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Synergistic Antithrombotic Effects of Argatroban and Ticlopidine In the Rat Venous Thrombosis Model
Thrombosis Research 92 (1998) 261–266
REGULAR ARTICLE
Synergistic Antithrombotic Effects of Argatroban and Ticlopidine In the Rat Venous Thrombosis Model Kaoru Morishita and Masahiro Iwamoto New Product Research Laboratories 2, Daiichi Pharmaceutical Co., Ltd., 1-16-13, Kita-Kasai, Edogawa-ku, Tokyo, 134-8630, Japan. (Received 11 May 1998 by Editor N. Sakuragawa; revised/accepted 3 August 1998)
Abstract Argatroban, a synthetic thrombin inhibitor, and ticlopidine, an anti-platelet agent, are major antithrombotic agents. We investigated the antithrombotic effects of a combination of argatroban and ticlopidine in the rat venous thrombosis model. Argatroban or ticlopidine inhibited thrombus formation in a dose-dependent manner; 50% inhibition (ED50) is obtained with 1.0 mg/kg/h (infusion) argatroban or 30 mg/kg (p.o.) ticlopidine. The combination of argatroban and ticlopidine inhibited thrombus formation in a dose-dependent manner; ED50 is obtained with 0.25 mg/kg/h argatroban plus 10 mg/kg ticlopidine and 0.5 mg/kg/h argatroban plus 3 mg/kg ticlopidine, whereas 0.5 mg/kg/h argatroban alone or 10 mg/kg ticlopidine alone had negligible effect (,20% inhibition). Isobole analysis showed that the antithrombotic effects of the combination of argatroban and ticlopidine involved synergism with potentiation. In contrast, the combination of argatroban and ticlopidine did not prolong the bleeding time synergistically. These data showed that the combination therapy of argatroban and ticlopidine should be clinically benefi-
Abbreviations: ADP, adenosine diphosphate; APTT, activated partial thromboplastin time; PT, prothrombin time; TT, thrombin time. P.O., per os. Corresponding author: Kaoru Morishita, New Product Research Laboratories 2, Daiichi Pharmaceutical Co., Ltd., 1-16-13, KitaKasai, Edogawa-ku, Tokyo, 134-8630, Japan. Phone: 181 (3) 3680 0151 ext. 3722; Fax: 181 (3) 5696 8718; E-mail: ,moriscjz@ daiichipharm.co.jp..
cial, but the different administration route may restrict the clinical usage. 1998 Elsevier Science Ltd. Key Words: Argatroban; Ticlopidine; Thrombin inhibitor; Anticoagulant; Experimental venous thrombosis; Bleeding time
A
rgatroban, a synthetic thrombin inhibitor [1,2], has been shown to be an effective anticoagulant agent in rat venous thrombosis models [3,4] and in rat cerebral artery thrombosis models [5,6]. Argatroban also has been used clinically for the treatment of chronic peripheral arterial obstructive disease and acute ischemic stroke in Japan [7]. Ticlopidine, an antiplatelet agent [8,9], has been shown to be benefit in reducing the rate of subsequent occurrence of stroke, myocardial infarction, or vascular death in patient who have had a recent thromboembolic stroke (CATS study) [10] and in preventing of recurrent transient ischemic attacks in comparison with aspirin (TASS study) [11]. Recently, new antiplatelet agents have been developed. Clopidogrel, which is chemically related to ticlopidine, reduced the combined risk of ischemic stroke, myocardial infarction, or vascular death (CAPRIE study) [12]. Integrelin, glycoprotein IIb/IIIa blocker, was effective in routine coronary interventions (IMPACT study) [13]. As anticoagulants inhibit fibrin formation and antiplatelet agents inhibit platelet aggregation, both drugs could cooperate to inhibit thrombus
0049-3848/98 $–see front matter 1998 Elsevier Science Ltd. Printed in the USA. All rights reserved. PII S0049-3848(98)00140-6
262
K. Morishita et al./Thrombosis Research 92 (1998) 261–266
formation. We do not know much about whether the antithrombotic effects of the combination of an anticoagulant and an antiplatelet agent involve additive synergism, synergism with potentiation, or antagonism. If the combination of these agents potentiated the antithrombotic effects synergistically without prolonging the bleeding time, which is a parameter of bleeding, the combination therapy could be clinically beneficial. Reduction of the dosage of each agent in the combination therapy could also be clinically beneficial because it could contribute to the reduction of other side effects of the agents and the expense of medication. To investigate the possibility of the combination therapy, we studied the antithrombotic effects of combinations of argatroban and ticlopidine using the rat venous thrombosis model [4]. We also examined whether the combinations of argatroban and ticlopidine prolong the bleeding time synergistically.
1. Materials and Methods 1.1. Reagents and Animals Argatroban was supplied by Mitsubishi Chemical Corporation. Ticlopidine was supplied by Daiichi Pharmaceutical Co. Ltd. PLATELIN Plus Activator and SIMPLASTIN were obtained from Organon Teknika Co., Durham, NC. All other reagents were of the highest grade commercially available. Male albino rats (Wistar) weighing 200– 250 g were used. Water and food pellets were available ad libitum.
1.2. Experimental Venous Thrombosis We used the endothelial damage-induced jugular vein thrombosis model, which was previously reported [4]. Rats were anesthetized with sodium pentobarbital. The jugular vein was exposed and isolated free from the surrounding tissues. The vessel was placed between two flexible plates over a length of 8 mm connected to a cooling unit (Immersion Cooler ECS-50, EYELA, Tokyo, Japan) and frozen at 2208C for 5 minutes. At the end of this procedure, the frozen plates were defrosted, and the jugular vein was detached. The blood flow was then reestablished in the injured vein by gentle rubbing. After 3–5 hours, the thrombus was carefully
dissected from the vessel and washed with saline. It was dissolved in 2% Na2CO3–0.1 N NaOH, and the protein concentration was measured by the Lowry method. The thrombus weight was expressed as the protein content of the thrombus. All experiments were done according to the guidelines for animal experimentation prepared by the Japanese Association for Laboratory Animal Science. The statistical evaluation was performed using the Kruscal-Wallis test and the Williams–Wilcoxon test. Values at p,0.05 were considered as statistically significant.
1.3. Determination of Bleeding Time Bleeding time was measured by the methods introduced by Hara [14]. The tails of anesthetized rats were severed 5 mm from the tip with a sharp razor blade. The bleeding time was measured by blotting the blood from the incision with a filter paper at intervals of 15 seconds.
1.4. Coagulation Assays The activated partial thromboplastin time (APTT), the prothrombin time (PT), and the thrombin time (TT) were measured with a Microcoagulometer (Greiner Electronics, AG, Langenthal, Switzerland). For APTT, 20 ml of plasma was mixed with 20 ml of PLATELIN Plus Activator in a process tube, and after incubation at room temperature for 2 minutes, coagulation was started with the addition of 20 ml of 20 mM CaCl2 solution; for PT, 20 ml of plasma was placed in the process tube, and the coagulation was started by adding 40 ml of SIMPLASTIN; for TT, 20 ml of plasma was placed in the process tube and the coagulation was started by adding 40 ml of thrombin (5 U/ml).
1.5. Platelet Aggregation Platelet-rich plasma (PRP) was prepared by centrifugation of citrated blood collected from each rat. Platelet aggregation was measured with 250 ml of PRP and 20 ml of 50 mM ADP using a PAM8C aggregometer (Mebanix Co., Tokyo, Japan).
K. Morishita et al./Thrombosis Research 92 (1998) 261–266
263
Fig. 1. Effect of argatroban and ticlopidine in the rat venous thrombosis model. Argatroban was infused intravenously for 1 hour. Infusion was started 20 minutes before the jugular vein was frozen. Ticlopidine was administered orally 2 and 24 hours before the freezing. Results are expressed as mean6SD. Numbers of rats are indicated in the parentheses. 1From reference 4. *p,0.01 vs. control, &p,0.05 vs. control. #p,0.05 vs. 0.5 mg/kg/hour argatroban alone. $p,0.05 vs. 10 mg/kg ticlopidine alone. A, argatroban; T, ticlopidine.
2. Results 2.1. Synergistic Antithrombotic Effects of Combination of Argatroban and Ticlopidine We investigated the antithrombotic effects of the combination of argatroban and ticlopidine compared with those of each agent alone on the rat venous thrombosis model. All data from the series of experiments showed that argatroban or ticlopidine alone or the combination of these two drugs inhibited the thrombus formation in a dose-dependent manner (Figure 1). To investigate whether the antithrombotic effects of the combination of argatroban and ticlopidine involved additive synergism, synergism with potentiation, or antagonism, we plotted isobolic curves for 50% inhibitory doses of these two drugs (Figure 2). A hyperbolic isobole which lies below the straight line connecting the points which represent the ID50 for the individual drugs is obtained from the figure [15]. This result showed that the antithrombotic effects of the combination of argatroban and ticlopidine involved synergism with potentiation. To confirm the synergistic effects of argatroban and ticlopidine, we reexamined the antithrombotic
effects of argatroban or ticlopidine alone or the combination of these drugs. The antithrombotic effects of 0.5 mg/kg/h argatroban110 mg/kg ticlopidine were significantly stronger than those of 0.5 mg/kg/h argatroban or 10 mg/kg ticlopidine alone (Figure 3A) and were comparable with those of 1.5 mg/kg/h argatroban or 100 mg/kg ticlopidine alone (Figure 3B). Therefore, we concluded that the antithrombotic effects of the combination of
Fig. 2. An isobolic curve for 50% inhibitory doses of argatroban and ticlopidine. Values are from Figure 1.
264
K. Morishita et al./Thrombosis Research 92 (1998) 261–266
Fig. 3. The synergistic antithrombotic effects of 0.5 mg/kg/hour argatroban and 10 mg/kg ticlopidine in the rat venous thrombosis model. Drugs were administered at the indicated doses (argatroban, mg/kg/h; ticlopidine, mg/kg). Results are expressed as mean6SD of seven rats. (A) *p,0.05 vs. 0.5 mg/kg/h argatroban plus 10 mg/kg ticlopidine, (B)*p,0.05 vs. control; **p,0.01 vs. control.
argatroban and ticlopidine involved synergism with potentiation.
2.2. Negligible Additive Effects on Coagulation Parameters and Platelet Aggregation Argatroban (0.5 mg/kg/hour) prolonged the coagulation parameters (APTT, PT, and TT), but ticlopidine did not inhibit the ADP-induced platelet
aggregation at a dose of 10 mg/kg (Table 1). Combination of 0.5 mg/kg/h argatroban and 10 mg/kg ticlopidine showed coagulation parameters comparable to those of 0.5 mg/kg/h argatroban alone and also platelet aggregation comparable with those of 10 mg ticlopidine alone. These data indicated that argatroban did not enhance the inhibitory effect on platelet aggregation of ticlopidine and that ticlopidine did not enhance the anticoagulant effects of argatroban. Therefore, synergism with
Table 1. Effects of argatroban and ticlopidine on plasma coagluation time and platelet aggregation in rats Platelet aggregation (%) Control Argatroban (0.5 mg/kg/hour) Ticlopidine (10 mg/kg) Argatroban (0.5 mg/kg/hour) 1 Ticlopidine (10 mg/kg)
Coagulation time (seconds) APTT
PT
TT
n
6862 5863 6464
2261 3763a 246 1
2161 3062a 2161
1361 94614a 1561
5 6 5
6762
3262a
2962b
79615a
6
Argatroban was infused intravenously for 30 min and/or ticlopidine was administered orally 2 and 24 hours before blood collection. Platelet aggregation was induced by the addition of 50 mM ADP. Data are expressed as means6SEM. n, number of rats. a p,0.01 when compared with control. b p,0.05 when compared with control.
K. Morishita et al./Thrombosis Research 92 (1998) 261–266
Table 2. Effects of argatroban and ticlopidine on bleeding time in rats
265
Argatroban (1.0 mg/kg/h) alone and a combination of 0.5 mg/kg/h argatroban and 10 mg/kg ticlopidine prolonged the bleeding time 45% and 38%, respectively, in the tail transection model (Table 2). The bleeding time at the dose of 0.5 mg/kg/h argatroban and 10 mg/kg ticlopidine was comparable with that of 1.0 mg/kg/h argatroban. These results showed that the combination of argatroban and ticlopidine did not prolong bleeding time synergistically.
combination of these drugs inhibited thrombus formation synergistically. The synergistic antithrombotic effects of argatroban and ticlopidine were not peculiar in this model, because the antithrombotic effects of argatroban were potentiated by G4120, an RGD-containing synthetic peptide (GPIIb/IIIa antagonist), in a hamster femoral vein platelet-rich thrombosis model [16] and by ozagrel, a thromboxane synthase inhibitor, in the rabbit arterial occlusion model [17]. Argatroban also enhanced the thrombolytic effects of tissue-type plasminogen activator in the various thrombosis models [18–20]. In the contrast of the synergistic antithrombotic effects of argatroban and ticlopidine, the combination of these drugs did not prolong the bleeding time synergistically. This means that bleeding, a major side effect of antithrombotic agents, would be less in the combination therapy. Reduction of the dosage of each agent in the combination therapy could also be clinically beneficial because it could contribute to the reduction of other side effects of the agents and the expense of medication. As argatroban and ticlopidine are administered intravenously and orally, respectively, the different administration route may restrict the clinical usage. In conclusion, the antithrombotic effects of the combination of argatroban and ticlopidine involved synergism with potentiation in the rat venous thrombosis model. In contrast, the combination of argatroban and ticlopidine did not prolong the bleeding tendency synergistically.
3. Discussion
We thank Drs. Toshihiko Kumada, Yoshifumi Watanabe, and Kiyoshi Tanabe for discussions and review of the manuscript.
Control Argatroban (1 mg/kg/hour) Ticlopidine (30 mg/kg) Argatroban (0.5 mg/kg/hour) 1 Ticlopidine (10 mg/kg)
Bleeding time (seconds)
n
244617 353629a 261622
9 10 8
336631b
10
Argatroban was infused intravenously for 30 minutes and/or ticlopidine was administered orally 2 and 24 hours before measuring bleeding time. Data are expressed as mean6SEM. n, number of rats. a p,0.01 when compared with control. b p,0.05 when compared with control.
potentiation was not ascribed to prolongation of coagulation parameters nor enhanced inhibition of platelet aggregation.
2.3. Effects of Combination of Argatroban and Ticlopidine on Bleeding Time
The antithrombotic effects of the combination of argatroban and ticlopidine involved synergism with potentiation in the rat venous thrombosis model. In contrast, the combination of argatroban and ticlopidine did not prolong the bleeding time synergistically. One of the reasons why the combination of argatroban and ticlopidine involved synergism with potentiation could be that both fibrin formation and platelet aggregation were involved in the thrombus formation in this model. We previously showed that platelets adhered to the damaged endothelium in the early stage, then fibrin clots with red blood cells formed on the platelets aggregates in this model [4]. Because argatroban should inhibit the formation of fibrin clots and ticlopidine should inhibit the formation of platelet aggregates, the
References 1. Kikumoto R, Tamao Y, Tezuka T, Tonomura S, Hara H, Ninomiya K, Hijikata A, Okamoto S. Selective inhibition of thrombin by (2R,4R)4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro8-quinolinyl111)sulfonyl]-l-arginyl)]-2-piperidinecarboxylic acid. Biochemistry 1984;23:85–90. 2. Okamoto S, Hijikata A, Kikumoto R, Tonomura S, Hara H, Ninomiya K, Maruyama A, Sugano M, Tamao Y. Potent inhibition of thrombin by the newly synthesized arginine derivative No. 805. The importance of stereostructure of its hydrophobic carboxamide portion. Biochem Biophys Res Commun 1981; 101:440–6.
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3. Kumada T, Abiko Y. Comparative study on heparin and a synthetic thrombin inhibitor No. 805 (MD-805*) in experimental antithrombin III-deficient animals. Thromb Res 1981;24: 285–98. 4. Morishita K, Wada M, Kumada T, Tanaka T, Tomikawa M, Iwamoto M. Effect of ticlopidine and other antithrombotics on the venous thrombosis induced by endothelial damage of jugular vein in rats. Thromb Res 1991;63:373–84. 5. Kawai H, Umemura K, Nakashima M. Effect of argatroban on microthrombi formation and brain damage in the rat middle cerebral artery thrombosis model. Jpn J Pharmacol 1995; 69:143–8. 6. Kawai H, Yuki S, Sugimoto J, Tamao Y. Effects of a thrombin inhibitor, argatroban, on ischemic brain damage in the rat distal middle cerebral artery occlusion model. J Pharmacol Exp Ther 1996;278:780–5. 7. Hursting MJ, Alford KL, Becker JC, Brooks RL, Joffrion JL, Knappenberger GD, Kogan PW, Kogan TP, McKinney AA, Schwarz RP Jr. Novastan (brand of argatroban): a smallmolecule, direct thrombin inhibitor. Semin Thromb Hemost 1997;23:503–16. 8. Ashida S, Abiko Y. Inhibition of platelet aggregation by a new agent, Ticlopidine. Thromb Haemost 1979;15:542–50. 9. Ashida S, Abiko Y. Mode of action of ticlopidine in inhibition of platelet aggregation in the rat. Thromb Haemost 1979;23:436–49. 10. Gent M, Blakely JA, Easton JD, Ellis DJ, Hachinski VC, Harbison JW, Panak E, Roberts RS, Sicurella J, Turpie AG. The Canadian American Ticlopidine Study (CATS) in thromboembolic stroke. Lancet 1989;1:1215–20. 11. Bellavance A. Efficacy of ticlopidine and aspirin for prevention of reversible cerebrovascular ischemic events. The Ticlopidine Aspirin Stroke Study. Stroke 1993;24:1452–7. 12. CAPRIE Steering Committee. Randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE). Lancet 1996;348:1329–39.
13. Tcheng JE, Harrington RA, Kottke-Marchant K, Kleiman NS, Ellis SG, Kereiakes DJ, Mick MJ, Navetta FI, Smith JE, Worley SJ, Miller JA, Joseph DM, Sigmon KN, Kitt MM, du Me´e CP, Califf RM, Topol EJ. Multicenter, randomized, double-blind, placebo-controlled trial of the platelet integrin glycoprotein IIb/ IIIa blocker Integrelin in elective coronary intervention. IMPACT Investigators. Circulation 1995;91:2151–7. 14. Hara T, Yokoyama A, Tanabe K, Ishihara H, Iwamoto M. DX-9065a, an orally active, specific inhibitor of factor Xa, inhibits thrombosis without affecting bleeding time in rats. Thromb Haemost 1995;74:635–9. 15. Gero A. Mechanisms of molecular drug action. In: Dipalma JR, editor. Drill’s Pharmacology in Medicine. New York: McGraw-Hill Co.; 1971. p. 67–98. 16. Imura Y, Stassen J-M, Vreys I, Lesaffre E, Gold HK, Collen D. Synergistic antithrombotic properties of G4120, a RGD-containing synthetic peptide, and argatroban, a synthetic thrombin inhibitor, in a hamster femoral vein platelet-rich thrombosis model. Thromb Haemost 1992;68:336–40. 17. Kawai H, Tamao Y. The combination of thrombin inhibitor and thromboxane synthase inhibitor on experimental thrombosis and bleeding. Thromb Res 1995;80:429–34. 18. Jang I-K, Gold HK, Leinbach RC, Fallon JT, Collen D. In vitro thrombin inhibitor enhances and sustains arterial recanalization with recombinant tissue-type plasminogen activator. Circ Res 1990;67:1552–61. 19. Nishiyama H, Umemura K, Saniabadi AR, Takiguchi Y, Uematsu T, Nakashima M. Enhancement of thrombolytic efficacy of tissuetype plasminogen activator by adjuvants in the guinea pig thrombosis model. Eur J Pharmacol 1994;264:183–90. 20. Mellott M, Connolly TM, York SJ, Bush LR. Prevention of reocclusion by MCI-9038, a thrombin inhibitor, following t-PA-induced thrombolysis in canine model of femoral arterial thrombosis. Thromb Haemost 1990;64:526–34.
REGULAR ARTICLE
Synergistic Antithrombotic Effects of Argatroban and Ticlopidine In the Rat Venous Thrombosis Model Kaoru Morishita and Masahiro Iwamoto New Product Research Laboratories 2, Daiichi Pharmaceutical Co., Ltd., 1-16-13, Kita-Kasai, Edogawa-ku, Tokyo, 134-8630, Japan. (Received 11 May 1998 by Editor N. Sakuragawa; revised/accepted 3 August 1998)
Abstract Argatroban, a synthetic thrombin inhibitor, and ticlopidine, an anti-platelet agent, are major antithrombotic agents. We investigated the antithrombotic effects of a combination of argatroban and ticlopidine in the rat venous thrombosis model. Argatroban or ticlopidine inhibited thrombus formation in a dose-dependent manner; 50% inhibition (ED50) is obtained with 1.0 mg/kg/h (infusion) argatroban or 30 mg/kg (p.o.) ticlopidine. The combination of argatroban and ticlopidine inhibited thrombus formation in a dose-dependent manner; ED50 is obtained with 0.25 mg/kg/h argatroban plus 10 mg/kg ticlopidine and 0.5 mg/kg/h argatroban plus 3 mg/kg ticlopidine, whereas 0.5 mg/kg/h argatroban alone or 10 mg/kg ticlopidine alone had negligible effect (,20% inhibition). Isobole analysis showed that the antithrombotic effects of the combination of argatroban and ticlopidine involved synergism with potentiation. In contrast, the combination of argatroban and ticlopidine did not prolong the bleeding time synergistically. These data showed that the combination therapy of argatroban and ticlopidine should be clinically benefi-
Abbreviations: ADP, adenosine diphosphate; APTT, activated partial thromboplastin time; PT, prothrombin time; TT, thrombin time. P.O., per os. Corresponding author: Kaoru Morishita, New Product Research Laboratories 2, Daiichi Pharmaceutical Co., Ltd., 1-16-13, KitaKasai, Edogawa-ku, Tokyo, 134-8630, Japan. Phone: 181 (3) 3680 0151 ext. 3722; Fax: 181 (3) 5696 8718; E-mail: ,moriscjz@ daiichipharm.co.jp..
cial, but the different administration route may restrict the clinical usage. 1998 Elsevier Science Ltd. Key Words: Argatroban; Ticlopidine; Thrombin inhibitor; Anticoagulant; Experimental venous thrombosis; Bleeding time
A
rgatroban, a synthetic thrombin inhibitor [1,2], has been shown to be an effective anticoagulant agent in rat venous thrombosis models [3,4] and in rat cerebral artery thrombosis models [5,6]. Argatroban also has been used clinically for the treatment of chronic peripheral arterial obstructive disease and acute ischemic stroke in Japan [7]. Ticlopidine, an antiplatelet agent [8,9], has been shown to be benefit in reducing the rate of subsequent occurrence of stroke, myocardial infarction, or vascular death in patient who have had a recent thromboembolic stroke (CATS study) [10] and in preventing of recurrent transient ischemic attacks in comparison with aspirin (TASS study) [11]. Recently, new antiplatelet agents have been developed. Clopidogrel, which is chemically related to ticlopidine, reduced the combined risk of ischemic stroke, myocardial infarction, or vascular death (CAPRIE study) [12]. Integrelin, glycoprotein IIb/IIIa blocker, was effective in routine coronary interventions (IMPACT study) [13]. As anticoagulants inhibit fibrin formation and antiplatelet agents inhibit platelet aggregation, both drugs could cooperate to inhibit thrombus
0049-3848/98 $–see front matter 1998 Elsevier Science Ltd. Printed in the USA. All rights reserved. PII S0049-3848(98)00140-6
262
K. Morishita et al./Thrombosis Research 92 (1998) 261–266
formation. We do not know much about whether the antithrombotic effects of the combination of an anticoagulant and an antiplatelet agent involve additive synergism, synergism with potentiation, or antagonism. If the combination of these agents potentiated the antithrombotic effects synergistically without prolonging the bleeding time, which is a parameter of bleeding, the combination therapy could be clinically beneficial. Reduction of the dosage of each agent in the combination therapy could also be clinically beneficial because it could contribute to the reduction of other side effects of the agents and the expense of medication. To investigate the possibility of the combination therapy, we studied the antithrombotic effects of combinations of argatroban and ticlopidine using the rat venous thrombosis model [4]. We also examined whether the combinations of argatroban and ticlopidine prolong the bleeding time synergistically.
1. Materials and Methods 1.1. Reagents and Animals Argatroban was supplied by Mitsubishi Chemical Corporation. Ticlopidine was supplied by Daiichi Pharmaceutical Co. Ltd. PLATELIN Plus Activator and SIMPLASTIN were obtained from Organon Teknika Co., Durham, NC. All other reagents were of the highest grade commercially available. Male albino rats (Wistar) weighing 200– 250 g were used. Water and food pellets were available ad libitum.
1.2. Experimental Venous Thrombosis We used the endothelial damage-induced jugular vein thrombosis model, which was previously reported [4]. Rats were anesthetized with sodium pentobarbital. The jugular vein was exposed and isolated free from the surrounding tissues. The vessel was placed between two flexible plates over a length of 8 mm connected to a cooling unit (Immersion Cooler ECS-50, EYELA, Tokyo, Japan) and frozen at 2208C for 5 minutes. At the end of this procedure, the frozen plates were defrosted, and the jugular vein was detached. The blood flow was then reestablished in the injured vein by gentle rubbing. After 3–5 hours, the thrombus was carefully
dissected from the vessel and washed with saline. It was dissolved in 2% Na2CO3–0.1 N NaOH, and the protein concentration was measured by the Lowry method. The thrombus weight was expressed as the protein content of the thrombus. All experiments were done according to the guidelines for animal experimentation prepared by the Japanese Association for Laboratory Animal Science. The statistical evaluation was performed using the Kruscal-Wallis test and the Williams–Wilcoxon test. Values at p,0.05 were considered as statistically significant.
1.3. Determination of Bleeding Time Bleeding time was measured by the methods introduced by Hara [14]. The tails of anesthetized rats were severed 5 mm from the tip with a sharp razor blade. The bleeding time was measured by blotting the blood from the incision with a filter paper at intervals of 15 seconds.
1.4. Coagulation Assays The activated partial thromboplastin time (APTT), the prothrombin time (PT), and the thrombin time (TT) were measured with a Microcoagulometer (Greiner Electronics, AG, Langenthal, Switzerland). For APTT, 20 ml of plasma was mixed with 20 ml of PLATELIN Plus Activator in a process tube, and after incubation at room temperature for 2 minutes, coagulation was started with the addition of 20 ml of 20 mM CaCl2 solution; for PT, 20 ml of plasma was placed in the process tube, and the coagulation was started by adding 40 ml of SIMPLASTIN; for TT, 20 ml of plasma was placed in the process tube and the coagulation was started by adding 40 ml of thrombin (5 U/ml).
1.5. Platelet Aggregation Platelet-rich plasma (PRP) was prepared by centrifugation of citrated blood collected from each rat. Platelet aggregation was measured with 250 ml of PRP and 20 ml of 50 mM ADP using a PAM8C aggregometer (Mebanix Co., Tokyo, Japan).
K. Morishita et al./Thrombosis Research 92 (1998) 261–266
263
Fig. 1. Effect of argatroban and ticlopidine in the rat venous thrombosis model. Argatroban was infused intravenously for 1 hour. Infusion was started 20 minutes before the jugular vein was frozen. Ticlopidine was administered orally 2 and 24 hours before the freezing. Results are expressed as mean6SD. Numbers of rats are indicated in the parentheses. 1From reference 4. *p,0.01 vs. control, &p,0.05 vs. control. #p,0.05 vs. 0.5 mg/kg/hour argatroban alone. $p,0.05 vs. 10 mg/kg ticlopidine alone. A, argatroban; T, ticlopidine.
2. Results 2.1. Synergistic Antithrombotic Effects of Combination of Argatroban and Ticlopidine We investigated the antithrombotic effects of the combination of argatroban and ticlopidine compared with those of each agent alone on the rat venous thrombosis model. All data from the series of experiments showed that argatroban or ticlopidine alone or the combination of these two drugs inhibited the thrombus formation in a dose-dependent manner (Figure 1). To investigate whether the antithrombotic effects of the combination of argatroban and ticlopidine involved additive synergism, synergism with potentiation, or antagonism, we plotted isobolic curves for 50% inhibitory doses of these two drugs (Figure 2). A hyperbolic isobole which lies below the straight line connecting the points which represent the ID50 for the individual drugs is obtained from the figure [15]. This result showed that the antithrombotic effects of the combination of argatroban and ticlopidine involved synergism with potentiation. To confirm the synergistic effects of argatroban and ticlopidine, we reexamined the antithrombotic
effects of argatroban or ticlopidine alone or the combination of these drugs. The antithrombotic effects of 0.5 mg/kg/h argatroban110 mg/kg ticlopidine were significantly stronger than those of 0.5 mg/kg/h argatroban or 10 mg/kg ticlopidine alone (Figure 3A) and were comparable with those of 1.5 mg/kg/h argatroban or 100 mg/kg ticlopidine alone (Figure 3B). Therefore, we concluded that the antithrombotic effects of the combination of
Fig. 2. An isobolic curve for 50% inhibitory doses of argatroban and ticlopidine. Values are from Figure 1.
264
K. Morishita et al./Thrombosis Research 92 (1998) 261–266
Fig. 3. The synergistic antithrombotic effects of 0.5 mg/kg/hour argatroban and 10 mg/kg ticlopidine in the rat venous thrombosis model. Drugs were administered at the indicated doses (argatroban, mg/kg/h; ticlopidine, mg/kg). Results are expressed as mean6SD of seven rats. (A) *p,0.05 vs. 0.5 mg/kg/h argatroban plus 10 mg/kg ticlopidine, (B)*p,0.05 vs. control; **p,0.01 vs. control.
argatroban and ticlopidine involved synergism with potentiation.
2.2. Negligible Additive Effects on Coagulation Parameters and Platelet Aggregation Argatroban (0.5 mg/kg/hour) prolonged the coagulation parameters (APTT, PT, and TT), but ticlopidine did not inhibit the ADP-induced platelet
aggregation at a dose of 10 mg/kg (Table 1). Combination of 0.5 mg/kg/h argatroban and 10 mg/kg ticlopidine showed coagulation parameters comparable to those of 0.5 mg/kg/h argatroban alone and also platelet aggregation comparable with those of 10 mg ticlopidine alone. These data indicated that argatroban did not enhance the inhibitory effect on platelet aggregation of ticlopidine and that ticlopidine did not enhance the anticoagulant effects of argatroban. Therefore, synergism with
Table 1. Effects of argatroban and ticlopidine on plasma coagluation time and platelet aggregation in rats Platelet aggregation (%) Control Argatroban (0.5 mg/kg/hour) Ticlopidine (10 mg/kg) Argatroban (0.5 mg/kg/hour) 1 Ticlopidine (10 mg/kg)
Coagulation time (seconds) APTT
PT
TT
n
6862 5863 6464
2261 3763a 246 1
2161 3062a 2161
1361 94614a 1561
5 6 5
6762
3262a
2962b
79615a
6
Argatroban was infused intravenously for 30 min and/or ticlopidine was administered orally 2 and 24 hours before blood collection. Platelet aggregation was induced by the addition of 50 mM ADP. Data are expressed as means6SEM. n, number of rats. a p,0.01 when compared with control. b p,0.05 when compared with control.
K. Morishita et al./Thrombosis Research 92 (1998) 261–266
Table 2. Effects of argatroban and ticlopidine on bleeding time in rats
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Argatroban (1.0 mg/kg/h) alone and a combination of 0.5 mg/kg/h argatroban and 10 mg/kg ticlopidine prolonged the bleeding time 45% and 38%, respectively, in the tail transection model (Table 2). The bleeding time at the dose of 0.5 mg/kg/h argatroban and 10 mg/kg ticlopidine was comparable with that of 1.0 mg/kg/h argatroban. These results showed that the combination of argatroban and ticlopidine did not prolong bleeding time synergistically.
combination of these drugs inhibited thrombus formation synergistically. The synergistic antithrombotic effects of argatroban and ticlopidine were not peculiar in this model, because the antithrombotic effects of argatroban were potentiated by G4120, an RGD-containing synthetic peptide (GPIIb/IIIa antagonist), in a hamster femoral vein platelet-rich thrombosis model [16] and by ozagrel, a thromboxane synthase inhibitor, in the rabbit arterial occlusion model [17]. Argatroban also enhanced the thrombolytic effects of tissue-type plasminogen activator in the various thrombosis models [18–20]. In the contrast of the synergistic antithrombotic effects of argatroban and ticlopidine, the combination of these drugs did not prolong the bleeding time synergistically. This means that bleeding, a major side effect of antithrombotic agents, would be less in the combination therapy. Reduction of the dosage of each agent in the combination therapy could also be clinically beneficial because it could contribute to the reduction of other side effects of the agents and the expense of medication. As argatroban and ticlopidine are administered intravenously and orally, respectively, the different administration route may restrict the clinical usage. In conclusion, the antithrombotic effects of the combination of argatroban and ticlopidine involved synergism with potentiation in the rat venous thrombosis model. In contrast, the combination of argatroban and ticlopidine did not prolong the bleeding tendency synergistically.
3. Discussion
We thank Drs. Toshihiko Kumada, Yoshifumi Watanabe, and Kiyoshi Tanabe for discussions and review of the manuscript.
Control Argatroban (1 mg/kg/hour) Ticlopidine (30 mg/kg) Argatroban (0.5 mg/kg/hour) 1 Ticlopidine (10 mg/kg)
Bleeding time (seconds)
n
244617 353629a 261622
9 10 8
336631b
10
Argatroban was infused intravenously for 30 minutes and/or ticlopidine was administered orally 2 and 24 hours before measuring bleeding time. Data are expressed as mean6SEM. n, number of rats. a p,0.01 when compared with control. b p,0.05 when compared with control.
potentiation was not ascribed to prolongation of coagulation parameters nor enhanced inhibition of platelet aggregation.
2.3. Effects of Combination of Argatroban and Ticlopidine on Bleeding Time
The antithrombotic effects of the combination of argatroban and ticlopidine involved synergism with potentiation in the rat venous thrombosis model. In contrast, the combination of argatroban and ticlopidine did not prolong the bleeding time synergistically. One of the reasons why the combination of argatroban and ticlopidine involved synergism with potentiation could be that both fibrin formation and platelet aggregation were involved in the thrombus formation in this model. We previously showed that platelets adhered to the damaged endothelium in the early stage, then fibrin clots with red blood cells formed on the platelets aggregates in this model [4]. Because argatroban should inhibit the formation of fibrin clots and ticlopidine should inhibit the formation of platelet aggregates, the
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