Abstract
Platelet activation is important in the regulation of hemostasis and thrombosis. Uncontrolled activation of platelets may lead to arterial thrombosis, which is a major cause of myocardial infarction and stroke. After activation, metabolism of arachidonic acid (AA) by 12-lipoxygenase (12-LOX) may play a significant role in regulating the degree and stability of platelet activation because inhibition of 12-LOX significantly attenuates platelet aggregation in response to various agonists. Protein kinase C (PKC) activation is also known to be an important regulator of platelet activity. Using a newly developed selective inhibitor for 12-LOX and a pan-PKC inhibitor, we investigated the role of PKC in 12-LOX-mediated regulation of agonist signaling in the platelet. To determine the role of PKC within the 12-LOX pathway, a number of biochemical endpoints were measured, including platelet aggregation, calcium mobilization, and integrin activation. Inhibition of 12-LOX or PKC resulted in inhibition of dense granule secretion and attenuation of both aggregation and αIIbβ3 activation. However, activation of PKC downstream of 12-LOX inhibition rescued agonist-induced aggregation and integrin activation. Furthermore, inhibition of 12-LOX had no effect on PKC-mediated aggregation, indicating that 12-LOX is upstream of PKC. These studies support an essential role for PKC downstream of 12-LOX activation in human platelets and suggest 12-LOX as a possible target for antiplatelet therapy.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants HL089457, HL081009] and the Molecular Libraries Initiative of the National Institutes of Health Roadmap for Medical Research [Grant MH081283].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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ABBREVIATIONS:
- AA
- arachidonic acid
- COX-1
- cyclooxygenase-1
- 12-LOX
- 12-lipoxygenase
- PLA2
- phospholipase A2
- PKC
- protein kinase C
- 12-HETE
- 12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid
- PAR
- protease-activated receptor
- Ro 31-8220
- 3-[1-(3-(amidinothio)propyl-1H-indol-3-yl)]-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX)
- PMA
- phorbol myristoyl acetate
- GPCR
- G protein-coupled receptor
- NCTT-956
- N-((8-hydroxy-5-nitroquinolin-7-yl)(thiophen-2-yl)methyl)propionamide
- ERK
- extracellular regulated kinase
- AP
- activating peptide
- FITC
- fluorescein isothiocyanate
- TxB2
- thromboxane B2
- cPLA2
- cytosolic phospholipase A2
- CVX
- convulxin
- 12-HpETE
- 12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid
- MAPK
- mitogen-activated protein kinase.
- Received September 9, 2011.
- Accepted December 8, 2011.
- U.S. Government work not protected by U.S. copyright
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