Abstract
Thrombin induces platelet activation through an early, reversible stage of platelet aggregation, which is followed by a later, irreversible stage of platelet aggregation. Without intervention, events leading to pathological platelet activation can result in vessel occlusion, acute coronary syndrome, and stroke. Therefore, a better understanding of events leading to platelet-mediated clot formation may provide insight into new therapeutic targets. Once activated, protease activated receptors (PARs) are essential in regulating events leading to platelet aggregation. We have determined a signaling cascade through PAR1, which involves phosphatidylinositol (PI) kinases, phosphatidylinositol bisphosphate (PIP2), and Rap1 activation (independent of P2Y12) in the formation of a stable platelet aggregate. The putative phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 was found to reduce basal and PAR-stimulated PIP2 levels by mass spectrometry and to inhibit PAR1-mediated stable platelet aggregation. Rap1 activation in platelets (during time points corresponding to the late, irreversible phase of aggregation) was found to require the PI signaling pathway. Perturbation of PI3K signaling by isoform-selective inhibitors had differential effects on Rap1 activation through PAR1 and PAR4. Hence, it is possible to disrupt lipid signaling pathways involved in stable clot formation without inhibiting early clot formation, offering a new potential target for antiplatelet therapy.
Footnotes
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This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants P50-HL081009, R01-HL084388, K99-HL089457, R00-HL089457] and the National Institutes of Health National Institute of General Medical Sciences [Grant U54-GM069338].
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M.H. and A.M.P. contributed equally to this work.
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ABBREVIATIONS: PAR, protease-activated receptor; CalDAG-GEF, calcium, diacylglycerol, guanine nucleotide exchange factor; PA, phosphatidic acid;, phosphatidylinositol; PI3K, phosphatidylinositol-3 kinase; PI5K, phosphatidylinositol-5 kinase; PIPns, phosphatidylinositol phosphates; PIP2, phosphatidylinositol bisphosphate; PIP3, phosphatidylinositol trisphosphate; PLD, phospholipase D; RapGEF, Rap1 guanine nucleotide exchange factor; DAG, diacylglycerol; PKC, protein kinase C; MS, mass spectrometry; TGX-115, 8-(2′-methylphenoxy)-2-morpholino-4-quinolone; PI-103, 3-(4-(4-morpholinyl)pyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl)phenol; 2-MeSAMP, 2-methylthio-AMP; AKTi X, 10-(4′-(N-diethylamino)butyl)-2-chlorophenoxazine; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride.
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↵1 Current affiliation: Division of Hematology, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania.
- Accepted May 29, 2009.
- Received March 31, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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