figure 2.

Comparison of aggregation and thrombus formation responses of 3KO (triple NOX [NADPH oxidase] knockout) and single NOX knockout mice (Nox1^−/−, Nox2^−/−, and Nox4^−/−). Aggregation in response to 3 µg/mL fibrillar Horm collagen (A) or 0.1 unit/mL human thrombin (B) was measured by turbidimetry. Representative aggregation traces (top) and quantitative analysis (bottom) are shown. For thrombus formation under flow (C), heparin- and D-phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK)–anticoagulated whole blood was incubated with 1 μM 3,3′-dihexyloxacarbocyanine iodide (DiOC6) for 10 min, and thrombus formation was tested on collagen at intermediate shear stress (1000 s⁻¹). Where indicated, WT (wild type) platelets were pretreated with 10 μM VAS2870 (VAS) for 10 min. Statistical analysis was performed using 1-way ANOVA with Bonferroni post test. ns indicates nonsignificant. *P<0.05, **P<0.01, ***P<0.001.

Figure 3.

Triple NOX (NADPH oxidase) 1, 2, and 4 deficiency impairs different components of the intracellular signaling of platelets. The intracellular signaling of 3KO (triple NOX knockout) platelets was tested by immunoblotting protein extracts from resting (R), 10 μg/mL collagen (C)-stimulated, or 0.1 u/mL thrombin (T)-stimulated platelets (A). Tyrosine phosphorylation (top) and PKC (protein kinase C)-dependent phosphorylation (bottom) were tested. While anti-ERK (extracellular receptor kinase) was used as a loading control (bottom). Densitometry analysis was performed using ImageJ 1.47v (Wayne Rasband, National Institutes of Health) and is expressed as the ratio between the immunoreactivity intensity above 60 kDa for 4G10 and phosphorylated-PKC substrate blots and the intensity of ERK1/2 bands in the loading control blots for the same condition. Integrin αIIbβ3 activation and P-selectin surface expression (ie, a marker of degranulation) were tested by flow cytometry with JON/A (B) and anti-CD62P antibodies (C), respectively. Platelets were resting (R), activated by 5 μg/mL CRP-XL (C [cross-linked collagen-related peptide]) or activated with 1 u/mL thrombin (T). Statistical analysis was performed by 1-way ANOVA with Bonferroni post test with n=4 for A and C and n=5 for B.). CD62P indicates P-selectin; CRP, CRP-XL; ns, nonsignificant; p-PKC, phosphorylated PKC substrates; and WT, wild type. *P<0.05, **P<0.01, ***P<0.001.

Figure 4.

Triple NOX (NADPH oxidase) 1, 2, and 4 deficiency attenuates platelet signaling by increasing cGMP levels. For cGMP quantification (A), WT (wild type) and 3KO (triple NOX knockout) mouse washed platelets were left unstimulated (resting [R]) or stimulated with 10 μg/mL collagen (C) or 0.25 u/mL thrombin (T) for 20 min before cell lysis was obtained by 3 freeze/thaw cycles. cGMP was then quantified in the lysates following supplier instructions and expressed in pmol per 109 platelets. For immunoblotting (B), the same conditions as above were tested. Densitometry analysis was performed using ImageJ 1.47v (Wayne Rasband, National Institutes of Health) and is expressed as the ratio between the immunoreactivity intensity for p-VASP (Ser238) and total VASP (vasodilator-stimulated phosphoprotein). Aggregation in response to 3 µg/mL fibrillar platelet aggregation in response by Horm collagen (C) or 0.1 unit/mL human thrombin (D) and collagen-induced thrombus formation under physiological flow (1000 s⁻¹) are shown in (E). Where indicated, platelets were preincubated with 1 μM KT5823 (KT), 5 μM Rp-8-pCPT-cGMPs (Rp), and 20 μM L-NG-monomethyl arginine (L-NMMA) or vehicle solution (0.1% dimethyl sulfoxide) for 30 min. For aggregation, representative traces (top) and quantitative analysis (bottom) are shown. For thrombus formation, representative pictures (left) and quantitative analysis (right) are shown. Data are analyzed by 1-way ANOVA with Bonferroni post test for A (n=5), C (n=4), D (n=4), and E (n=5), while nonparametric Kruskal-Wallis assay with Dunn post-test for B (n=4). ns indicates nonsignificant; p-VASP, phosphorylated VASP; and R, rest. *P<0.05, **P<0.01, ***P<0.001.

Figure 5.

Triple NOX (NADPH oxidase) 1, 2, and 4 deficiency protects against pulmonary thromboembolism (A) and arterial thrombosis (B) without affecting hemostasis (C) in vivo. Pulmonary thromboembolism was induced by intravenous injection of a 0.4-mg/kg collagen and 60 μg/kg epinephrine into the vena cava (A). Time for thromboembolic death was measured and plotted (mean±SEM, n=7). Carotid occlusion was induced by application of 5% w/v ferric chloride (B). Occlusion times detected by Doppler ultrasound scanning for 3KO (triple NOX knockout) and WT (wild type) animals are plotted (mean±SEM, n=6). Bleeding time upon tail-tip transection is shown in C (mean±SEM plot, n=6). As data are not homoscedastic in A and B (ie, significantly different SD), statistical analysis was performed by nonparametric Mann-Whitney U test. Data in C are normally distributed and homoscedastic; therefore, parametric Student t test was used for the statistical analysis. ns indicates nonsignificant. *P<0.05, **P<0.01, ***P<0.001.

Figure 6.

Triple NOX (NADPH oxidase) 1, 2, and 4 deficiency does not affect the procoagulant activity of platelets (A) or coagulation response in plasma (B and C). Phosphatidylserine externalization (A) was tested by flow cytometry. Platelets were resting (R) or activated by 5 μg/mL CRP-XL (C [cross-linked collagen-related peptide]) or activated by 1 u/mL thrombin (T) in the absence of shear stress for 40 min and then fixed (1% PFA) and labeled with FITC-annexin V. Representative histograms are shown in top (WT [wild type] in blue, 3KO [triple NOX knockout] in red, and unstimulated in dotted black), and statistical analysis is shown bottom (n=5). Statistical analysis was performed by 1-way ANOVA with Bonferroni post test. Thrombin generation in platelet-poor plasma in response to either 6 pM tissue factor (B) or 10 μg/mL kaolin (C) is shown (representative thrombin generation peaks in the left; mean±SEM for the area under the curve in the right; n=6 for tissue factor and n=8 for kaolin). For both B and C, the statistical analysis was performed by unpaired Student t test. CRP indicates CRP-XL; FC, flow cytometry; FITC, fluorescein isothiocyanate; and ns, nonsignificant. *P<0.05, **P<0.01, ***P<0.001.