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This study was performed to examine the antithrombotic effect of DP-1904, a synthetic inhibitor of thromboxane synthetase, on He-Ne laser-induced thrombus formation in an attempt to clarify the role of arachidonic acid metabolites in thrombogenesis in rats. DP-1904 inhibited thrombus formation in arterioles in a dose-dependent manner, but this inhibition was transient and disappeared 6 hours after oral administration of DP-1904. Inhibition was not observed in venules. Platelet function ex vivo, platelet adhesion and thrombin and collagen-induced platelet aggregation, was not affected significantly by *神 戸 学 院 大 学 栄 養 学 部 生 理 学 研 究 室 〔〒651 -21神 戸 市 西 区 伊 川 谷 町 有 瀬 〕:Correspondence to:Junichiro Yamamoto, Laboratory of Physiology, Faculty of Nutrition, Kobe-Gakuin University, Kobe 651-21, Japan. 受 付:1993.5.24,受 理:1993.11.1 10 日本血栓止血学会誌 第5巻 第1号 DP-1904. These results suggested that 1) DP-1904 was an excellent antithrombotic drug and 2) arachidonic acid metabolites contributed to thrombosis in rats and their role in rat arterioles was different from that in venules. Introduction Thrombus formation is triggered by injury to endothelial cells, followed by platelet adhesion, aggregation and fibrin deposition. It is thought therefore that platelet activation may play a key role in primary thrombus formation both in arterioles and in venules. Thromboxane A2 (TxA2), a powerful inducer of platelet aggregation, is an arachidonic acid metabolite which is produced in platelets and which promotes platelet thrombus formation. In contrast, prostacyclin (PGI2), an arachidonic acid metabolite produced in endothelial cells, is a powerful inhibitor of platelet aggregation. It is thought that the balance of these two metabolites is important to control platelet aggregatlion in vivo. Aspirin is widely used clinically to shift this balance to the antithrombotic side. However, the antithrombotic effect of aspirin in vivo is not as powerful as that expected from in vitro studies, because a cyclooxygenase inhibitor like aspirin prevents not only TxA2 synthesis in platelets but also PGI2 synthesis in endothelial cells. In contrast, a selective thromboxane synthetase inhibitor such as DP-1904 would prevent TxA2 synthesis but not PGI2 synthesis resulting in a decrease in the ratio of TxA2/PGI2 and then inhibition of the thrombus formation. It is logical, therefore, to expect that a thromboxane synthetase inhibitor would be an excellent antithrombotic Burg. Antithrombotic effect of thromboxane synthetase inhibitors has been reported in some kinds of thrombus model1)5). In contrast with these reports, it has been also reported that thromboxane synthetase inhibitors do not have antithrombotic effect in other thrombosis model6)7). So, antithrombotic effect of thromboxane synthetase inhibitors in vivo is not clear yet. The purpose of the present study was to examine the antithrombotic effect of DP-1904 on He-Ne laser-induced thrombus, platelet thrombus, formation in rat mesenteric microvessels. The effect of this drug ex vivo on platelet adhesion, platelet aggregation and coagulation activities was also examined. Materials and Methods Male Wister/ST rats (8-15 weeks, 220-360 g body weight, SLC Inc, Japan) were used. DP-1904, a synthetic thromboxane synthetase inhibitor (donated from Daiichi Pharmaceutical Co. Ltd., Japan) was dissolved in water. The agent (10,50,100 or 200mg/kg body weight) was administrated orally, and experiments were started at 1,2,3 and 6 hours after administration. Experimental Microthrombus Formation by He-Ne Laser Irradiation Thrombi were formed according to the method of Kovacs et al., with slight modification8)•`10). Rats were anesthetized with sodium pentobarbital (60mg/kg, i.m.) and 1% Evans blue (14.2mg/kg, E. Merck) was injected into the jugular veins. An intestinal loop was exposed through a hypogastric incision and was continuously irrigated with Tyrode solution at 3TC. The mesentery was spread out flat on a self-constructed object table, mounted on an Olympus BH 2 microscope equipped with ultralong working distant objective (Olympus ULWD CDPIan 20, x 20). An Oring was placed on the mesentery to stop vessel movement. Arterioles (20•`25 ISHII et al.: Antithrombotic effect of TX synthetase inhibitor 11 p m outer diameter) and venules (30-35ƒÊm) exhibiting fast blood flow, no curves and no branches were selected. An He-Ne laser beam (Neo-50MS, Nihon Kagaku Eng. Co Ltd., Japan) was introduced into the microscope by a dichroic mirror and was directed through the optical path of the microscope to the center of the microvessels. The diameter of the laser spot on the focal plane was 15 p m. Power of 15.8mW and 21.0mW were used for arterioles and venules, respectively. A 5-second pulse of irradiation was repeated every 30 seconds until an occlusive thrombus was formed. The number of irradiations necessary to form an occlusive thrombus was counted. More than three thrombi were formed in arterioles and venules in each experiment. Platelet Adhesion Platelet adhesion was performed according to the method of Salzman with slight modification". Blood was collected from the abdominal aorta with a plastic syringe at 1, 2,3 or 6 hours after administration of the agent. Native blood was passed through a glass beads column at a constant speed using a syringe-pump (Igaku Shoin Kikai Co. Ltd., Japan). The number of platelets in blood anticoagulated with EDTA before and after passing through the column was counted using an automatic cell counter (Sysmexmicrocell counter CC-180 A, Toa Medical Electronics Co., Ltd.). Platelet Aggregation Citrated blood (9 vols blood: 1vol citrate) was kept at room temperature for 15 minutes prior to testing. Platelet aggregation was measured by the impedance method. Five-hundred microliters Locke solution were added to 500 pl blood and warmed for about 4 min at 3TC. Then, ten microliters thrombin (final conc. 1 p/ml) or 20 pl collagen (final conc. 20 pg/ml) were added to the mixture and platelet aggregation was measured at 3 min. Whole Blood Clotting Time Whole blood clotting time was measured by adding 30 pl of CaCl2 to 300 pl citrated blood using a coagulometer, Amelung KC 1 A. Statistics Values were expressed as mean•} SEM. Unpaired Wilcoxon's two-sample test was used for the laser irradiation experiment, and unpaired Student's t-test was used for other measurements.

Antithrombotic Effect of Thromboxane Synthetase Inhibitor, DP-1904, on Laser-Induced Thrombogenesis in Rat Mesenteric Microvessels