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


OBJECTIVE
The 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.


RESULTS
ROS 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.


CONCLUSIONS
Together 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.
Department of Experimental Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland.
T G
TG
Walsh
Department of Experimental Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland ; Faculty of Health Sciences, Curtin University, Perth, Australia.
M C
MC
Berndt
Department of Experimental Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland.
N
N
Carrim
Department of Molecular Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin, Ireland.
J
J
Cowman
Department of Molecular Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin, Ireland.
D
D
Kenny
Department of Experimental Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland ; Faculty of Health Sciences, Curtin University, Perth, Australia.
P
P
Metharom
D000818:Animals; D001158:Arteries / Q000378:metabolism*; D002352:Carrier Proteins / Q000494:pharmacology; D005452:Fluoresceins / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D010640:Phenothiazines / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D017382:Reactive Oxygen Species; D013927:Thrombosis / Q000378:metabolism*; D013928:Thromboxane A2 / Q000378:metabolism
C110400:2',7'-dichlorodihydrofluorescein diacetate; C584787:2-acetylphenothiazine; D002352:Carrier Proteins; D005452:Fluoresceins; D008562:Membrane Glycoproteins; D010455:Peptides; D010640:Phenothiazines; D010980:Platelet Membrane Glycoproteins; D017382:Reactive Oxygen Species; C435908:collagen-related peptide; C098341:platelet membrane glycoprotein VI; D013928:Thromboxane A2; D009247:NADH, NADPH Oxidoreductases; C488842:Cybb protein, mouse; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C000614711:NOX1 protein, mouse
Glycoprotein VI; NAPDH oxidase; Platelets; Reactive oxygen species; Thrombus