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.

Protein disulfide isomerase‐A1 regulates intraplatelet reactive oxygen species–thromboxane A2‐dependent pathway in human platelets

“…This finding could be vital in preventing atherothrombosis, given the success of aspirin as a major antiplatelet drug limiting the production and release of TxA 2 . PDIA1‐based platelet pharmacology warrants future studies, given that recent studies highlighted the role of Nox1 function in platelets 43,54,55 …”

Platelet-derived extracellular vesicles express NADPH oxidase-1 (Nox-1), generate superoxide and modulate platelet function

“…Initially, Nox-2 was shown to be relevant to collagen receptor (GPVI)-mediated responses and regulate thrombus formation in male mice [ 8 ]. This has been disputed by us [ 9 , 10 ] and others [ 11 , 12 ] that showed that Nox-1 is the relevant isotype in GPVI-mediated responses, whilst Nox-2 is dispensable for thrombus formation. Such discrepant result across groups may be due to sex disparities amongst studies, as the initial report of Delaney et al used male Nox-1-deficient mice [ 8 ], whilst we have used female mice, noting that the Nox-1 gene is on the X chromosome [ 9 ].…”

Diabetes and Thrombosis: A Central Role for Vascular Oxidative Stress

“…The signaling pathway activated by CD36 includes tyrosine kinase- and protein kinase C-dependent activation of NOX2 and generation of reactive oxygen species (ROS), ultimately counteracting the negative regulatory function of the cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Recent studies from our laboratory highlighted the involvement of both NOX1 and NOX2 in the signaling of ox-LDL [ 17 ] and confirmed the negative modulation of the cyclic nucleotide pathways by NOXs [ 18 ]. In addition to enzymatic ROS sources, HG causes metabolic overload in platelet mitochondria, which results in the leakage of electrons from the respiration chain and the release of ROS [ 32 ].…”

“…Work in our laboratory showed that platelets from DM patients with poor glycemic control express significantly higher levels of the pro-oxidant enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) ( Figure 1 ). NOXs have been described as important positive regulators of platelet activity [ 17 , 18 , 19 , 20 ]. In view of the pro-thrombotic role of platelet NOXs both in vitro and in vivo ( Figure 2 ), the upregulation of NOX1 in DM patients contributes significantly to platelet hyperresponsiveness.…”