Biochemical Pharmacology, Vol. 54, pp. 259-268, 1997. 0 1997 Elsevier Science Inc. All rights reserved. ELSEVIER ISSN 0006-2952/ 97/ $17.00 + 0.00 PII SOOOS-2952(97)00179-2 Thromboxane A, Synthase Inhibition and Thromboxane A2 Receptor Blockade by 2-[(+Cyanophenyl)amino]-3-chloro-l,+ nap~~thal~nedione (NQdY 15) in Rat Platelets Ton~S~~~ Chg, * Hyun-Mee Kim, * Ki-Seon Lee, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP * Lee-Yong KM, * Woong-Chon Mar, i: Chung-Kyu Ryu$ and C/tang-Kiu Moon* 8 zyxwvutsrqponmlkjihgfedcba *COLLEGE OF PHARMACY, SEOUL NATIONAL UNIVERSITY, SEOUL; JFNA-ruRAL PRODUCTS RESEARCH INSTITUTE, SEOULNATIONAL UNIVERSITY, SEOUL; AND SCOLLEGE OF PHARMACY,EWHA WOMAN’S UNIVERSITY, SOUL, KOREA A BSTRA CT. The effects of 2*[( 4.acetyIphenyl)amino].3,chloro~ 1,4~naphthalenedione (NQ-Y 15), a synthetic 1,4,na!~hthoquinone derivative, on platelet activity and its mechanism of action were investi- gated. NQ-Y15 caused a concentration-dependent inhibition of the aggregation induced by thrombin, collagen, arachidonic acid (AA), and A23187. The tcsO values of NQ-Yl5 on thrombin (0.1 U/ mL)-, collagen (10 p,g/ mL)-, AA (5C FM)-, and A23187 (2 CM&induced aggregation were 36.2 ? 1.5, 6.7 t: 0.7, 35.4 5 1.7, and 93.1 2 1.4 FM, respectively. NQ-YlS also inhibited thrombin-, collagen-, AA-, and A23187-stimulated serotonin secretion in a concentration-dependent manner. However, a high concentration (100 CM) of NQ-Y 15 showed no significant inhibitory effect on ADP-induced primary aggregation, which is independent of thromboxane A, (TXA,) production in rat platelets. In fura-a-loaded platelets, the elevation of intracellular free calcium concentration stimulated by AA, thrombin, and 4.bromo-A23187 was inhibited by NQ-Y15 in a concentration-dependent manner. The formation of TXA, caused by AA, thrombin, and collagen was inhibited significantly by %‘Q-Y15. NQ-Y15 inhibited TXA , synthase in intact rat platelets, since this agent reduced the conversion of prostaglandin (PG) H, to TXA,. Similarly, NQ#Y15 selectively inhibited the TXA, synthase activity in human platelet microsomes, whereas it had no effect on activity of phospholipase A,, cyclooxygenase, and PGI, synthase in vitro. NQ-Yl5 inhibited platelet aggregation induced by the endoperoxide analogue U46619 in human platelets, indicating TXA , receptor antagonism, possibly of a competitive nature. These results suggest that the antiplatelet effect of NQ-Yl5 is due to a combination of TXA, synthase inhibition with TXA , receptor blockade, and that it may be useful as an antithrombotic agent. BI~~-IEM PHARMA~~L 54;2: 259-268, 1997. 63 1997 Eisevier Science Inc. KEY WORDS. 1,4-naphthalenedione derivative; platelet: aggregation; secretion; thromboxane A, synthase; thromboxane A, receptor Platelet-vessel wall interact ions represent important factors in the development of thrombosis and atherosclerosis [l, Z]. When blood vessels are damaged, platelet aggregation occurs rapidly to form hemostatic plugs or arterial thrombi at sites of vessel injury or in regions where blood flow is disturbed. These thrombi are the source of thromboembolic complications of atherosclerosis, heart attacks, strokes, and Q Grresponding author: Dr. C. ‘K. Moon, College of Pharmacy, Seoul National University, San 56-1 Sbilim-Dong Kwanak-Ku, Seoul 151-742, Korea. Tel. 82-02-880-7843; FAX 82-02-884-4580. i’Abbre&kns: A23187, 6S~(6~,8~,9~,1lu)-5-(methylamino)-2-((3,9,1 I- trimethyl-8-( l-methyl-2-oxo-2*( l.~~pyrrol-2-yl)ethyl)~l,7-dioxaspiro(5,5) undec-2-yl)methyl)-4-betlzooxazo ecarboxylic acid; AA, arachidonic acid; [Ca2 ‘I,, intracellular free calciurr. concentration; COX, cyclooxygenase; l%HPETE, 15(S)-hydroperoxyeictaa_5Z,8Z,l lZ,13E+etraenoic acid; NQ- Y15, 2~[(4-cyanophenyl)amino]-3-chloro-l,4-naphthalenedione; PC, prostaglandin; PL, phospholipase; RIA, radioimmunoassay; TX, throm- boxane; and U46619,9,11-dioxy-%,I l~~epoxymethano~rostaglandin H,. Received 24 September 1996; accepted 14 February 1997. peripheral vascular disease. Platelet adhesion to subendo- thelial components, such as collagen, activates signalling pathways that lead to TXA, formation and secretion of platelet granule contents. Both substances cause platelet aggregation [3]. The morphological evidence that arterial thrombi are largely composed of platelet aggregates has led many investigators to postulate that platelet aggregation is a major pathogenic mechanism in arterial thrombosis [4]. Thus, inhibition of platelet function represents a promising approach for the prevention of thrombosis. AA” is a membrane-derived fatty acid that is metabolized by COX to PG endoperoxide intermediates, such as PGH,. In platelets, endoperoxides are further metabolized to TXA , by TXA, y s nthase. TXA , is a potent inducer of platelet aggregation and a vasoconstrictor [5]. Levels of this mediator are increased in several thrombotic disorders 161, Therefore, agents that inhibit the formation or the action