Anti-platelet effects of clopidogrel in rat middle cerebral artery thrombosis model

Thrombosis Research,

Vol. 80, No. 3, pp. 209-216,1995 Copyright 8 1995 Elsevier Science Ltd F’r&din the USA. AI1 rights reserved ocl49-384tu95 $9.50 + .oo

Pergamon 0049-3848(95)00169-7

ANTI-PLATELET EFFECTS OF CLOPIDOGREL CEREBRAL ARTERY THROMBOSIS

IN RAT MODEL

MIDDLE

K. Umemura, H. Ishihara and M. Nakashima

Department of Pharmacology, Hamamatsu University Japan

School of Medicine, Hamamatsu, 43 l-3 1,

(Received 1 March 1995 by Editor Y Ikeda; revisetiaccepfed

Abstract

21 August 1995)

We have developed a model whereby the middle cerebral artery (MCA) in an experimental animal can be occluded by photochemical reaction between rose bengal and green light which causes endothelial injury followed by platelet adhesion, aggregation and formation of a platelet and fibrin-rich thrombus at the site of photochemical reaction. Using this model, we investigated the effect of clopidogrel, an analogue of ticlopidine which is a potent inhibitor of adenosine S’- diphosphate (ADP)-induced platelet aggregation. Oral clopidogrel (3-10mg/kg) inhibited ex-vivo platelet aggregation induced by ADP, thrombin or the thromboxane A2 mimetic, and U46619, when platelets had been primed with low concentration of phorbol myristate acetate. At these doses, clopidogrel significantly (P
Cerebral infarction is well known to bc related to the activation of platelets and coagulation cascade. The subsequent stroke in patients with transient ischaemic attacks can be prevented by Key words; clopidogrcl, platelet- and fibrin-rich thrombosis, middle cerebral artery Correspondence to: Dr. K. Umemura, Department of Pharmadogy, Hamamalsu University School of Medicine. 3600 Handa-cho Hamamatsu, 43 1-3 1 Japan Tel: 053-435-227 1 Pax: ()534.752270

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platelet activation inhibitors. Canadian American Ticlopidine Study (CATS; 1) and Ticlopidine Aspirin Stroke Study (TASS; 2), double blind trials of ticlopidine, have shown that ticlopidine is effective in stroke prevention. Consequently ticlopidine is widely used in the treatment of stroke (l-4). Clopidogrel (57), which has thienopyridine structure of ticlopidine is a more potent inhibitor of ADP-induced platelet aggregation than ticlopidine, and is expected to produce beneficial effect in ischaemic cerebral diseases. We have developed a model whereby the middle cerebral artery (MCA) in an experimental animal can be occluded by a thrombus induced by photochemical reaction between rose bengal and green light which causes endothelial injury followed by platelet adhesion, aggregation and formation of a platelet and fibrin-rich thrombus at the site of photochemical reaction (8,9). Using this model, we aimed to investigate the protective effect of clopidogrel on thrombotic MCA occlusion and see if this results in reduction of the size of cerebral ischaemic damage.

MATERIALS

AND METHODS

Materials Thrombin is purchased from Mochida, Japan. Rose Bengal is from Wako Japan. PMA and ADP is from sigma. U46619 is from Funakoshi, Japan. Clopidogrel is donated from Daiichi Pharmaceutical Co. Ltd. Animals and drug administration Male Wistar rats weighing 230-260g were used. Clopidogrel was dissolved in distilled water and administrated 2 times a day for 2 days. The last administration preceded experiments by 12 hours. Rats were anesthetized by intraperitoneal injection of 5OmgIkg of sodium pentobarbital. Platelet aggregation Blood samples were collected in 3.15% trisodium citrate solution (9:1 v/v) from aorta after anaesthesia. Platelet-rich plasma for ADP-induced platelet aggregation was obtained by centrifugation at 1OOrpm for 7min. Washed platelets (4x10s cells/ml) in Tris-buffer (pH7.4) for thrombin and U46619-induced aggregation were prepared (10) basically according to the method of Beanziger and Ma.jerus (11). Platelet aggregation was monitored at 37°C on an aggregometer (NBS hematracer model 601, Niko bioscience, Japan) in the presence of 1SmM CaCl2 with constant stirring at 100()rpm. Results are expressed as percentage of inhibition which is calculated (a-b)XlOO/a; a: mean maximum aggregation of control animals, b: each maximum aggregation of clopidogrel treated animals. As in the case of U46619-induced platelet aggregation, platelets had been primed with low concentration of PMA (OSnM) 1 min before the addition of the agonist (12). Animal nrenaration for the middle cerebral arterv thrombosis Wistar male rats weighing 240-260g were used. Body temperature of the animals was maintained at 37.5”C with a heating-pad. The MCA thrombosis model in the rat has been described previously (8,9). In brief, under pentobarbital anaesthesia and spontaneous respiration, a catheter for the administration of rose bengal was inserted into the femoral vein. The scalp and temporalis muscle were folded over and a subtemporal craniotomy was performed using a dental drill under an operating microscope to open a 3 mm diameter oval bonny window, which can be observed only the MCA.

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Photo-irradiation The 3 mm diameter oval window was irradiated with green light (wave length 540 nm) and the entire irradiated segment including the proximal end of the lenticulostriate branch became thrombotically occluded. Photo-illumination was achieved by using a xenon lamp (L4887: Hamamatsu Photon&, Hamamatsu, Japan) with a heat absorbing filter and a green filter. The irradiation was directed by a 3 mm diameter optic fiber mounted on a micromanipulator. The probe (1 mm in diameter) of a laser Doppler flowmeter (ALF 2100 Advance, Japan) was placed on the MCA for measuring the MCA blood flow. When a steady baseline blood flow was obtained, rose bengal (20 mg/kg) was injected intravenously. Photo-illumination was continued for a further 8 minutes. The blood flow in the MCA was continuously monitored for 15 minutes after rose bengal injection. The MCA was considered to be occluded when the blood flow had completely stopped as indicated by the flow monitor. The time taken from the injection of rose bengal to the cessation of blood flow was recorded as the MCA occlusion time. When the MCA blood flow continued beyond the 15 minutes observation period, the occlusion time was taken as 15 minutes (maximum). Determination of size of cerebral ischaemic damage 24 hours after MCA occlusion About 24 hours after surgery, rats were sacrificed by an overdose of pentobarbital and brains were quickly taken out. The cerebrum was separated from the other parts of the brain and w&q cut into six slices of lmm thick slices using a microslicer (D.S.K. DTK-300, Kyoto, Japan). Each slice was incubated in 1% tetratrihydrochloride (TTC, Katayama, Nagoya, Japan) solution at room temperature for 30 min, and photographed the slices. For each slice, the area of infarction was measured using a computerized image analysis system (Videoplane, Germany), and size of cerebral ischaemic damage divided by the whole area was expressed as the ratio of infarction. Statistical analvsis Data are expressed as mean * SE. Statistical analysis was made with unpaired Student’s t-test. For comparisons between groups, the analysis of ANOVA was applied. For the incidence of thrombolysis, groups were compared using Fisher’s Exact test. p
RESULTS Platelet Anerecation Figure 1 shows the inhibitory effect of clopidogrel on platelet aggregation induced by ADP. Administration of 3 mg/kg clopidogrel produced about a significant inhibition (P
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FIG. 1 Inhibitory effect of clopidogrel on ADP-induced platelet aggregation. PRP was stimulated by ADP with constant stirring at 37 “C. Final concentration of ADP were 5pM (open column) and IOpM (closed column). Values are mean + S.E. of 3-5 experiments. ***P
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FIG. 2 Inhibitory effect of clopidogrel on thrombin-induced platelet aggregation. Washed platelets were stimulated by thrombin with constant stirring at 37 “C. Final concentrations of thrombin were 0.2U/ml (open column) and 0.5U/ml (solid column). Values are mean + SE. of 5-6 experiments. ***P
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FIG. 3 Inhibitory effect of clopidogrel on U46619-inducedplatelet aggregationprimed by PMA. Washed plateletswere stimulatedby U46619 with constant stirring at 37 “C. Final concentration of U46619 was 1pM. Platelet was primed with OSnM PMA lmin before the addition of U46619. Values are meanf S.E. of 3 experiments. **P
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FIG. 4 Effect of clopidogrel on percentage of infarcted area 24 hours after induction of cerebral ischemia by thrombotic occlusion of the MCA. Results are expressed as infarcted ratio, which is infarcted areadivided by the whole area. Values are meank S.E. of 10 experiments but in the control group values of 9 animals were available. ** p
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0.01). The time taken from the injection of rose bengal to the cessation of MCA blood flow in control group was 290? 25.2 seconds (n=9), and the patency of MCA within 8 min of irradiation was 0% (O/9). The size of cerebral ischaemic damage in control group was 16.9 f 2.2% (Figure 4). The MCA patency with 3mg/kg clopidogrel treated group was 70% (7/l& P&O1 vs control group) and the time taken from the injection of rose bengal to the cessation of MCA blood flow (3/1(J) was 240 f 15.3 seconds. The size of cerebral ischaemic damage was 8.0 & 1.8%, which was significantly (P
DISCUSSION In this study, thrombotic occlusion of the rat middle cerebral artery was achieved by photochemical reaction between rose bengal and green light which cause endothelial injury followed by platelet adhesion to the damaged vessel and formation of a thrombus at the site of photochemical reaction (8, 9). Against the thrombotic occlusion of MCA, we tested the new ticlopidine analogue, clopidogrel which produced striking inhibitory effect on ex-vivo ADPinduced platelet aggregation. The prolongation of the time taken from the injection of rose bengal to the cessation of MCA blood flow was used as an index of antithrombotic effect of clopidogrel in vivo. The marked inhibitory effect of clopidogrel on the thrombotic occlusion of MCA, suggests that ADP-induced platelet activation contributes to the MCA thrombosis in this model. Further, observations 24 hours after the operation, showed that clopidogrel reduced the size of ischaemic cerebral damage. To know how much platelet-aggregation inhibition by clopidogrel is necessary to prolong the time to achieve thrombotic occlusion of MCA, inhibitory effect of clopidogrel on platelet aggregation induced by some agonists were monitored. In this study, ADP-induced aggregation was the most sensitive to clopidogrel rather than thrombin or U46619. Clopidogrel is supposed to inhibit platelet aggregation induced by some other agonists by blocking the amplification of platelet activation by released ADP. As agonist concentration increases, platelet aggregation becomes less ADP-dependent and the compound less effective. Thrombin-induced aggregation is Ca2+ dcpcndent at low concentration, but the aggregation sift to Ca *+ independent by increasing its concentration. It is known that only released ADP play a ma.jor role in the amplification of platelet response to thrombin, whereas thromboxane A2 (TXA2) does not seem to play any role through the experiment of platelet aggregation in the presence of ADP scavenging system, creatine phosphate/creatinc phosphokinase (CP/CPK) (5). As the platelet of Fawn-Hooded rat is devoid of ADP in their dense granules and therefore lacking in ADP release, no aggregation was obtained at low concentration of thrombin (13). High concentration of thrombin is supposed to activate intracellular signal transduction independent of Ca*+, and clopidogrel was less effective in this Condition. As rat platelet does not aggregate by the challenge of U4661Y, TXA2 mimetic, it is necessary to prime with low concentration of PMA to make platelet aggregate by U4661Y. And complctc inhibition by vapiprost, which is TXA2 receptor antagonist, of PMA primed aggregation of ~466 1Y indicates that aggregation is mediated by TXA;! receptor. Clopidogrel inhibited this

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aggregation in a dose dependent manner. Clopidogrel prolonged the time to thrombotic occlusion of MCA in a dose dependent manner and the patency after irradiation was greatly improved. The size of infarcted area 24 hours after irradiation also was reduced as platelet inhibition and thrombotic occlusion improved by clopidogrel. It is concluded that 3- 10mg/kg clopidogrel inhibited platelet aggregation induced by some agonists and this results in prolongation of the time taken from the injection of rose bengal to the cessation of MCA blood flow following reduction of the size of cerebral ischaemic damage. This prolongation is based on the inhibitory effect of clopidogrel on ADP-induced platelet aggregation.

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