The role of Nox1 and Nox2 in GPVI-dependent platelet activation and thrombus formation

Abstract

BackgroundActivation of the platelet-specific collagen receptor, glycoprotein (GP) VI, induces intracellular reactive oxygen species (ROS) production; however the relevance of ROS to GPVI-mediated platelet responses remains unclear.ObjectiveThe objective of this study was to explore the role of the ROS-producing NADPH oxidase (Nox)1 and 2 complexes in GPVI-dependent platelet activation and collagen-induced thrombus formation.Methods and resultsROS production was measured by quantitating changes in the oxidation-sensitive dye, H2DCF-DA, following platelet activation with the GPVI-specific agonist, collagen related peptide (CRP). Using a pharmacological inhibitor specific for Nox1, 2-acetylphenothiazine (ML171), and Nox2 deficient mice, we show that Nox1 is the key Nox homolog regulating GPVI-dependent ROS production. Nox1, but not Nox2, was essential for CRP-dependent thromboxane (Tx)A2 production, which was mediated in part through p38 MAPK signaling; while neither Nox1 nor Nox2 was significantly involved in regulating CRP-induced platelet aggregation/integrin αIIbβ3 activation, platelet spreading, or dense granule and α-granule release (ATP release and P-selectin surface expression, respectively). Ex-vivo perfusion analysis of mouse whole blood revealed that both Nox1 and Nox2 were involved in collagen-mediated thrombus formation at arterial shear.ConclusionTogether these results demonstrate a novel role for Nox1 in regulating GPVI-induced ROS production, which is essential for optimal p38 activation and subsequent TxA2 production, providing an explanation for reduced thrombus formation following Nox1 inhibition.

NOX2 is critical for heterotypic neutrophil-platelet interactions during vascular inflammation

“…Compared with WT mice, NOX2 KO mice showed no defects in neutrophil rolling and adhesion to the TNF-a-inflamed endothelium, but exhibited nearly complete inhibition of neutrophil-platelet interactions on the inflamed venules ( Figure 1B-E and supplemental Videos 1 and 2). Consistent with recent studies, 14 there were no differences in the number of circulating blood cells between WT and NOX2 KO mice (Table 1). These results indicate that intravascular NOX2 is critical for neutrophil-platelet association during vascular inflammation.…”

“…14 We found that the surface expression of GPVI and GPIba was also similar in WT and NOX2 KO platelets (supplemental Figure 2A-B). In addition, we observed that NOX2 deletion significantly decreased the surface expression of P-selectin and aIIbb3 integrin in response to 0.025 but not 0.05 U/mL thrombin ( Figure 3A and supplemental Figure 2C), suggesting that other signaling pathways induced by the increased concentration of thrombin compensate for the defective function of the KO platelets.…”

“…12 However, NOX5 is expressed only in the vasculature of humans. Compared with platelets that produce low amounts of ROS through NOX1 and NOX2, 13,14 neutrophils rapidly generate much larger amounts of ROS via NOX2 after cell activation. 15 The NOX2 enzyme consists of membrane subunits (p22 phox and gp91 phox ) and cytosolic components (p47 phox , p67 phox , p40 phox , and small GTPase Rac1/2), and generates O 2 ·-by transferring 1 electron from NADPH to molecular oxygen.…”

Current State and Novel Approaches of Antiplatelet Therapy

“…[10][11][12][13][14] Signaling immediately downstream of GPIb-IX-V/GPVI involves activation of Src and Syk kinase-dependent tyrosine phosphorylation pathways and Src/Syk-dependent and Src/ Syk-independent generation of intracellular reactive oxygen species (ROS). [17][18][19][20] The cytoplasmic domains of GPIb-IX-V and GPVI directly bind to signaling or adaptor proteins, including phosphatidylinositol 3-kinase (via the p85 subunit) 21,22 and constitutively phosphorylated Lyn (via a proline-rich sequence of GPVI), 23 respectively. In the case of GPVI, ligand-induced receptor cross-linking enables active Lyn to phosphorylate Syk associated with the immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic domain of Fc receptor γ-chain, initiating canonical Syk-dependent signaling pathways.…”

Magnetic microparticles post-synthetically coated by hyaluronic acid as an enhanced carrier for microfluidic bioanalysis

“…The behaviour of the tested particles in the microfluidic channel was monitored during the experiment using an AM-7013MZT microscope (Dino-Lite Digital Microscope, Naarden, the Netherlands). Representative images were evaluated using ImageJ analysis software (version 1.42) from the National Institutes of Health (Bethesda, Md., USA) [30].…”