Antiplatelet effects of KW-7, a new inhibitor of cyclic nucleotide phosphodiesterases
Introduction
Numerous evidence indicate that platelets contribute significantly to the pathogenesis of arterial thromboembotic diseases, such as acute coronary syndrome and ischemic stroke, which are the major causes of death in developed countries Schwortz et al., 1990, Ross, 1993. Antiplatelet drugs, such as aspirin and ticlopidine, have been shown to be beneficial in the treatment of thromboembotic diseases Coller, 1992, Antiplatelet Trialists' Collaboration, 1994a, Antiplatelet Trialists' Collaboration, 1994b, Antithrombotic Trialists' Collaboration, 2002. However, current antiplatelet drugs still have considerable limitation in their mode of action and efficacy. Greater understanding of platelet function in molecular levels will probably lead to the development of new more potent drugs Gresele and Agnelli, 2002, Bhatt and Topol, 2003.
Following endothelial damage or disruption of atherosclerotic plaque in blood vessels, platelets rapidly adhere to newly exposed extracellular matrix. Adhesion is followed by platelet activation and release proaggregatory substances, such as ADP and thromboxane A2, which thereby recruiting more circulating platelets, resulting in the formation of a platelet-rich thrombus. On the other hand, blood platelets are under tight negative control, mainly through prostacyclin and nitric oxide released from vascular endothelial cells, which increase platelet cyclic AMP and cyclic GMP levels and lead to inhibition of platelet activation Siess, 1989, Schwarz et al., 2001.
Benzylisoquinolines present a class of the most common alkaloids in higher plants that show various biological activities such as antiplatelet, vasorelaxing, analgesic, and anticancer effects. In the search of new antiplatelet agents, 40 newly synthetic benzylisoquinoline derivatives were tested for their antiplatelet activities. Among them, 8,9-dimethoxyl-1-(4-methoxy-phenyl)-5,6-dihydro-pyrrolo[2,1-a] isoquinoline-2,3-dione (KW-7, Fig. 1) showed the most potent inhibitory effect on platelet aggregation elicited by various platelet stimulators (Kuo et al., 2003). In the present study, the mechanism of action of KW-7 was further investigated. Our data suggest that the antiplatelet effect of KW-7 is cyclic AMP-dependent, and is through inhibition of platelet phosphodiesterases.
Section snippets
Preparation of washed rabbit platelets
Blood anticoagulated with ethylenediaminetetraacetic acid (EDTA) was collected from New Zealand rabbits. Rabbit platelet suspension was prepared according to the procedure previously described (Wu et al., 2000). The platelets, after washing, were finally suspended in Tyrode's solution containing Ca2+ (1 mM), glucose (11.1 mM) and bovine serum albumin (3.5 mg/ml) at a concentration of 3×108 platelets/ml.
Measurement of platelet aggregation
Platelet aggregation was measured turbidimetrically with a light-transmission aggregometer
Effect of KW-7 on the aggregation of washed rabbit platelets
In rabbit washed platelets, KW-7 completely inhibited arachidonic acid (AA, 100 μM)-, collagen (10 μg/ml)-, platelet activating factor (PAF, 1 ng/ml)-, and thrombin (0.1 U/ml)-induced platelet aggregation in a concentration-dependent manner with IC50 values of 10.4±0.1, 10.9±2.0, 32.9±1.8, and 32.7±0.4, respectively (n=4). In contrast, the IC50 values of the non-selective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) on platelet aggregation caused by these four stimulators are
Discussion
Elevation of intracellular cyclic AMP and cyclic GMP levels, either by stimulation of adenylate or guanylate cyclase or by inhibition of phosphodiesterases, is the most potent mechanism of platelet inhibition Haslam et al., 1999, Schwarz et al., 2001. Similar to the cyclic nucleotide-elevating agents Rao et al., 1980, Radomski et al., 1987, Sargeant and Sage, 1994, KW-7 also prevented platelet aggregation, dispersed formed platelet aggregates and blunted the increase of intracellular free
Acknowledgments
This work was supported by grants from the National Science Council of Taiwan (NSC 91-2320-B-037-059).
References (28)
Platelet prostanoid receptors
Pharmacol. Ther.
(1996)- et al.
Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells
J. Biol. Chem.
(1990) - et al.
Novel approaches to the treatment of thrombosis
Trends Pharmacol. Sci.
(2002) - et al.
Prostaglandins: specific inhibition of platelet adhesion to collagen and relationship with cyclic AMP level
Prostaglandins
(1982) - et al.
YC-1, a novel activator of platelet guanylate cyclase
Blood
(1994) - et al.
Antiplatelet activity of synthetic pyrrolo-benzylisoquinolines
Bioorg. Med. Chem. Lett.
(2003) - et al.
Cyclic AMP and cyclic GMP phosphodiesterase inhibition by an antiplatelet agent, 6-[(3-methylene-2-oxo-5-phenyl-5-tetrahydrofuranyl)methoxy]quinolinone (CCT-62)
Eur. J. Pharmacol.
(1998) - et al.
The influence of epinephrine on prostacyclin (PGI2) induced dissociation of ADP aggregated platelets
Prostaglandins
(1980) - et al.
Calcium signaling in platelets and other nonexcitable cells
Pharmacol. Ther.
(1994) - et al.
Taming platelets with cyclic nucleotides
Biochem. Pharmacol.
(2001)