NR4A1 Acts as a Novel Regulator of Platelet Activation and Thrombus Formation

Abstract

Background: Mounting evidence indicates that nuclear receptors play a critical regulatory role in platelet pathophysiology and thrombotic disorders. Although NR4A (the nuclear receptor subfamily 4 group A) plays an important role in cardiovascular pathophysiology, the expression profile and biological function of NR4A member 1 (NR4A1) in platelets have never been reported.

Methods: We evaluated the functions and the underlying mechanisms of NR4A1 in platelet activation and thrombus formation using platelet-specific NR4A1-deficient mice and NR4A1-specific agonists. Using a hyperlipidemic mouse model and platelets from patients with hypercholesterolemia, we explored the influence of hypercholesterolemia on NR4A1 expression and the effects of NR4A1-specific agonists on platelet hyperreactivity induced by hypercholesterolemia.

Results: NR4A1 was expressed in both human and mouse platelets. Platelet-specific NR4A1 deletion accelerated FeCl₃-induced carotid arterial occlusive thrombus formation, enhanced collagen/epinephrine-induced pulmonary thromboembolism, and exacerbated microvascular microthrombi obstruction and infarct expansion in an acute myocardial infarction model. NR4A1-deficient platelets exhibited enhanced agonist-induced aggregation responses, integrin α_IIbβ₃ activation, dense granule release, α-granule release, platelet spreading, and clot retraction. Consistently, pharmacological activation of NR4A1 by specific agonists decreased platelet activation in both mouse and human platelets. Mechanistically, CAP1 (adenylyl cyclase-associated protein 1) was identified as the direct downstream interacting protein of NR4A1. NR4A1 deletion decreased cAMP levels and phosphorylation of VASP (vasodilator-stimulated phosphoprotein), while NR4A1-specific agonists increased cAMP levels and phosphorylation of VASP in platelets. Importantly, NR4A1 expression in platelets was upregulated in the setting of hypercholesterolemia, which was derived from its upregulation in megakaryocytes in a reactive oxygen species-dependent manner. Platelets from hypercholesterolemic patients and mice exhibited hyperreactivity. However, NR4A1-specific agonists significantly inhibited the activation of hypercholesterolemic platelets to the levels of healthy control platelets.

Conclusions: We provide the first evidence that nuclear receptor NR4A1 negatively regulates platelet activation and thrombus formation. NR4A1 may serve as a novel therapeutic target for managing thrombosis-based cardiovascular diseases, especially with hypercholesterolemia.

Keywords: blood platelets; hypercholesterolemia; platelet activation; receptors, cytoplasmic and nuclear; thrombosis.

Figure 1.

NR4A1 (nuclear receptor subfamily 4 group A member 1) is present in human and mouse platelets. A and B, Detection of NR4A1 mRNA (A) and protein (B) in human platelets. Human lung, kidney, and brain tissues served as positive controls (n=5 donors per group). GAPDH was used as an internal control. C, Validation of the NR4A1 antibody using an NR4A1 blocking peptide to block the antibody epitope (n=5 independent experiments). D, Detection of NR4A1 in human platelets using flow cytometry without or with permeabilization (Permea; n=6 per group). Data were analyzed by 1-way ANOVA followed by Tukey multiple comparisons test. E, Representative images of NR4A1 expression by immunofluorescence staining in human platelets and co-staining with the key platelet marker CD42b (n=5 donors per group). Primary antibody omission served as the negative control. F and G, Detection of Nr4a1 mRNA (F) and protein (G) in mouse platelets. Mouse lung, kidney, and brain tissues served as positive controls (n=5 mice per group). GAPDH was used as an internal control. H, NR4A1 protein expression by immunoblotting in platelets from Nr4a1^+/+ and Nr4a1^−/− mice (n=5 independent experiments). I, Detection of NR4A1 in mouse platelets using flow cytometry without or with permeabilization (n=6 per group). Data were analyzed by 1-way ANOVA followed by Tukey multiple comparisons test. J, Representative images of NR4A1 expression by immunofluorescence staining in mouse platelets and co-staining with the key platelet marker CD41 (n=5 mice per group). Primary antibody omission served as the negative control. All data are presented as mean±SD. FITC-A indicates fluorescein isothiocyanate-area.

Figure 2.

NR4A1 (nuclear receptor subfamily 4 group A member 1), but not NR4A2, is upregulated in platelets under hypercholesterolemia (HTC). A through C, Representative images (A) and quantification of NR4A1 protein (B) and NR4A2 protein (C) expression in platelets from healthy subjects and patients with hypercholesterolemia (n=25 donors per group). D and E, Quantification of NR4A1 mRNA (D) and NR4A2 mRNA (E) expression in platelets from healthy subjects and patients with HTC (n=25 donors per group). F, Quantification of NR4A1 expression in platelets from healthy subjects and patients with HTC by flow cytometry without or with permeabilization (Permea) using an anti-C-terminal antibody (n=6 donors per group). G through I, Representative images (G) and quantification of NR4A1 protein (H) and NR4A2 protein (I) expression in platelets from ApoE^−/− mice fed a high-fat diet (HFD) or a control diet (CD; n=6 per group). J and K, Quantification of NR4A1 mRNA (J) and NR4A2 mRNA (K) expression in platelets from ApoE^−/− mice fed an HFD or a CD (n=6 per group). L, Quantification of NR4A1 expression in platelets from ApoE^−/− mice fed an HFD or a CD by flow cytometry without or with permeabilization using an anti-C-terminal antibody (n=6 per group). Data are presented as mean±SD and were analyzed by Student t test (B, D, E, F, H, I, K, and L) or Mann-Whitney U test (C and J). FITC-A indicates fluorescein isothiocyanate-area.

Figure 3.

A high-fat diet (HFD) increases the expression of NR4A1 (nuclear receptor subfamily 4 group A member 1) in mouse megakaryocytes (MKs) in vivo, and oxLDL (oxidized low-density lipoprotein) increases the expression of NR4A1 in primary mouse MKs in vitro in a reactive oxygen species-dependent manner. A, Representative images of NR4A1 expression by immunofluorescence staining in MKs isolated from mice fed an HFD or a control diet (CD) for 8 weeks, costained with the key marker CD41 in 5 independent experiments). B and C, Representative images and quantification of NR4A1 protein (B) and mRNA (C) expression in MKs isolated from mice fed an HFD or a CD for 8 weeks (n=6 per group). D and E, Representative images and quantification of NR4A1 protein (D) and mRNA (E) expression in primary mouse MKs treated with various concentrations of oxLDL for 24 hours (n=6 per group). F and G, Representative images and quantification of NR4A1 protein (F) and mRNA (G) expression in mouse MKs treated with oxLDL (50 μg/mL) in the absence or presence of 10 mmol/L N-acetyl cysteine (NAC) or 100 μmol/L edaravone (EDA) for 24 hours (n=6 per group). H and I, Representative images and quantification of NR4A1 protein (H) and mRNA (I) expression in mouse MKs treated with various concentrations of H₂O₂ for 12 hours (n=6 per group). Data are presented as mean±SD and were analyzed by Student t test (B and C), 1-way ANOVA followed by Dunnett multiple comparisons test (D, E, H, and I), or 1-way ANOVA followed by Tukey multiple comparisons test (F and G). DAPI indicates 4′,6-diamidino-2-phenylindole.

Figure 4.

Platelet-specific deficiency of NR4A1 (nuclear receptor subfamily 4 group A member 1) shortens bleeding time and enhances thrombus formation in vivo. A, Bleeding time for Nr4a1^fl/flPF4Cre⁻ and Nr4a1^fl/flPF4Cre⁺ mice. Data are presented with geometric mean±geometric SD (n=40 mice per group). Data were analyzed by Mann-Whitney U test. B, Percentage of bleeding time ≥15 or <15 minutes for Nr4a1^fl/flPF4Cre⁻ and Nr4a1^fl/flPF4Cre⁺ mice. Data were analyzed by χ² test. C, Representative images of blood flow through the left carotid vascular corroded by 10% FeCl₃. The first occlusion time in Nr4a1^fl/flPF4Cre⁻ and Nr4a1^fl/flPF4Cre⁺ mice was analyzed (n=16 mice per group). D, Representative images of hematoxylin and eosin (HE)-stained paraffin-embedded sections of left carotid vascular 5 minutes after 10% FeCl₃ corrosion. The ratio of thrombus area to lumen area in Nr4a1^fl/flPF4Cre⁻ and Nr4a1^fl/flPF4Cre⁺ mice was quantified (n=10 mice per group). E, Images of the mouse lungs injected with Evans blue solution into the heart 5 minutes after the injection of the collagen-epinephrine mix. F, Images of HE-stained paraffin-embedded sections of the lungs from mice receiving the injection of the collagen-epinephrine mix. The ratio of the embolism area to the lung area was quantified (n=9 mice per group). Data are presented as mean±SD and were analyzed by Student t test (C and D) or Mann-Whitney U test (F).

Figure 5.

Platelet-specific deficiency of NR4A1 (nuclear receptor subfamily 4 group A member 1) exacerbates microvascular microthrombi obstruction and infarct expansion. A, Immunofluorescence of the peripheral infarct zone from Nr4a1^fl/flPF4Cre⁻ and Nr4a1^fl/flPF4Cre⁺ mouse hearts post-acute myocardial infarction (AMI). CD41 areas per field were quantified (n=10 mice per group). B, Representative images of Masson trichrome-stained hearts of Nr4a1^fl/flPF4Cre⁻ and Nr4a1^fl/flPF4Cre⁺ mice on day seven post-AMI. Ratio of infarct size to left ventricular (LV) area (n=10 mice per group). C, Representative images of M-mode echocardiography of Nr4a1^fl/flPF4Cre⁻ and Nr4a1^fl/flPF4Cre⁺ mice on day seven post-AMI. Echocardiographic cardiac function was quantified (n=10 mice per group). All data are presented as mean±SD and were analyzed by Student t test. FS indicates fractional shortening; LVEF, left ventricular ejection fraction.

Figure 6.

Platelet-specific deficiency of NR4A1 (nuclear receptor subfamily 4 group A member 1) enhances platelet activation in vitro. A through G, Representative platelet aggregation tracings and quantification of light transmission, as well as representative ATP release tracings and quantification of luminance of washed Nr4a1^fl/flPF4Cre⁻ and Nr4a1^fl/flPF4Cre⁺ platelets stimulated with thrombin (A and B), collagen (C and D), U46619 (E and F), or ADP (G; n=6 per group). H through K, Quantification of P-selectin surface exposure and JON/A-binding of washed Nr4a1^fl/flPF4Cre⁻ and Nr4a1^fl/flPF4Cre⁺ platelets stimulated with various concentrations of thrombin (H and I) or ADP (J and K; n=6 per group). L, Representative pictures of washed Nr4a1^fl/flPF4Cre⁻ and Nr4a1^fl/flPF4Cre⁺ platelets that spread on fibrinogen and stained with phalloidin. Quantification of the areas of spread platelets (μm²; n=6 per group). M, Representative images and quantification of clot retraction stimulated with 1.0 U/mL thrombin in Nr4a1^fl/flPF4Cre⁻ and Nr4a1^fl/flPF4Cre⁺ platelets (n=7 per group). Data are presented as mean±SD and were analyzed by Mann-Whitney U test (A and H), Student t test (B, C, D, E, F, G, I, J, K, and L), or repeated measures 2-way ANOVA followed by Bonferroni multiple comparisons test (M).

Figure 7.

Cytosporone B (Csn-B) inhibits activation of mouse and human platelets in vitro. Mouse and human platelets were pretreated with vehicle (Veh) or various concentrations of Csn-B for 15 minutes. A, Representative platelet aggregation tracings and quantification of light transmission of washed mouse platelets stimulated with thrombin (n=6 per group). B, Representative ATP release tracing and quantification of luminance of washed mouse platelets stimulated with thrombin (n=6 per group). C and D, P-selectin surface exposure (C) and JON/A-binding (D) of washed mouse platelets activated by 0.1 U/mL thrombin. E, Representative images and quantification of mouse platelet clot retraction stimulated with 1.0 U/mL thrombin (n=7 per group). F, Representative platelet aggregation tracings and quantification of light transmission of washed human platelets stimulated with thrombin (n=6 per group). G, Representative ATP release tracings and quantification of luminance of washed human platelets stimulated with thrombin (n=6 per group). H and I, P-selectin surface exposure (H, n=6 per group) and PAC-1-binding (I, n=6 per group) of washed human platelets activated with 0.1 U/mL thrombin. J, Representative images and quantification of human platelet clot retraction stimulated with 1.0 U/mL thrombin (n=7 per group). Data are presented as mean±SD and were analyzed by 1-way ANOVA followed by Dunnett multiple comparisons test (A, B, F, and G), Student t test (C, D, H, and I), or repeated measures 2-way ANOVA followed by Bonferroni multiple comparisons test (E and J).

Figure 8.

NR4A1 (nuclear receptor subfamily 4 group A member 1) interacts with CAP1 (adenylyl cyclase-associated protein 1), promotes cAMP generation, and phosphorylates of VASP (vasodilator-stimulated phosphoprotein) in platelets. A, Rabbit anti-NR4A1 monoclonal antibody was used to immunoprecipitate NR4A1 from the lysates of resting and thrombin-stimulated platelets from healthy humans or wild-type mice (n=5 independent experiments). Rabbit IgG was used as the negative control. Then, the presence of CAP1 was detected with rabbit anti-CAP1 polyclonal antibody by Western blot analysis. B, Rabbit anti-CAP1 polyclonal antibody was used to immunoprecipitate CAP1 from the lysates of resting and thrombin-stimulated platelets from healthy humans or wild-type mice (n=5 independent experiments). Rabbit IgG was used as the negative control. Then, the presence of NR4A1 was detected with rabbit anti-NR4A1 monoclonal antibody by Western blot analysis. C, The lysates of healthy human and wild-type mouse platelets treated with cytosporone B (Csn-B) or vehicle (Veh) were used for co-immunoprecipitation with rabbit anti-NR4A1 monoclonal antibody (n=5 independent experiments). Rabbit IgG was used as the negative control. Then, the amount of CAP1 was detected with rabbit anti-CAP1 polyclonal antibody by Western blot analysis. D and E, cAMP levels (D) and phosphorylation of VASP at Ser157 (E) in Veh or 30 μmol/L Csn-B-pretreated washed human platelets stimulated with various concentrations of ADP. Representative images and quantification of the ratio of p-VASP to t-VASP of 6 independent experiments using platelets from different donors. F and G, cAMP levels (F) and phosphorylation of VASP at Ser157 (G) in Veh or 30 μmol/L Csn-B-pretreated washed human platelets stimulated with various concentrations of epinephrine (EPI). Representative images and quantification of the ratio of p-VASP to t-VASP of 6 independent experiments using platelets from different donors. H and I, cAMP levels (H) and phosphorylation of VASP at Ser157 (I) in Veh, 0.6 μmol/L prostaglandin I₂ (PGI₂), or 30 μmol/L Csn-B-pretreated washed human platelets stimulated with 10 μmol/L ADP. Representative images and quantification of the ratio of p-VASP to t-VASP of 6 independent experiments using platelets from different donors. J and K, cAMP levels (J) and phosphorylation of VASP at Ser157 (K) in washed Nr4a1^fl/flPF4Cre⁻ (fl/fl) and Nr4a1^fl/flPF4Cre⁺ (ΔPlt) platelets stimulated with various concentrations of PGI₂. Representative images and quantification of the ratio of p-VASP to t-VASP (n=6 per group). Data are presented as mean±SD and were analyzed by Student t test (D, E, F, G, J, and K) or 1-way ANOVA followed by Tukey multiple comparisons test (H and I).