Differential Roles of the NADPH-Oxidase 1 and 2 in Platelet Activation and Thrombosis

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 (GPCR)-mediated platelet activation induced by thrombin and thromboxane A2 analog U46619, but were not affected in platelet activation induced by collagen-related peptide (CRP), a glycoprotein VI (GPVI) agonist. In contrast, NOX2−/− platelets showed potent inhibition of CRP-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 CRP, whereas NOX2−/− platelets showed reduced ROS generation induced by CRP or thrombin. Reduced ROS generation in NOX1/2 deficient platelets is associated with impaired activation of Syk and phospholipase Cγ2 (PLCγ2), 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. WT 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/PLCγ/calcium signaling pathway.

Response by Liu and Hu to Letter Regarding Article, “Class III PI3K Positively Regulates Platelet Activation and Thrombosis via PI(3)P-Directed Function of NADPH Oxidase”

“…5,6 However, it is notable that the inhibitory effects of NOX1 and NOX2 knockout on ROS generation and platelet aggregation diminish at high concentrations of agonists. 2 Consistent with the findings that VPS34 functions through NOX, VPS34 deficiency also causes an obvious inhibition of ROS production and platelet aggregation with low dose agonists stimulation (thrombin, 0.025 U/mL or collagen, 0.5 μg/mL), but the inhibition diminishes on the raise of the agonist dosage (thrombin, 0.05 U/mL or collagen, 1 μg/mL). Yet high concentrations of agonists (collagen, 30 μg/mL 3 ; collagen, 4 μg/mL 5 ; collagen, 2 μg/mL 6 ; and CRP [collagen-related peptide], 1 μg/mL 4 ) is the condition tested by the opposing studies (including the chronic granulomatous disease patient study 5,6 ) to evidence the insignificant role of NOX in platelet activation.…”

“…With the importance of NOXs in platelet function emerging and their regulation mechanism unveiling, such as suggested by the study of Delaney et al 2 and ours, 1 we agree with the authors that it is imperative for future studies to further reveal the regulation network involving NOX isoforms and VPS34 in the context of platelet activation and thrombosis.…”

“…Hence, although the recent study using NOX1 and NOX2 deficient mice has proven that these 2 isoforms are selectively important for ROS generation and platelet activation, 2 there are previous reports showing that (1) murine platelets lacking NOX subunits, such as NOX2 or p47Phox, display normal ROS generation and aggregation responses to thrombin and collagen 3,4 ; and (2) human platelets deficient of NOX2 (from chronic granulomatous disease) also exhibit normal aggregation responses. 5,6 However, it is notable that the inhibitory effects of NOX1 and NOX2 knockout on ROS generation and platelet aggregation diminish at high concentrations of agonists.…”

“…The NOX family consists of 7 catalytic homologues, 7,8 in addition to NOX1 and NOX2, NOX4 is also detected in platelets. 2 More recently, NOX5 is found expressed in platelets from patients with chronic granulomatous disease 9 although it may not present in rodent platelets. 8 Unfortunately, except for NOX1 and NOX2, neither the individual role of NOX isoforms (eg, NOX4) in platelet activation nor the possible mutual compensational mechanism among them is currently understood.…”

Thrombotic Regulation From the Endothelial Cell Perspectives

“…[49][50][51] The general sources of ROS in vascular system include dihydronicotinamide adenine dinuclectide phosphate oxidases, xanthine oxidase, uncoupled endothelial nitric oxide synthase, and leakage of activated oxygen from mitochondria during oxidative respiration. 50 ROS regulates EC biology.…”