The anti-platelet effects of apocynin in mice are not mediated by inhibition of NADPH oxidase activity.
Apocynin, or a (myelo)peroxidase-derived product thereof, is a powerful inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Apocynin has also been shown to prevent aggregation of platelets in response to agonists such as collagen and thrombin. The aims of this study were to establish whether NADPH oxidase activity is required for aggregation of murine platelets to collagen and other agonists and whether the anti-aggregatory effects of apocynin are due to an inhibitory action against this enzyme. Washed platelets were isolated from male C57BL6 (wild-type), Nox2-deficient (Nox2(-/y )), and p47phox-deficient (p47phox(-/-)) mice for assessment of aggregation and NADPH oxidase subunit (Nox2, p47phox) expression. Collagen and U46619 elicited aggregation of murine platelets, and these responses were inhibited by apocynin at concentrations ≥100 μM. By contrast, aggregations to a direct protein kinase C activator, phorbol-12,13-dibutyrate, were insensitive to apocynin. Immunoblotting of platelet protein homogenates from wild-type mice with anti-Nox2 or p47phox antibodies revealed strong bands at 58 and 50 kDa, respectively. While expression of these immunoreactive bands was greatly diminished in platelets from Nox2(-/y ) and p47phox(-/-) mice, collagen still elicited aggregations that were similar to those observed in platelets from wild-types. Moreover, apocynin was an equally effective inhibitor of aggregation in platelets from all three mouse strains. In conclusion, these data suggest that NADPH oxidase-derived reactive oxygen species play no role in the aggregation response of washed murine platelets to collagen. Thus, our observation that apocynin is a powerful inhibitor of platelet aggregation raises further questions about the selectivity of this drug as an NADPH oxidase inhibitor.
Dharmarajah
Janahan
J
Department of Pharmacology, Monash University, Clayton, Victoria, Australia.
Arthur
Jane F
JF
Sobey
Christopher G
CG
Drummond
Grant R
GR
D000098:Acetophenones / Q000494:pharmacology*; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D002478:Cells, Cultured; D008297:Male; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000172:deficiency / Q000235:genetics; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*
D000098:Acetophenones; D008562:Membrane Glycoproteins; D010975:Platelet Aggregation Inhibitors; C056165:acetovanillone; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1
J Thromb Haemost. 2005 Aug;3(8):1725-36
16102039
FEBS J. 2008 Jul;275(13):3249-77
18513324
FEBS Lett. 1992 Jul 6;305(3):254-6
1299626
Blood. 2005 Oct 15;106(8):2757-60
15976180
Antioxid Redox Signal. 2008 Oct;10(10):1713-65
18707220
Clin Biochem. 2005 Jul;38(7):607-13
15922319
Free Radic Biol Med. 2009 Nov 1;47(9):1239-53
19628035
Free Radic Biol Med. 2008 Jul 15;45(2):158-66
18452718
Blood Rev. 2009 Mar;23(2):61-5
18644663
J Pharmacol Exp Ther. 2006 May;317(2):791-6
16452393
Free Radic Biol Med. 1990;8(3):251-8
2160411
J Biol Chem. 2002 Aug 2;277(31):28051-7
12023963
Hypertension. 2008 Feb;51(2):211-7
18086956
Am J Respir Cell Mol Biol. 1994 Jul;11(1):95-102
8018341
Arterioscler Thromb Vasc Biol. 2002 Dec 1;22(12):2003-9
12482826
Pharmacol Ther. 2008 Dec;120(3):254-91
18804121
Thromb Res. 2004;114(5-6):447-53
15507277
Naunyn Schmiedebergs Arch Pharmacol. 2009 Aug;380(2):193-204
19337723
J Cell Biol. 1999 Feb 22;144(4):745-54
10037795
Cardiovasc Res. 2008 Nov 1;80(2):271-9
18596059
Blood. 1996 Mar 1;87(5):2005-10
8634451
Arch Biochem Biophys. 2007 Jan 15;457(2):134-41
17166480
Atherosclerosis. 2009 Aug;205(2):427-32
19217621
J Biol Chem. 1994 Jul 29;269(30):19256-61
8034687
Free Radic Biol Med. 2006 Jul 15;41(2):193-201
16814099
Naunyn Schmiedebergs Arch Pharmacol. 2009 Nov;380(5):399-406
19730822
Antioxid Redox Signal. 2009 Oct;11(10):2607-19
19438290
Antioxid Redox Signal. 2004 Aug;6(4):691-8
15242549
Thromb Haemost. 2002 Aug;88(2):186-93
12195687
Thromb Res. 2001 Sep 1;103(5):399-409
11553372
Pharmacol Rep. 2007 Jul-Aug;59(4):428-36
17901572
Platelets. 2008 Feb;19(1):1-8
18231933