TY - JOUR
T1 - The anti-platelet effects of apocynin in mice are not mediated by inhibition of NADPH oxidase activity
AU - Dharmarajah, Janahan
AU - Arthur, Jane Frances
AU - Sobey, Christopher Graeme
AU - Drummond, Grant Raymond
PY - 2010
Y1 - 2010
N2 - 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 muM. 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.
AB - 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 muM. 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.
UR - http://www.springerlink.com/content/761776r73745745p/fulltext.pdf
M3 - Article
SN - 0028-1298
VL - 382
SP - 377
EP - 384
JO - Naunyn-Schmiedeberg's Archives of Pharmacology
JF - Naunyn-Schmiedeberg's Archives of Pharmacology
IS - 4
ER -