Differential roles of the NADPH-oxidase 1 and 2 in platelet activation and thrombosis

M. Keegan Delaney, Kyungho Kim, Brian Estevez, Zheng Xu, Aleksandra Stojanovic-Terpo, Bo Shen, Masuko Fukai, Jaehyung Cho, Xiaoping Du

Research output: Contribution to journalArticle

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Abstract

Objective - Reactive oxygen species (ROS) are known to regulate platelet activation; however, the mechanisms of ROS production during platelet activation remain unclear. Platelets express different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs). Here, we investigated the role of NOX1 and NOX2 in ROS generation and platelet activation using NOX1 and NOX2 knockout mice. Approach and Results - NOX1-/Y platelets showed selective defects in G-protein-coupled receptor-mediated platelet activation induced by thrombin and thromboxane A2 analog U46619, but were not affected in platelet activation induced by collagen-related peptide, a glycoprotein VI agonist. In contrast, NOX2-/- platelets showed potent inhibition of collagen-related peptide-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet activation. Consistently, production of ROS was inhibited in NOX1-/Y platelets stimulated with thrombin, but not collagen-related peptide, whereas NOX2-/- platelets showed reduced ROS generation induced by collagen-related peptide or thrombin. Reduced ROS generation in NOX1/2-deficient platelets is associated with impaired activation of Syk and phospholipase Cγ2, but minimally affected mitogen-activated protein kinase pathways. Interestingly, laser-induced arterial thrombosis was impaired but the bleeding time was not affected in NOX2-/- mice. Wild-type thrombocytopenic mice injected with NOX2-/- platelets also showed defective arterial thrombosis, suggesting an important role for platelet NOX2 in thrombosis in vivo but not hemostasis. Conclusions - NOX1 and NOX2 play differential roles in different platelet activation pathways and in thrombosis. ROS generated by these enzymes promotes platelet activation via the Syk/phospholipase Cγ2/calcium signaling pathway.

Original languageEnglish (US)
Pages (from-to)846-854
Number of pages9
JournalArteriosclerosis, thrombosis, and vascular biology
Volume36
Issue number5
DOIs
StatePublished - May 1 2016

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Platelet Activation
Thrombosis
Blood Platelets
Reactive Oxygen Species
Thrombin
Phospholipases
NADPH oxidase 1
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid
Thromboxane A2
Bleeding Time
Calcium Signaling
G-Protein-Coupled Receptors
Mitogen-Activated Protein Kinases
Hemostasis
NADP
Knockout Mice
Glycoproteins
Oxidoreductases
Protein Isoforms
Lasers

Keywords

  • NADPH oxidase
  • blood platelets
  • platelet activation
  • reactive oxygen species
  • thrombosis

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

Cite this

Differential roles of the NADPH-oxidase 1 and 2 in platelet activation and thrombosis. / Delaney, M. Keegan; Kim, Kyungho; Estevez, Brian; Xu, Zheng; Stojanovic-Terpo, Aleksandra; Shen, Bo; Fukai, Masuko; Cho, Jaehyung; Du, Xiaoping.

In: Arteriosclerosis, thrombosis, and vascular biology, Vol. 36, No. 5, 01.05.2016, p. 846-854.

Research output: Contribution to journalArticle

Delaney, MK, Kim, K, Estevez, B, Xu, Z, Stojanovic-Terpo, A, Shen, B, Fukai, M, Cho, J & Du, X 2016, 'Differential roles of the NADPH-oxidase 1 and 2 in platelet activation and thrombosis', Arteriosclerosis, thrombosis, and vascular biology, vol. 36, no. 5, pp. 846-854. https://doi.org/10.1161/ATVBAHA.116.307308
Delaney, M. Keegan ; Kim, Kyungho ; Estevez, Brian ; Xu, Zheng ; Stojanovic-Terpo, Aleksandra ; Shen, Bo ; Fukai, Masuko ; Cho, Jaehyung ; Du, Xiaoping. / Differential roles of the NADPH-oxidase 1 and 2 in platelet activation and thrombosis. In: Arteriosclerosis, thrombosis, and vascular biology. 2016 ; Vol. 36, No. 5. pp. 846-854.
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abstract = "Objective - Reactive oxygen species (ROS) are known to regulate platelet activation; however, the mechanisms of ROS production during platelet activation remain unclear. Platelets express different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs). Here, we investigated the role of NOX1 and NOX2 in ROS generation and platelet activation using NOX1 and NOX2 knockout mice. Approach and Results - NOX1-/Y platelets showed selective defects in G-protein-coupled receptor-mediated platelet activation induced by thrombin and thromboxane A2 analog U46619, but were not affected in platelet activation induced by collagen-related peptide, a glycoprotein VI agonist. In contrast, NOX2-/- platelets showed potent inhibition of collagen-related peptide-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet activation. Consistently, production of ROS was inhibited in NOX1-/Y platelets stimulated with thrombin, but not collagen-related peptide, whereas NOX2-/- platelets showed reduced ROS generation induced by collagen-related peptide or thrombin. Reduced ROS generation in NOX1/2-deficient platelets is associated with impaired activation of Syk and phospholipase Cγ2, but minimally affected mitogen-activated protein kinase pathways. Interestingly, laser-induced arterial thrombosis was impaired but the bleeding time was not affected in NOX2-/- mice. Wild-type thrombocytopenic mice injected with NOX2-/- platelets also showed defective arterial thrombosis, suggesting an important role for platelet NOX2 in thrombosis in vivo but not hemostasis. Conclusions - NOX1 and NOX2 play differential roles in different platelet activation pathways and in thrombosis. ROS generated by these enzymes promotes platelet activation via the Syk/phospholipase Cγ2/calcium signaling pathway.",
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AU - Delaney, M. Keegan

AU - Kim, Kyungho

AU - Estevez, Brian

AU - Xu, Zheng

AU - Stojanovic-Terpo, Aleksandra

AU - Shen, Bo

AU - Fukai, Masuko

AU - Cho, Jaehyung

AU - Du, Xiaoping

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N2 - Objective - Reactive oxygen species (ROS) are known to regulate platelet activation; however, the mechanisms of ROS production during platelet activation remain unclear. Platelets express different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs). Here, we investigated the role of NOX1 and NOX2 in ROS generation and platelet activation using NOX1 and NOX2 knockout mice. Approach and Results - NOX1-/Y platelets showed selective defects in G-protein-coupled receptor-mediated platelet activation induced by thrombin and thromboxane A2 analog U46619, but were not affected in platelet activation induced by collagen-related peptide, a glycoprotein VI agonist. In contrast, NOX2-/- platelets showed potent inhibition of collagen-related peptide-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet activation. Consistently, production of ROS was inhibited in NOX1-/Y platelets stimulated with thrombin, but not collagen-related peptide, whereas NOX2-/- platelets showed reduced ROS generation induced by collagen-related peptide or thrombin. Reduced ROS generation in NOX1/2-deficient platelets is associated with impaired activation of Syk and phospholipase Cγ2, but minimally affected mitogen-activated protein kinase pathways. Interestingly, laser-induced arterial thrombosis was impaired but the bleeding time was not affected in NOX2-/- mice. Wild-type thrombocytopenic mice injected with NOX2-/- platelets also showed defective arterial thrombosis, suggesting an important role for platelet NOX2 in thrombosis in vivo but not hemostasis. Conclusions - NOX1 and NOX2 play differential roles in different platelet activation pathways and in thrombosis. ROS generated by these enzymes promotes platelet activation via the Syk/phospholipase Cγ2/calcium signaling pathway.

AB - Objective - Reactive oxygen species (ROS) are known to regulate platelet activation; however, the mechanisms of ROS production during platelet activation remain unclear. Platelets express different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs). Here, we investigated the role of NOX1 and NOX2 in ROS generation and platelet activation using NOX1 and NOX2 knockout mice. Approach and Results - NOX1-/Y platelets showed selective defects in G-protein-coupled receptor-mediated platelet activation induced by thrombin and thromboxane A2 analog U46619, but were not affected in platelet activation induced by collagen-related peptide, a glycoprotein VI agonist. In contrast, NOX2-/- platelets showed potent inhibition of collagen-related peptide-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet activation. Consistently, production of ROS was inhibited in NOX1-/Y platelets stimulated with thrombin, but not collagen-related peptide, whereas NOX2-/- platelets showed reduced ROS generation induced by collagen-related peptide or thrombin. Reduced ROS generation in NOX1/2-deficient platelets is associated with impaired activation of Syk and phospholipase Cγ2, but minimally affected mitogen-activated protein kinase pathways. Interestingly, laser-induced arterial thrombosis was impaired but the bleeding time was not affected in NOX2-/- mice. Wild-type thrombocytopenic mice injected with NOX2-/- platelets also showed defective arterial thrombosis, suggesting an important role for platelet NOX2 in thrombosis in vivo but not hemostasis. Conclusions - NOX1 and NOX2 play differential roles in different platelet activation pathways and in thrombosis. ROS generated by these enzymes promotes platelet activation via the Syk/phospholipase Cγ2/calcium signaling pathway.

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