THROMBOSIS RESEARCH Vol. It. pp. I 157-llti. @ PergamonPressLtd. 1978. Primedin Great Britain.
AXTI-THROMBOTIC ACTION OF TICLOPIDINE, A NEW PLATELET AGGREGATIOE; INHIBITOR Munehiro Tomikawa, Shin-ichiro Ashida, Koji Kakihata and Yasushi Abiko Laboratory of Biochemistry, Research Institute, Daiichi Seiyaku Co., Ltd. Edogawa-ku, Tokyo 132, Japan
(Received
16.2.1978:
Accepted
by
in revised
Editor
K.X.
forrrl 13.4.1978. Brinkhous)
Anti-thrombotic action of Ticlopidine, 5-(2-chlorobenzyl)-4,5,6,7tetrahydrothieno[3,2-Clpyrldine hydrochloride, was studied by using a lactic acidosis-induced pulmonary thrombosis model in rats. Ticlopidine was highly effective in preventing thrombus formation at an oral dose as low as 50 mg/kg, and the action was well retained even after repeated daily administration of the compound at 100 mglkg for 4 weeks. No difference was observed between control and the treated rats in morphology of thrombi formed, which were composed of platelet aggregates, fibrin and coagulated protein-like substances. Ticlopidine was also effective in preventing ADP- and collageninduced acute pulmonary thromboembolic death in mice, collageninduced transient thrombocytopenia in rats and platelet aggregation in ,l-aminopropionitrile-treated rats. Ticlopidine was found to be superior to aspirin and dipyridamole in the treatment of these thrombosis models.
INTRODUCTION Pathogenesis of thrombosis is thought to involve many factors, among which platelet aggregation and adhesion play a great part of initial events in thrombus formation. Inhibition of platelet aggregation is thus one of the most promising approaches to prevention of thrombosis, if a satisfactory inhibitor of platelet aggregation is available. Ticlopidine (1) is a potent, long-lasting inhibitor of platelet aggregation induced by a wide variety of stimuli including thromboxane A2-like substance and others (2). The present paper describes anti-thrombotic effect of Ticlopidine in several thrombosis models and in comparison with the effect of aspirin and dipyridamole.
1157
1138
TICLOPIDIXE
XXD
PLXTELETS
MATERIALS AND ?lETHODS Ticlopidine, S-(Z-chlorobenzyl)-4,S,6,7-tetrahydrothieno[3,2-C]pyridine hydrochloride, was supplied by PARCOR (Toulouse, France). Test animals used were male ddY mice weighing 17.0-19.5 g, male Wistar SLC rats weighing 220-250 g and male Wistar-Imamichi rats weighing 180-200 g. Acute pulmonary thrombosis in rats. According to the method described previously (3), pulmonary thrombosis was induced in Wistar-SLC rats by slow infusion of 0.7 M lactic acid (14 ml/kg) in 2 hr into the femoral vein under pentobarbital anesthesia. The severity of thrombosis was expressed by a total thrombus count in 10 microscopic fields of a lung section under 200 fold magnification averaged from 4 paraffin sections of the lung lobes (left, right superior, right middle and right inferior lobes). Thrombus count was measured on the samples obtained immediately after the lactic acid infusion. Acute thromboembolic death in mice. Acute pulmonary thromboembolism was induced in mice by a rapid intravenous injection of ADP (400 mg/lO ml saline/ kg) in 3 set or soluble collagen solution (25 mg/lO ml/kg) in 2 set, in reference to the reports by Nordijy and Chandler (4) and Nishizawa et al. (5), respectively. About 85 % of control mice died within 3 min after the injection. The collagen solution was prepared as follows. 2.5 mg Of acidsoluble collagen (Sigma Chemical Co., Type IV, calf skin) was dissolved in 1 ml of 0.05 M acetic acid containing 2 p CaC12 and 5 % glucose (pH 1.7). The solution was neutralized (pH 7.4) with S N NaOH, incubated at 37'C for 2 hr, then at 23-25°C for 3 hr and was used as a working solution. These procedures were important to give reproducible results. Endotoxin shock in mice. Endotoxin shock was induced in mice according to Fleming et al. (6). Escherichia coli endotoxin (Difco Lab., E. coli 026: B6) was,intravenously injected at 25 mg/lO ml saline/kg. Mortality rate was calculated 24 hr after the injection. About 80 % of control mice died under these conditions. Transient thrombocytopenia in rats (intravascular platelet aggregation). Insoluble collagen (Sigma Chemical Co., Type I, bovine achilles tendon) was homogenized with 0.025 M Tris-HCl (pH 7.4) containing 0.13 M NaCl. The homogenate was centrifuged at 200 g for 5 min, and the resultant supernatant fine suspension was diluted to 75 % of transmittance at 420 nm with the Trissaline. The collagen suspension (20 ml/kg) was rapidly injected into the tail vein of Wistar-Imamichi rats in 3 sec. Before and exactly 2 min after the injection, blood samples were taken from the tail vein for measuring platelet count. Degree of thrombocytopenia was expressed by the decrease (X) in platelet count after the collagen injection as reported previously (7). ,&-Aminopropionitrile-induced platelet hyper-reactivity. According to Pollock and Heath (8), ,&-aminopropionitrile (I-APN) was mixed into drinking water (2 mg/ml) and given ad libitum to Wistar-Imamichi rats for 7 days. The rats received p-APN in a total amount of 88 mg/rat/day. Control rats received water instead of a-APN solution. Two weeks thereafter titrated platelet-rich plasmas (PRP) were collected and tested for ADP- and collagen-induced platelet aggregation and clot retraction as described previously (2). Tests of blood coagulation and fibrinolysis.
Plasma recalcification
Vol.l2,Ko.6
TICLOPTDIXE
22.9 PLATELETS
1159
time and euglobulin lysis time were measured according to the methods described previously (3). Thrombelastography was performed with non-anticoagulated whole blood according to Hartert (9).
RESDLTS Effect on Lactic Acid-Induced Acute Pulmonary Thrombosis in Rats Ticlopidine or aspirin was given to rats at an oral dose of 50-200 mg/kg before the lactic acid infusion, and their protective action on thrombus formation was studied histologically. As shown in Table 1, Ticlopidine was effective in inhibiting thrombus formation in this model. Aspirin also prevented thrombus formation but was rather less effective than Ticlopidine in comparison of the results obtained with oral dose of 50 mg/kg. After repeated daily administration at 100 mg/kg for 4 weeks, Ticlopidine was found to retain well its anti-thrombotic action. Histological examination revealed no difference between the control and the treated rats in morphology of thrombi formed, which were composed of platelet aggregates, fibrin and coagulated protein-like substances as reported previously (3). TABLE 1 Prophylactic Effect of Ticlopidine and Aspirin on Lactic Acid-Induced Pulmonary Thrombosis in Rats Dose (mg/kg,po) Control 50 200
Aspirin Thrombus count Inhibition f S.E. (%)
Ticlopidine Inhibition Thrombus count f S.E. (%)
70.5 f10.5 (6) 53.6 f 8.6 (6) 28.2 f 5.5 (5)
70.5 f10.5 (6) 26.6 * 9.7 (5) 32.4 f 5.8 (5)
62.3* 54.0*
65.3 f 4.4 (9) 32.1 i 9.5 (6) 25.9 f 9.8 (5)
50.9** 60.4**
Control 100 (single dose) 100 (daily doses for 4 weeks)
24.0 60.0**
Data were analyzed by Student's t-test. * p(O.05, ** p
AXTI-THROMBOTIC ACTION OF TICLOPIDINE, A NEW PLATELET AGGREGATIOE; INHIBITOR Munehiro Tomikawa, Shin-ichiro Ashida, Koji Kakihata and Yasushi Abiko Laboratory of Biochemistry, Research Institute, Daiichi Seiyaku Co., Ltd. Edogawa-ku, Tokyo 132, Japan
(Received
16.2.1978:
Accepted
by
in revised
Editor
K.X.
forrrl 13.4.1978. Brinkhous)
Anti-thrombotic action of Ticlopidine, 5-(2-chlorobenzyl)-4,5,6,7tetrahydrothieno[3,2-Clpyrldine hydrochloride, was studied by using a lactic acidosis-induced pulmonary thrombosis model in rats. Ticlopidine was highly effective in preventing thrombus formation at an oral dose as low as 50 mg/kg, and the action was well retained even after repeated daily administration of the compound at 100 mglkg for 4 weeks. No difference was observed between control and the treated rats in morphology of thrombi formed, which were composed of platelet aggregates, fibrin and coagulated protein-like substances. Ticlopidine was also effective in preventing ADP- and collageninduced acute pulmonary thromboembolic death in mice, collageninduced transient thrombocytopenia in rats and platelet aggregation in ,l-aminopropionitrile-treated rats. Ticlopidine was found to be superior to aspirin and dipyridamole in the treatment of these thrombosis models.
INTRODUCTION Pathogenesis of thrombosis is thought to involve many factors, among which platelet aggregation and adhesion play a great part of initial events in thrombus formation. Inhibition of platelet aggregation is thus one of the most promising approaches to prevention of thrombosis, if a satisfactory inhibitor of platelet aggregation is available. Ticlopidine (1) is a potent, long-lasting inhibitor of platelet aggregation induced by a wide variety of stimuli including thromboxane A2-like substance and others (2). The present paper describes anti-thrombotic effect of Ticlopidine in several thrombosis models and in comparison with the effect of aspirin and dipyridamole.
1157
1138
TICLOPIDIXE
XXD
PLXTELETS
MATERIALS AND ?lETHODS Ticlopidine, S-(Z-chlorobenzyl)-4,S,6,7-tetrahydrothieno[3,2-C]pyridine hydrochloride, was supplied by PARCOR (Toulouse, France). Test animals used were male ddY mice weighing 17.0-19.5 g, male Wistar SLC rats weighing 220-250 g and male Wistar-Imamichi rats weighing 180-200 g. Acute pulmonary thrombosis in rats. According to the method described previously (3), pulmonary thrombosis was induced in Wistar-SLC rats by slow infusion of 0.7 M lactic acid (14 ml/kg) in 2 hr into the femoral vein under pentobarbital anesthesia. The severity of thrombosis was expressed by a total thrombus count in 10 microscopic fields of a lung section under 200 fold magnification averaged from 4 paraffin sections of the lung lobes (left, right superior, right middle and right inferior lobes). Thrombus count was measured on the samples obtained immediately after the lactic acid infusion. Acute thromboembolic death in mice. Acute pulmonary thromboembolism was induced in mice by a rapid intravenous injection of ADP (400 mg/lO ml saline/ kg) in 3 set or soluble collagen solution (25 mg/lO ml/kg) in 2 set, in reference to the reports by Nordijy and Chandler (4) and Nishizawa et al. (5), respectively. About 85 % of control mice died within 3 min after the injection. The collagen solution was prepared as follows. 2.5 mg Of acidsoluble collagen (Sigma Chemical Co., Type IV, calf skin) was dissolved in 1 ml of 0.05 M acetic acid containing 2 p CaC12 and 5 % glucose (pH 1.7). The solution was neutralized (pH 7.4) with S N NaOH, incubated at 37'C for 2 hr, then at 23-25°C for 3 hr and was used as a working solution. These procedures were important to give reproducible results. Endotoxin shock in mice. Endotoxin shock was induced in mice according to Fleming et al. (6). Escherichia coli endotoxin (Difco Lab., E. coli 026: B6) was,intravenously injected at 25 mg/lO ml saline/kg. Mortality rate was calculated 24 hr after the injection. About 80 % of control mice died under these conditions. Transient thrombocytopenia in rats (intravascular platelet aggregation). Insoluble collagen (Sigma Chemical Co., Type I, bovine achilles tendon) was homogenized with 0.025 M Tris-HCl (pH 7.4) containing 0.13 M NaCl. The homogenate was centrifuged at 200 g for 5 min, and the resultant supernatant fine suspension was diluted to 75 % of transmittance at 420 nm with the Trissaline. The collagen suspension (20 ml/kg) was rapidly injected into the tail vein of Wistar-Imamichi rats in 3 sec. Before and exactly 2 min after the injection, blood samples were taken from the tail vein for measuring platelet count. Degree of thrombocytopenia was expressed by the decrease (X) in platelet count after the collagen injection as reported previously (7). ,&-Aminopropionitrile-induced platelet hyper-reactivity. According to Pollock and Heath (8), ,&-aminopropionitrile (I-APN) was mixed into drinking water (2 mg/ml) and given ad libitum to Wistar-Imamichi rats for 7 days. The rats received p-APN in a total amount of 88 mg/rat/day. Control rats received water instead of a-APN solution. Two weeks thereafter titrated platelet-rich plasmas (PRP) were collected and tested for ADP- and collagen-induced platelet aggregation and clot retraction as described previously (2). Tests of blood coagulation and fibrinolysis.
Plasma recalcification
Vol.l2,Ko.6
TICLOPTDIXE
22.9 PLATELETS
1159
time and euglobulin lysis time were measured according to the methods described previously (3). Thrombelastography was performed with non-anticoagulated whole blood according to Hartert (9).
RESDLTS Effect on Lactic Acid-Induced Acute Pulmonary Thrombosis in Rats Ticlopidine or aspirin was given to rats at an oral dose of 50-200 mg/kg before the lactic acid infusion, and their protective action on thrombus formation was studied histologically. As shown in Table 1, Ticlopidine was effective in inhibiting thrombus formation in this model. Aspirin also prevented thrombus formation but was rather less effective than Ticlopidine in comparison of the results obtained with oral dose of 50 mg/kg. After repeated daily administration at 100 mg/kg for 4 weeks, Ticlopidine was found to retain well its anti-thrombotic action. Histological examination revealed no difference between the control and the treated rats in morphology of thrombi formed, which were composed of platelet aggregates, fibrin and coagulated protein-like substances as reported previously (3). TABLE 1 Prophylactic Effect of Ticlopidine and Aspirin on Lactic Acid-Induced Pulmonary Thrombosis in Rats Dose (mg/kg,po) Control 50 200
Aspirin Thrombus count Inhibition f S.E. (%)
Ticlopidine Inhibition Thrombus count f S.E. (%)
70.5 f10.5 (6) 53.6 f 8.6 (6) 28.2 f 5.5 (5)
70.5 f10.5 (6) 26.6 * 9.7 (5) 32.4 f 5.8 (5)
62.3* 54.0*
65.3 f 4.4 (9) 32.1 i 9.5 (6) 25.9 f 9.8 (5)
50.9** 60.4**
Control 100 (single dose) 100 (daily doses for 4 weeks)
24.0 60.0**
Data were analyzed by Student's t-test. * p(O.05, ** p
