Antiplatelet effects of KW-7, a new inhibitor of cyclic nucleotide phosphodiesterases

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Abstract

The antiplatelet effect of a new synthetic compound, 8,9-dimethoxyl-1-(4-methoxy-phenyl)-5,6-dihydro-pyrrolo[2,1-a]isoquinoline-2,3-dione (KW-7), was determined in rabbit platelets. KW-7 concentration-dependently prevented platelet aggregation caused by arachidonic acid, collagen, platelet-activating factor, and thrombin. KW-7 induced a substantial increase in cyclic AMP levels and a smaller increase in cyclic GMP levels in platelets. In platelet homogenates, KW-7 inhibited both cyclic AMP- and cyclic GMP-phosphodiesterase activities. The antiplatelet effect of KW-7 was reversed by SQ22536 (an inhibitor of adenylate cyclase) and H89 (an inhibitor of protein kinase A) but not by ODQ (an inhibitor of soluble guanylate cyclase). These data suggest that the antiplatelet effect of KW-7 is cyclic AMP-dependent, and is through inhibition of platelet phosphodiesterases. In addition, KW-7 inhibited arachidonic acid-stimulated thromboxane production; this effect was associated with an increase in prostaglandin D2 levels indicating KW-7 is also an inhibitor of thromboxane synthase. The dual inhibition of KW-7 on phosphodiesterase and thromboxane synthase might provide an attractive target in developing antiplatelet drugs.

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).

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