Original Paper Haemostasis 1992;22:147-152
J. Yamamoto I. Ishii Y. Sasaki Y. Nagamatsu T. Matsuda E. Ando
Antithrombotic Effect of Ticlopidine on He-IMe Laser-Induced Thrombus Formation in Rat Mesenteric Microvessels
Laboratory of Physiology, Faculty of Nutrition, Kobe-Gakuin University, Kobe, Japan
Abstract The inhibitory effects of ticlopidine and acetylsalicylic acid (ASA) on thrombus formation in rat mesenteric microvessels were studied. The results were compared with the effect of the drugs on platelet aggregation in citrated whole blood. He-Ne laser-induced thrombus formation in arterioles and in venules was significantly inhibited by 100 mg/kg ticlopidine. In con trast, a higher dose of ASA (300 mg/kg) was needed to inhibit thrombus formation and the effects of ASA were observed only in arterioles and not in venules. In addition, although the inhibition by ASA in arterioles was not strong it followed a dose-dependent manner, suggesting that the differential effect of ASA on platelets and endothelium may not be evident in vivo. Ticlopidine and ASA strongly inhibited ADP-induced whole blood platelet aggregation, but not collagen- or throm bin-induced platelet aggregation.
Introduction A thrombus is formed as the result of inter actions between blood components, the vessel wall and blood flow. In studies of laserinduced thrombosis, these mechanisms are examined under approximately physiological conditions and the method is especially useful
Received: June 5, 1991 Accepted in revised form: November 19, 1991
to evaluate the antithrombotic effect of drugs. The antithrombotic effect of antiplatelet drugs has been reported in a variety of animal models [1-4]. For example, Weichert et al. [5] used argon and ruby laser-induced thrombus formation to investigate the effect of ticlopi dine on hemostasis in venules. However, the
Prof. J. Yamamoto Laboratory of Physiology Faculty of Nutrition Kobe-Gakuin University Kobe 651-21 (Japan)
© 1992 S. Karger AG, Basel 0301-0147/92/ 0223—0147$2.75/0
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Keywords Thrombosis Platelet Ticlopidine Laser Rat
148
Materials and Methods Male Wistar ST strain rats aged 8-9 weeks were used. Ticlopidine (Daiichi Pharmaceutical Co. Ltd., Japan) and ASA (Nacalai Tesque, Inc., Japan) were dissolved in 0.5% Tween 80. Experimental Microthrombus Formation by the He-Ne Laser Method The microthrombus was formed according to Kovacs et al. [8], but with slight modifications [7], Rats were anesthetized with sodium pentobarbital (60 mg/kg, i.m.) and 1.5% Evans blue (E. Merck) was injected via the jugular vein (21.6 mg/kg). An intesti nal loop, exposed through a hypogastric incision and continuously irrigated with Tyrode solution at 37 °C, was spread out flat on a self-constructed object table, which was mounted on the microscope table of an Olympus BH2 microscope equipped with long work ing distant objectives (Olympus ULWD CDPlan 20, X 20). An O-ring of 1.5 g was secured to a cover glass and was placed on the mesentery to stop vessel move ment and to observe microvessels. Arterioles and ve nules with an outer diameter of 20-25 and 25-35 pm, respectively, exhibiting fast blood flow in the fat-free portion, were selected to form microthrombi. A He-Ne laser beam (Neo-15MS; 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 microves sels. The diameter of the laser spot on the focal plane was 20 pm with a power of 17.0 mW. A 5-second irra diation period was repeated every 30 s until an occlu sive thrombus was formed. The number of laser irra diations necessary to induce an occlusive thrombus formation was counted. Thrombi were formed in three venules and in three arterioles in each rat. The mean values in the arterioles and venules of an individual are calculated from three respective values. Ticlopidine (50, 100, 200 or 300 mg/kg), ASA (50, 200 or 300 mg/kg) or 0.5% Tween 80 (control) was administered orally and irradiation of the mesenteric microvessels was begun 2 or 17 h later. The increase in number of irradiations necessary to form an occlusive thrombus provided an index of the inhibitory effect of each drug. Platelet Aggregation Platelet aggregation in whole blood was measured using the impedance method with a Chronolog C-500 aggregometer. Blood was collected from the abdominal aorta into 3.2% sodium citrate (blood volumexitrate volume = 9:1) immediately after the microthrombus
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mechanisms of thrombus formation using different lasers are not consistent. Argon laer irradiation does not produce occlusive thrombi in venules, though it can produce fragile occlusive thrombi in arterioles. Plate let aggregates formed by argon lasers are eas ily disrupted by flowing blood and pass into the circulation as microemboli [5]. In con trast, He-Ne irradiation readily stimulates the formation of occlusive thrombi in both arteri oles and venules. In addition, the action of ruby lasers appears to be different from that of either argon or He-Ne lasers. Ruby lasers destroy erythrocytes and produce plasma pro tein precipitates. ADP released from the dam aged erythrocytes and the precipitate induce platelet thrombus formation [5, 6]. Furthermore, a natural thrombus formed in arteries or arterioles is mainly composed of platelets, whilst a thrombus formed in veins or venules is composed of erythrocytes and platelets entrapped by fibrin. A laser-induced thrombus is mainly composed of platelets. It would be reasonable, therefore, to use arteri oles, not venules, for evaluating antithrom botic drugs in laser-induced thrombosis. Nev ertheless, a primary thrombus may consist predominantly of platelets even in a red thrombus formed in veins or venules. The properties of the endothelial cells of veins or venules may be different from those of arter ies or arterioles and the nature of the vessel wall may govern thrombus formation. We have previously observed a variable effect of exercise on thrombus formation in arterioles and venules [7], In the present study, we have examined the antithrombotic effect of ticlopidine and acetylsalicylic acid (ASA), on He-Ne laser-in duced thrombus formation in arterioles and venules. We have compared these results with the effect of the drugs on platelet aggregation in whole blood in vitro.
Fig. 1. Antithrombotic effect of ticlopidine on la ser-induced thrombus formation in rat mesenteric ar terioles and venules. Irradiation was begun 2 h (a) or 17 h (b) after oral administration of ticlodipine or Tween 80 (control). Ordinate shows number of irradia tions necessary to form an occlusive thrombus. □ = Arteriole; ■ = venule; n = 8 animals in each group; * p < 0.05; ** p < 0.01.
Fig. 2. Effect of ASA on laser-induced thrombus formation in rat mesenteric arterioles and venules. Irradiation was begun 2 h (a) or 17 h (b) after adminis tration of ASA or Tween 80 (control). □ = Arteriole; ■ = venule; n = 8 animals in each group; * p < 0.05.
formation experiment. Blood samples were at room temperature for 30 min until used. To 0.5 ml of pre warmed Locke solution 0.5 ml of citrated blood was added. The mixture was allowed to stand until the tem perature rose to 37 °C (approx. 4 min). Twenty micro liters of agonist, i.e. collagen (1 mg/ml; Horm), throm bin (100 U/ml in 50 mMTris-HCl containing 100 mM NaCl, pH 7.3; Mochida Pharmaceutical Co. Ltd., Ja pan) or ADP (10 m M in 50 mM Tris-HCl, pH 6.8, stored at - 80 ° C; Sigma) were added to the mixture and aggregation was recorded for 6 min. The reaction was observed in a plastic cuvette at 1,000 rpm. Colla gen-induced platelet aggregation was started 30 min after the blood was collected, then thrombin- and ADP-induced platelet aggregation tests followed se quentially. Whole blood dotting time was measured by adding 30 pi of 0.117 M CaCl2 to 300 pi citrated blood using a KC 1A Amelung Coagulometer. Blood cell count was measured using an automatic cell counter (Sysmex Microcellcounter CC-180A, Toa Medical Electronics Co. Ltd., Japan).
Statistics Wilcoxon’s two-sample test was employed in analy sis of the number of irradiations, and unpaired Stu dent’s t test was used for that of platelet aggregation, body weight and of blood cell count. Statistical signifi cance was examined against each control. Values are expressed as mean ± SE.
Antithrombotic Effect o f Ticlopidine and ASA Results are shown in figures 1 and 2. Ticlo pidine showed a dose-dependent antithrom botic effect in arterioles as well as in venules (fig. 1). However, this inhibition was stronger in arterioles than in venules. ASA showed sig nificant antithrombotic effect in arterioles when 300 mg/kg was administered, but not in venules (fig. 2).
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Results
Table 1. Effect of ticlopidine and ASA on coagula tion and blood cell counts
E
to
E ■C o ) cto
—
12 1
X
T
10-
0o° V
/y
Control
j
WBCT
2 h 160.1 ±5.1 133.3 ± 17.9 118.6 ± 13.3 3 h 148.4 + 5.5 128.0 ± 11.0 17 h 124.1 ±7.1 133.1 ±9.7 102.0 ± 10.2
RBC
2h 3h 17 h
807 ± 11 810 ± 32 793 ± 12
759 ± 16* 754 ±19 738 ±14*
780±6
2h 3h 17 h
26 ±2 39 ± 3 35 ± 1
28 ±5 25 ±2** 48±3*
31 ±3
2h 3h 17 h
76 ± 3 81 ±3 79±6
87 ±4** 76±2 81 ±4
WBC
Dose of ticlopidine and A SA (mg/kg)
Fig. 3. Antiaggregatory effect of ticlopidine and
ASA on ADP-induced platelet aggregation. Impedance change 2 h (a), 3 h (b) and 17 h (c) after administration of ticlopidine or ASA. Values are expressed as impe dance change 6 min after addition of ADP (AO/6 min), n = 6 animals in each group; * p < 0.05; ** p < 0.01.
Antiaggregatory Effect o f Ticlopidine and ASA Ticlopidine and ASA inhibited ADP-in duced aggregation in a time-dependent man ner (fig. 3). Neither ticlopidine nor ASA in hibited thrombin- or collagen-induced plate let aggregation (data not shown). Effect o f Ticlopidine and ASA on Coagulation and Blood Cell Counts Results are shown in table 1. Neither ticlo pidine nor ASA affected whole blood clotting time, but some significant effects on erythro cyte, leukocyte and platelet counts were ob served. These changes might be relevant in the context of the mild to severe neutropenia observed in some patients treated with ticlo pidine but they were not examined further in the present study.
150
Ticlopidine ASA
PLT
-
748 ±13*
-
45 ± 5 70± 3 -
84 ±3
These parameters were measured prior to platelet aggregation test using the same blood samples. Blood was collected 2, 3 or 17 h after oral administration of ticlopidine (200 mg/kg), ASA (200 mg/kg) or Tween 80. WBCT = whole blood clotting time (s); RBC = erythrocytes (X 104/pl); WBC = leukocytes (X 102/gl); PLT = platelets (X 104/pl); n = 6 animals in each group; * p < 0.05; ** p < 0.01.
Discussion It is thought that platelet thrombus forma tion in vivo is initiated by injury to endothe lial cells, followed by adhesion, aggregation of platelets, and fibrin deposition. A primary thrombus may be mainly composed of plate lets both in arterioles and in venules. He-Ne laser irradiation injures endothelial cells to form a thrombus mainly composed of plate lets in arterioles and in venules [8]. Therefore, He-Ne laser-induced thrombus formation provides a useful method for testing antiag gregatory drugs in vivo [9]. In the present study this method was used to assess the anti thrombotic effects of ticlopidine and ASA.
Yamamoto/Ishii/Sasaki/N agamatsu/ Matsuda/Ando
Antithrombotic Effect of Ticlopidine
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a
be explained. In the present whole blood ag gregation studies, however, antiplatelet activ ity of ASA was demonstrated at 200 mg/kg but in contrast, in the He-Ne laser model, higher concentrations (300 mg/kg) were re quired to inhibit in vivo thrombus formation. These data tend to suggest that the differential effects of ASA on endothelium and platelets may not be apparent in vivo. In vitro, ticlopidine and ASA inhibited ADP-induced whole blood platelet aggrega tion, but not collagen- and thrombin-induced aggregation. These data are in keeping with the concept that ADP plays a key role in thrombogenesis in rats [16]. Furthermore, the antiaggregatory effect was almost parallel to the antithrombotic effect in arterioles, but not in venules. These results tend to support ear lier findings [7] and suggest that the proper ties of endothelial cells of arterioles may differ from those of venules. The present results demonstrated that the minimal effective doses of ticlopidine and ASA in rats were approximately 100 times greater than those required in man [17, 18]. However, ticlopidine appeared to be more potent than ASA as an antithrombotic agent in the animal model and the data supported the view that ticlopidine might be more effec tive than ASA in man.
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Ticlopidine inhibited thrombus formation in arterioles and in venules. A statistically sig nificant effect was noted at 100 mg/kg in arte rioles and in venules. Dose-dependent inhibi tion was more clearly observed in arterioles than in venules. This tendency was also ob served in animals given ASA, although a sta tistically significant effect could be observed only at higher doses of the drug (300 mg/kg). The minimal effective dose of ticlopidine in arterioles was similar to that reported by Ashida et al. [10], although a lower concentra tion (10 mg/kg) was found to be effective by Seiffge and Weithmann [11], In addition, the present effective dose in venules was similar to that reported by others using different tech niques [ 1, 3, 4, 12, 13] . It should be empha sized, however, that the antithrombotic effect of ticlopidine in models based on venous sta sis [12, 13] might be influenced by inhibition of blood coagulation, where coagulation is normally enhanced by coexisting platelets [14], In the present studies, ASA inhibited thrombus formation in arterioles at a dose of 300 mg/kg, but not at 200 mg/kg. These re sults were consistent with those of a rat, arte riovenous shunt model [2], but were markedly different from those seen using an argon-laser model, where 10 mg/kg of ASA significantly inhibited thrombus formation [11], It is clear that the effects of ASA are especially depen dent on the particular animal model. The efficacy of ASA as an antithrombotic drug is not firmly established. It has been sug gested that inhibition of cyclo-oxygenase by ASA simultaneously prevents the formation of aggregatory thromboxane A2 in platelets and that of antiaggregatory prostacyclin in vascular cells [15]. Vascular cells and platelets might be differentially sensitive to the effects of ASA and thus, the deceptive results from clinical trials of aspirin as an antithrombotic agent, the so-called ‘aspirin dilemma’, could
1 Tomikawa M, Ashida S, Kakihata K, Abiko Y: Anti-thrombotic action of ticlopidine, a new platelet aggre gation inhibitor. Thromb Res 1978; 12:1157-1164. 2 Ashida S, Sakuma K, Abiko Y: Anti thrombotic effects of ticlopidine, acetylsalicylic acid and dipyrida mole in vascular shunt model in rats. Thromb Res 1980; 17:663671. 3 Kumada T, Ishihara M, Ogawa H, Abiko Y: Experimental model of ve nous thrombosis in rats and effect of some agents. Thromb Res 1980; 18: 189-203. 4 Weichert W, Pauliks V, Breddin HK: Laser-induced thrombi in rat mesenteric vessels and antithrom botic drugs. Haemostasis 1983; 13: 61-71. 5 Weichert W, Breddin HK, Staubesand J: Application of a laser-in duced endothelial injury model in the screening of antithrombotic drugs. Semin Thromb Hemost 1988; 14(suppl): 106-114. 6 Hovig T, McKenzie FN, Arfors K-E: Measurement of the platelet re sponse to laser-induced microvascular injury. Thromb Diath Haemorrh 1974;32:695-703.
7 Yamamoto J, Iizumi H, Hirota R, Shimonaka K, Nagamatsu Y, Horie N, Morita S: Effect of physical train ing on thrombotic tendency in rats: decrease in thrombotic tendency measured by the He-Ne laser-in duced thrombus formation method. Haemostasis 1989;19:260-265. 8 Kovâcs IB, Tigyi-Sebes A, Trombitas K, Gôrôg P: Evans blue: an ideal energy-absorbing material to pro duce intravascular microinjury by He-Ne gas laser. Microvasc Res 1975;10:107-124. 9 Kovâcs IB, Gôrôg P: Laser-induced thrombosis test suitable for pharma cological screening studies. Micro vasc Res 1979;18:403-412. 10 Ashida S, Ishihara M, Ogawa H, Abiko Y: Protective effect of ticlo pidine on experimentally induced peripheral arterial occlusive disease in rats. Thromb Res 1980; 18:55— 67. 11 Seiffge D, Weithmann KU: Surpris ing effects of the sequential adminis tration of pentoxifylline and low dose acetylsalicylic acid on throm bus formation. Thromb Res 1987; 46:371-383.
12 Millet J, Theveniaux J, Pascal M: A new experimental model of venous thrombosis in rats involving partial stasis and slight endothelium altera tions. Thromb Res 1987;45:123— 133. 13 Bernat A, Vallee E, Maffrand JP, Roncucci R: Antithrombotic effect of ticlopidine in a platelet-indepen dent model of venous thrombosis. Thromb Res 1985;37:279-285. 14 Gôrôg P, Kovâcs IB: Coagulation of flowing native blood: advantages over stagnant clotting test (submit ted). 15 de Gaetano G, Cerletti C, Bertele V: Pharmacology of antiplatelet drugs and clinical trials on thrombosis prevention. Lancet 1982;ii:974— 977. 16 Maffrand JP, Bemat A, Pelebassee D, Defreyn G, Cazenave JP, Gorden JL: ADP plays a key role in thrombogenesis in rats. Thromb Haemost 1988;59:225-230. 17 Hardisty RM, Powling MJ, Nakes TJC: The action of ticlopidine on human platelet. Thromb Haemostas 1990;64:150-155. 18 Ratnatunga CP, Edomondson SF, Rees GM, Kovâcs IB: Aspirin inhib its shear-induced platelet reaction involving thrombin generation. Cir culation, in press.
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References
J. Yamamoto I. Ishii Y. Sasaki Y. Nagamatsu T. Matsuda E. Ando
Antithrombotic Effect of Ticlopidine on He-IMe Laser-Induced Thrombus Formation in Rat Mesenteric Microvessels
Laboratory of Physiology, Faculty of Nutrition, Kobe-Gakuin University, Kobe, Japan
Abstract The inhibitory effects of ticlopidine and acetylsalicylic acid (ASA) on thrombus formation in rat mesenteric microvessels were studied. The results were compared with the effect of the drugs on platelet aggregation in citrated whole blood. He-Ne laser-induced thrombus formation in arterioles and in venules was significantly inhibited by 100 mg/kg ticlopidine. In con trast, a higher dose of ASA (300 mg/kg) was needed to inhibit thrombus formation and the effects of ASA were observed only in arterioles and not in venules. In addition, although the inhibition by ASA in arterioles was not strong it followed a dose-dependent manner, suggesting that the differential effect of ASA on platelets and endothelium may not be evident in vivo. Ticlopidine and ASA strongly inhibited ADP-induced whole blood platelet aggregation, but not collagen- or throm bin-induced platelet aggregation.
Introduction A thrombus is formed as the result of inter actions between blood components, the vessel wall and blood flow. In studies of laserinduced thrombosis, these mechanisms are examined under approximately physiological conditions and the method is especially useful
Received: June 5, 1991 Accepted in revised form: November 19, 1991
to evaluate the antithrombotic effect of drugs. The antithrombotic effect of antiplatelet drugs has been reported in a variety of animal models [1-4]. For example, Weichert et al. [5] used argon and ruby laser-induced thrombus formation to investigate the effect of ticlopi dine on hemostasis in venules. However, the
Prof. J. Yamamoto Laboratory of Physiology Faculty of Nutrition Kobe-Gakuin University Kobe 651-21 (Japan)
© 1992 S. Karger AG, Basel 0301-0147/92/ 0223—0147$2.75/0
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Keywords Thrombosis Platelet Ticlopidine Laser Rat
148
Materials and Methods Male Wistar ST strain rats aged 8-9 weeks were used. Ticlopidine (Daiichi Pharmaceutical Co. Ltd., Japan) and ASA (Nacalai Tesque, Inc., Japan) were dissolved in 0.5% Tween 80. Experimental Microthrombus Formation by the He-Ne Laser Method The microthrombus was formed according to Kovacs et al. [8], but with slight modifications [7], Rats were anesthetized with sodium pentobarbital (60 mg/kg, i.m.) and 1.5% Evans blue (E. Merck) was injected via the jugular vein (21.6 mg/kg). An intesti nal loop, exposed through a hypogastric incision and continuously irrigated with Tyrode solution at 37 °C, was spread out flat on a self-constructed object table, which was mounted on the microscope table of an Olympus BH2 microscope equipped with long work ing distant objectives (Olympus ULWD CDPlan 20, X 20). An O-ring of 1.5 g was secured to a cover glass and was placed on the mesentery to stop vessel move ment and to observe microvessels. Arterioles and ve nules with an outer diameter of 20-25 and 25-35 pm, respectively, exhibiting fast blood flow in the fat-free portion, were selected to form microthrombi. A He-Ne laser beam (Neo-15MS; 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 microves sels. The diameter of the laser spot on the focal plane was 20 pm with a power of 17.0 mW. A 5-second irra diation period was repeated every 30 s until an occlu sive thrombus was formed. The number of laser irra diations necessary to induce an occlusive thrombus formation was counted. Thrombi were formed in three venules and in three arterioles in each rat. The mean values in the arterioles and venules of an individual are calculated from three respective values. Ticlopidine (50, 100, 200 or 300 mg/kg), ASA (50, 200 or 300 mg/kg) or 0.5% Tween 80 (control) was administered orally and irradiation of the mesenteric microvessels was begun 2 or 17 h later. The increase in number of irradiations necessary to form an occlusive thrombus provided an index of the inhibitory effect of each drug. Platelet Aggregation Platelet aggregation in whole blood was measured using the impedance method with a Chronolog C-500 aggregometer. Blood was collected from the abdominal aorta into 3.2% sodium citrate (blood volumexitrate volume = 9:1) immediately after the microthrombus
Yamamoto/Ishii/Sasaki/Nagamatsu/ Matsuda/Ando
Antithrombotic Effect of Ticlopidine
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mechanisms of thrombus formation using different lasers are not consistent. Argon laer irradiation does not produce occlusive thrombi in venules, though it can produce fragile occlusive thrombi in arterioles. Plate let aggregates formed by argon lasers are eas ily disrupted by flowing blood and pass into the circulation as microemboli [5]. In con trast, He-Ne irradiation readily stimulates the formation of occlusive thrombi in both arteri oles and venules. In addition, the action of ruby lasers appears to be different from that of either argon or He-Ne lasers. Ruby lasers destroy erythrocytes and produce plasma pro tein precipitates. ADP released from the dam aged erythrocytes and the precipitate induce platelet thrombus formation [5, 6]. Furthermore, a natural thrombus formed in arteries or arterioles is mainly composed of platelets, whilst a thrombus formed in veins or venules is composed of erythrocytes and platelets entrapped by fibrin. A laser-induced thrombus is mainly composed of platelets. It would be reasonable, therefore, to use arteri oles, not venules, for evaluating antithrom botic drugs in laser-induced thrombosis. Nev ertheless, a primary thrombus may consist predominantly of platelets even in a red thrombus formed in veins or venules. The properties of the endothelial cells of veins or venules may be different from those of arter ies or arterioles and the nature of the vessel wall may govern thrombus formation. We have previously observed a variable effect of exercise on thrombus formation in arterioles and venules [7], In the present study, we have examined the antithrombotic effect of ticlopidine and acetylsalicylic acid (ASA), on He-Ne laser-in duced thrombus formation in arterioles and venules. We have compared these results with the effect of the drugs on platelet aggregation in whole blood in vitro.
Fig. 1. Antithrombotic effect of ticlopidine on la ser-induced thrombus formation in rat mesenteric ar terioles and venules. Irradiation was begun 2 h (a) or 17 h (b) after oral administration of ticlodipine or Tween 80 (control). Ordinate shows number of irradia tions necessary to form an occlusive thrombus. □ = Arteriole; ■ = venule; n = 8 animals in each group; * p < 0.05; ** p < 0.01.
Fig. 2. Effect of ASA on laser-induced thrombus formation in rat mesenteric arterioles and venules. Irradiation was begun 2 h (a) or 17 h (b) after adminis tration of ASA or Tween 80 (control). □ = Arteriole; ■ = venule; n = 8 animals in each group; * p < 0.05.
formation experiment. Blood samples were at room temperature for 30 min until used. To 0.5 ml of pre warmed Locke solution 0.5 ml of citrated blood was added. The mixture was allowed to stand until the tem perature rose to 37 °C (approx. 4 min). Twenty micro liters of agonist, i.e. collagen (1 mg/ml; Horm), throm bin (100 U/ml in 50 mMTris-HCl containing 100 mM NaCl, pH 7.3; Mochida Pharmaceutical Co. Ltd., Ja pan) or ADP (10 m M in 50 mM Tris-HCl, pH 6.8, stored at - 80 ° C; Sigma) were added to the mixture and aggregation was recorded for 6 min. The reaction was observed in a plastic cuvette at 1,000 rpm. Colla gen-induced platelet aggregation was started 30 min after the blood was collected, then thrombin- and ADP-induced platelet aggregation tests followed se quentially. Whole blood dotting time was measured by adding 30 pi of 0.117 M CaCl2 to 300 pi citrated blood using a KC 1A Amelung Coagulometer. Blood cell count was measured using an automatic cell counter (Sysmex Microcellcounter CC-180A, Toa Medical Electronics Co. Ltd., Japan).
Statistics Wilcoxon’s two-sample test was employed in analy sis of the number of irradiations, and unpaired Stu dent’s t test was used for that of platelet aggregation, body weight and of blood cell count. Statistical signifi cance was examined against each control. Values are expressed as mean ± SE.
Antithrombotic Effect o f Ticlopidine and ASA Results are shown in figures 1 and 2. Ticlo pidine showed a dose-dependent antithrom botic effect in arterioles as well as in venules (fig. 1). However, this inhibition was stronger in arterioles than in venules. ASA showed sig nificant antithrombotic effect in arterioles when 300 mg/kg was administered, but not in venules (fig. 2).
149
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Results
Table 1. Effect of ticlopidine and ASA on coagula tion and blood cell counts
E
to
E ■C o ) cto
—
12 1
X
T
10-
0o° V
/y
Control
j
WBCT
2 h 160.1 ±5.1 133.3 ± 17.9 118.6 ± 13.3 3 h 148.4 + 5.5 128.0 ± 11.0 17 h 124.1 ±7.1 133.1 ±9.7 102.0 ± 10.2
RBC
2h 3h 17 h
807 ± 11 810 ± 32 793 ± 12
759 ± 16* 754 ±19 738 ±14*
780±6
2h 3h 17 h
26 ±2 39 ± 3 35 ± 1
28 ±5 25 ±2** 48±3*
31 ±3
2h 3h 17 h
76 ± 3 81 ±3 79±6
87 ±4** 76±2 81 ±4
WBC
Dose of ticlopidine and A SA (mg/kg)
Fig. 3. Antiaggregatory effect of ticlopidine and
ASA on ADP-induced platelet aggregation. Impedance change 2 h (a), 3 h (b) and 17 h (c) after administration of ticlopidine or ASA. Values are expressed as impe dance change 6 min after addition of ADP (AO/6 min), n = 6 animals in each group; * p < 0.05; ** p < 0.01.
Antiaggregatory Effect o f Ticlopidine and ASA Ticlopidine and ASA inhibited ADP-in duced aggregation in a time-dependent man ner (fig. 3). Neither ticlopidine nor ASA in hibited thrombin- or collagen-induced plate let aggregation (data not shown). Effect o f Ticlopidine and ASA on Coagulation and Blood Cell Counts Results are shown in table 1. Neither ticlo pidine nor ASA affected whole blood clotting time, but some significant effects on erythro cyte, leukocyte and platelet counts were ob served. These changes might be relevant in the context of the mild to severe neutropenia observed in some patients treated with ticlo pidine but they were not examined further in the present study.
150
Ticlopidine ASA
PLT
-
748 ±13*
-
45 ± 5 70± 3 -
84 ±3
These parameters were measured prior to platelet aggregation test using the same blood samples. Blood was collected 2, 3 or 17 h after oral administration of ticlopidine (200 mg/kg), ASA (200 mg/kg) or Tween 80. WBCT = whole blood clotting time (s); RBC = erythrocytes (X 104/pl); WBC = leukocytes (X 102/gl); PLT = platelets (X 104/pl); n = 6 animals in each group; * p < 0.05; ** p < 0.01.
Discussion It is thought that platelet thrombus forma tion in vivo is initiated by injury to endothe lial cells, followed by adhesion, aggregation of platelets, and fibrin deposition. A primary thrombus may be mainly composed of plate lets both in arterioles and in venules. He-Ne laser irradiation injures endothelial cells to form a thrombus mainly composed of plate lets in arterioles and in venules [8]. Therefore, He-Ne laser-induced thrombus formation provides a useful method for testing antiag gregatory drugs in vivo [9]. In the present study this method was used to assess the anti thrombotic effects of ticlopidine and ASA.
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be explained. In the present whole blood ag gregation studies, however, antiplatelet activ ity of ASA was demonstrated at 200 mg/kg but in contrast, in the He-Ne laser model, higher concentrations (300 mg/kg) were re quired to inhibit in vivo thrombus formation. These data tend to suggest that the differential effects of ASA on endothelium and platelets may not be apparent in vivo. In vitro, ticlopidine and ASA inhibited ADP-induced whole blood platelet aggrega tion, but not collagen- and thrombin-induced aggregation. These data are in keeping with the concept that ADP plays a key role in thrombogenesis in rats [16]. Furthermore, the antiaggregatory effect was almost parallel to the antithrombotic effect in arterioles, but not in venules. These results tend to support ear lier findings [7] and suggest that the proper ties of endothelial cells of arterioles may differ from those of venules. The present results demonstrated that the minimal effective doses of ticlopidine and ASA in rats were approximately 100 times greater than those required in man [17, 18]. However, ticlopidine appeared to be more potent than ASA as an antithrombotic agent in the animal model and the data supported the view that ticlopidine might be more effec tive than ASA in man.
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Ticlopidine inhibited thrombus formation in arterioles and in venules. A statistically sig nificant effect was noted at 100 mg/kg in arte rioles and in venules. Dose-dependent inhibi tion was more clearly observed in arterioles than in venules. This tendency was also ob served in animals given ASA, although a sta tistically significant effect could be observed only at higher doses of the drug (300 mg/kg). The minimal effective dose of ticlopidine in arterioles was similar to that reported by Ashida et al. [10], although a lower concentra tion (10 mg/kg) was found to be effective by Seiffge and Weithmann [11], In addition, the present effective dose in venules was similar to that reported by others using different tech niques [ 1, 3, 4, 12, 13] . It should be empha sized, however, that the antithrombotic effect of ticlopidine in models based on venous sta sis [12, 13] might be influenced by inhibition of blood coagulation, where coagulation is normally enhanced by coexisting platelets [14], In the present studies, ASA inhibited thrombus formation in arterioles at a dose of 300 mg/kg, but not at 200 mg/kg. These re sults were consistent with those of a rat, arte riovenous shunt model [2], but were markedly different from those seen using an argon-laser model, where 10 mg/kg of ASA significantly inhibited thrombus formation [11], It is clear that the effects of ASA are especially depen dent on the particular animal model. The efficacy of ASA as an antithrombotic drug is not firmly established. It has been sug gested that inhibition of cyclo-oxygenase by ASA simultaneously prevents the formation of aggregatory thromboxane A2 in platelets and that of antiaggregatory prostacyclin in vascular cells [15]. Vascular cells and platelets might be differentially sensitive to the effects of ASA and thus, the deceptive results from clinical trials of aspirin as an antithrombotic agent, the so-called ‘aspirin dilemma’, could
1 Tomikawa M, Ashida S, Kakihata K, Abiko Y: Anti-thrombotic action of ticlopidine, a new platelet aggre gation inhibitor. Thromb Res 1978; 12:1157-1164. 2 Ashida S, Sakuma K, Abiko Y: Anti thrombotic effects of ticlopidine, acetylsalicylic acid and dipyrida mole in vascular shunt model in rats. Thromb Res 1980; 17:663671. 3 Kumada T, Ishihara M, Ogawa H, Abiko Y: Experimental model of ve nous thrombosis in rats and effect of some agents. Thromb Res 1980; 18: 189-203. 4 Weichert W, Pauliks V, Breddin HK: Laser-induced thrombi in rat mesenteric vessels and antithrom botic drugs. Haemostasis 1983; 13: 61-71. 5 Weichert W, Breddin HK, Staubesand J: Application of a laser-in duced endothelial injury model in the screening of antithrombotic drugs. Semin Thromb Hemost 1988; 14(suppl): 106-114. 6 Hovig T, McKenzie FN, Arfors K-E: Measurement of the platelet re sponse to laser-induced microvascular injury. Thromb Diath Haemorrh 1974;32:695-703.
7 Yamamoto J, Iizumi H, Hirota R, Shimonaka K, Nagamatsu Y, Horie N, Morita S: Effect of physical train ing on thrombotic tendency in rats: decrease in thrombotic tendency measured by the He-Ne laser-in duced thrombus formation method. Haemostasis 1989;19:260-265. 8 Kovâcs IB, Tigyi-Sebes A, Trombitas K, Gôrôg P: Evans blue: an ideal energy-absorbing material to pro duce intravascular microinjury by He-Ne gas laser. Microvasc Res 1975;10:107-124. 9 Kovâcs IB, Gôrôg P: Laser-induced thrombosis test suitable for pharma cological screening studies. Micro vasc Res 1979;18:403-412. 10 Ashida S, Ishihara M, Ogawa H, Abiko Y: Protective effect of ticlo pidine on experimentally induced peripheral arterial occlusive disease in rats. Thromb Res 1980; 18:55— 67. 11 Seiffge D, Weithmann KU: Surpris ing effects of the sequential adminis tration of pentoxifylline and low dose acetylsalicylic acid on throm bus formation. Thromb Res 1987; 46:371-383.
12 Millet J, Theveniaux J, Pascal M: A new experimental model of venous thrombosis in rats involving partial stasis and slight endothelium altera tions. Thromb Res 1987;45:123— 133. 13 Bernat A, Vallee E, Maffrand JP, Roncucci R: Antithrombotic effect of ticlopidine in a platelet-indepen dent model of venous thrombosis. Thromb Res 1985;37:279-285. 14 Gôrôg P, Kovâcs IB: Coagulation of flowing native blood: advantages over stagnant clotting test (submit ted). 15 de Gaetano G, Cerletti C, Bertele V: Pharmacology of antiplatelet drugs and clinical trials on thrombosis prevention. Lancet 1982;ii:974— 977. 16 Maffrand JP, Bemat A, Pelebassee D, Defreyn G, Cazenave JP, Gorden JL: ADP plays a key role in thrombogenesis in rats. Thromb Haemost 1988;59:225-230. 17 Hardisty RM, Powling MJ, Nakes TJC: The action of ticlopidine on human platelet. Thromb Haemostas 1990;64:150-155. 18 Ratnatunga CP, Edomondson SF, Rees GM, Kovâcs IB: Aspirin inhib its shear-induced platelet reaction involving thrombin generation. Cir culation, in press.
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References
