The reduction of platelet thrombi on damaged vessel wall by a thromboxane synthetase inhibitor in rabbits

THROMBOSIS RESEARCH 27; 501-511, 1982 0049-3848/82/170501-11$03.00/O Printed in the USA. Copyright (c) 1982 Pergamon Press Ltd. All rights reserved.

THE REDUCTION OF PLATELET THROMBI ON DAMAGED VESSEL WALL BY A THROMBOXANE SYNTHETASE INHIBITOR IN RABBITS

Elizabeth R. Hall, Yao-Chang Chen, Teh Ho and Kenneth K. Wu Department of Medicine, Coagulation and Thrombosis Unit Rush-Presbyterian-St. Luke's Medical Center Chicago, Illinois, U.S.A. (Received 20.1.1982; in revised form 17.4.1982. Accepted by Editor J.B. Smith) ABSTRACT The role of thromboxane A2 (TXA2) in platelet-vessel wall interaction was investigated using 1-benzylimidazole (l-BI), a selective thromboxane synthetase inhibitor. l-B1 (0.9 mM) will reduce the aggregatory response of rabbit platelets to 0.2 mM arachidonate by 50% and their production of TXA2 by 84%. The effect of l-B1 on platelet thrombus formation was evaluated in vivo on New Zealand white male rabbits using the autologous indium-111 labeled platelet technique. After injection of autologous lllIn-platelets, 10 cm of the abdominal aorta was de-endothelialized with a balloon catheter. Three hours later the animals were sacrificed and injured and uninjured segments of the aorta removed. The radioactivity and dry weight of the tissue were determined. The radioactivity/gm of tissue was greater for the injured tissue than for the uninjured tissue. l-B1 at 10 mg/kg reduced the specific platelet accumulation at the injured site (n=5; 4.8 + 0.3 x 105 cpm/gm) compared to the controls (n=lO; 11.7 f 2.1 x 105 cpm/gm). Platelet accumulation on the injured tissue was further reduced by increasing the dosage to 30 mg/kg. Thirty minutes after l-B1 administration (30 mg/kg), platelets were less sensitive to arachidonate-induced aggregation (a 67% decrease) and TKA2 production was decreased 82%. Alterations in platelet sensitivity persisted for up to 3 hours. These findings indicate that TKA2 plays an important role in platelet-vessel wall interaction.

INTRODUCTION Formation of platelet thrombus on damaged vessel wall is governed by major humoral and cellular factors. Recently it has been indicated that prosta-

Key Words:

Thromboxane A2, Thromboxane Synthetase, Platelets, De-endothelialization, 1-Benzylimidazole

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glandins have a profound effect on platelet aggregate formation (1). Prostacyclin, which is synthesized from endoperoxides by vascular tissues (both the endothelium and to a lesser extent by the smooth muscle cells), is a potent inhibitor of platelet aggregation (2-4). Thromboxane A2 synthesized in platelets induces platelet aggregation (5). Regulation of platelet aggregation by these two compounds is probably mediated through their action on adenylate cyclase. Prostacyclin stimulates adenylate cyclase and elevates platelet cyclic AMP level (6), whereas TXA2 suppresses cyclic AMP elevation induced by PG12 (7). These observations led Moncada, Vane and their colleagues to postulate that the balance between PG12 and TXA2 plays an important physiological role in hemostasis (8). When vascular endothelium is damaged, production of prostacyclin at the injury site is reduced and platelets come in contact with subendothelial components, i.e. collagen and basement membrane. This results in the activation of platelet membrane phospholipases and the liberation of arachidonic acid, which is converted to the endoperoxides PGG2 and PGH2. PGH2 is further converted to TXA2 by thromboxane synthetase. TXA2 promotes the platelet release reaction and aggregate formation. As prostacyclin production is compromised, this leaves the aggregating effects of TXA2 unopposed and leads to platelet thrombus formation on the damaged vessel wall. Because of the pivotal role of TXA2 in platelet activation and platelet-vessel wall interaction, a thromboxane synthetase inhibitor, which would specifically block the conversion of the endoperoxides PGG2 and PGH2 into TXA2 might be expected to have a therapeutic potential for controlling thrombi formation. Although several compounds have been reported to inhibit thromboxane synthesis and arachidonate-induced platelet aggregation, very little has been reported about their activity in the intact animal. Imidazole was first reported to be a selective inhibitor of thromboxane synthetase by Needleman et al and Moncada et al (9,lO). Subsequently, Tai and Yuan demonstrated that the potency of imidazole could be enhanced by substitution of the l-position (12). 1-Benzylimidazole (l-BI) was among the most potent inhibitors that they tested. l-Benzylimidazole has also been reported to have an in vivo antiplatelet effect in dogs (12). In the present study, l-B1 at two doses, 10 mg/kg and 30 mg/kg, was investigated for its effect on platelet-vessel wall interaction in rabbits. Our findings indicate that l-B1 is effective in preventing platelet aggregate formation on injured aortic wall. METHODS Arachidonic acid was purchased from NuChek-Prep, Inc., Elysian, MN. Prostaglandin standards, thromboxane B2 and U46619 [(15S)-hydroxy-11, 9(epoxymethanal)-prosta-52, 13E dienoic acid] were kindly supplied by Dr. Udo Axen of the Upjohn Company, Kalamazoo, MI. l-Benzylimidazole was obtained from the Aldrich Chemical Company, Inc., Milwaukee, WI. Acid-citrate dextrose (ACD) solution (80 mg citric acid, 224 mg anhydrous sodium citrate and 120 mg anhydrous dextrose per 10 ml solution) was obtained from Squibb and Sons, Inc., Princeton, NJ. ACD-saline solution was prepared by mixing 1 part of ACD with 6 parts of normal saline. lllIn-chloride (2 mCi/ml) was obtained from Mediphysics, Emeryville, CA. 8-Hydroxyquinoline (oxine), adenosine diphosphate (ADP), bovine serum albumin (BSA) gamma globulin and other reagent grade chemicals were obtained from Sigma Chemical Company, St. Louis, MO. Collagen prepared from equine tendons was obtained from Hormon-Chemi, Munich, Germany. 5F Fogarty catheters were obtained from Edwards Laboratories, Inc., Santa Ana, CA. Norit "A" charcoal was purchased from Amend Drug Company, Irvington, NJ.

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Platelet Aggregation Platelet aggregation was performed with a Payton aggregometer according to the turbidometric procedure of Born (13). Blood was obtained from New Zealand White rabbits by cardiac puncture, mixed with 3.8% sodium citrate (1:9 v/v), and centrifuged for 10 min at 220 xg to obtain platelet-rich plasma (PRP) . The remaining sample was further centrifuged at 1000 xg for 20 min to prepare platelet-poor plasma (PPP), Platelet concentration of the PRP was adjusted to 4 x 108/ml with autologous PPP. 0.5 ml of PRP was preincubated in the aggregometer for 5 min before arachidonic acid (0.2 mM) was added. The mixure was stirred at 370C for 3 min and ensuing aggregation was monitored. l-BI, at various concentrations, was preincubated with the platelets 5 min before the addition of an equal amount of arachidonic acid as had been added to the controls. Platelet aggregations were compared by determining the maximum change in light transmission within a 3 min period in response to arachidonate. Thromboxane B? Measurement Since TXA2 is extremely short-lived and readily converted into TXB2 in aqueous media, the platelet production of TXA2 was assayed by measuring TXB2. PRP samples were aggregated as described for 3 min. These samples were then acidified to pH 3.0 with 1 N HCl. The acidified mixture was kept at 1oC for 3 min and then centrifuged for 5 min at 13,000 xg. The supernatant was neutralized with 1 M Tris buffer and diluted 500 fold in 50 mM Tris HCl buffer, pH 8.0, containing 0.1% gelatin. The TXB2 content of the samples was determined by radioimmunoassay as described by Tai and Yuan (14). Highly specific antibodies were obtained by immunizing rabbits with TXB2-BSA conjugates. The cross reactivity of the anti-TXB2 was less than 0.1% with arachidonic acid, prostaglandin endoperoxides and other prostaglandins. Separation of bound from free antigen was achieved by using gamma globulin-coated charcoal. The sensitivity of the assay was 20 pg per 200 ~1 (i.e., BIB0 = 80%). Preparation of lllIn-labeled Platelets Rabbit platelets were labeled with lllIn-oxine by a modification of a previously described method (15,16). Labeling procedures were carried out under sterile conditions. In brief, blood was collected from rabbits by cardiac puncture and divided into two samples: a) 20 ml were mixed with 4 ml ACD solution and b) 9 ml with 1 ml 3.8% sodium citrate. Blood samples were centrifuged at 220 xg for 10 min to prepare platelet-rich plasma (PRP) and the remainder further centrifuged at 1000 xg for 20 min to obtain platelet-poor plasma (PPP). ACD-PRP was washed twice with 10 ml ACD saline and then incubated with lllIn-oxine complex at room temperature for 20 min. The mixture was centrifuged and the latelet pellet washed once with 8 ml ACD-PPP to remove residual unbound PllI*. The pellet was suspended in 4 ml citrate-PPP. Platelet concentration was measured by phase microscopy and total radioactivity determined with a gamma-spectrometer in each sample. Platelets were examined by phase microscopy in each sample and platelet aggregates were undetected. Labeled platelets exhibited a normal aggregation response to ADP (3 I_IM) and collagen (5 Ug/ml). The survival time of the labeled platelets was determined in three normal, uninjured rabbits according to the procedure of Heaton et al (16). Mean initial recovery was 84% and mean survival time was 3.11

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days, which was similar to the rabbit normal values reported previously (17). De-endothelialization of Abdominal Aorta Aortic intimal injury was induced by the balloon catheter technique described by Spaet et al (18). Rabbits were anesthetized with an intravenous injection of sodium pentobarbital (25-30 mgfkg). The abdominal aorta was denuded of endothelium by passing a 5F Fogarty catheter into the aorta via the femoral artery. When the tip of the catheter reached the point of aortic bifurcation, the balloon was inflated to a pressure of about 735 mm Hg. While the pressure was maintained, the balloon catheter was passed upward to a level of 10 cm from the bifurcation and down to the original position; this was repeated six times. The balloon was then allowed to collapse, the femoral artery ligated and the wound closed. The intima was diffusely denuded in each of six rabbits, as evaluated by the infusion of Evans blue (19). Surgical procedures were performed by the same personnel with consistent results. Experiments All experimentswere performed on male New Zealand White rabbits (Z-3.5 kg). Blood samples were drawn 2% hrs prior to injury and labeled as dedescribed. Three groups of rabbits were used: Group 1 (n=lO) received the vehicle only (saline, 5.0 ml), Group 2 (n=5) received 10 mg/kg 1-BI, and Group 3 (n=5) received 30 mg/kg l-BI. After the balloon catheter had been placed at the bifurcation, autologous lllIn-labeledplatelets and l-B1 or vehicle were injected via separate marginal veins. Ten min later the balloon catheter was inflated and intimal damage along a-.10cm abdominal aortic segment was created. Three hours following injury a titrated blood sample was obtained, 1000 units of heparin were injected intravenously to prevent postmortem clotting and the animal was killed by an overdose of barbiturate. The aorta was separated and dissected from the top end of the thoracic aorta just below the arch, to the lower end of the abdominal aorta 1 cm above the bifurcation which corresponded to the starting point of balloon catheter injury. Residual tissues attached to the outer surface of the aorta were gently removed. The lower 10 cm of injured tissue and the upper 5 cm of uninjured tissue were then gently rinsed twice with normal saline to remove any blood from the lumen of the vessel, placed in a dessicator to dry for 48 hrs, and weighed. The titrated blood sample was treated similarly and its radioactivity and its weight measured. Radioactivity and percentage of injected radioactivity per gram of dry weight of tissue or blood were calculated. The data were expressed as mean 2 standard deviation (S.D.) and analyzed by a multiple comparison procedure described by Dunnett (20,21). RESULTS In vitro platelet function was altered by l-B1 in a concentration dependent manner. The concentration which inhibited 50% of arachidonic acid-induced platelet aggregation (IC50) was 0.9 + 0.2 mM (n=5>. Rabbit PRP was preincubated with either l-B1 or vehicle for 5 min at 37OC prior to the addition of a minimal dose of arachidonic acid to get maximal aggregation (0.2 mM). At 0.9 mM l-BI, TXB2 production was inhibited 83.7%, while at 2.7 mM l-B1 TXB2 production was inhibited by 93.2% (Table 1).

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TABLE1 Decrease of Thromboxane B2 Production In the Presence of l-Benzylimidazole

l-BI (mM)

ng TXB2/108 platelets

% Inhibition

0.0

251 f.lO*

---------_

0.9

41 f 17

83.7

2.7

17 + 3

93.2

*Values represent the mean f S.D. of four experiments. l-B1 was subsequently tested for its ex vivo effect on platelet aggregation and TXB2 formation. The animals were treated with l-B1 (30 mg/kg), which was estimated to give rise to a plasma level of 5.1 mM. Arterial blood samples were drawn prior to the intravenous injection of l-B1 and at 30 min and 3 hrs afterward. Thirty min after l-B1 administration, the aggregation response of the platelets to 0.2 mM arachidonic acid was inhibited by 66.5 + 8.8% (n=5) and the production of TXA2 by 82 + 4%. Alterations in TXB2 production per= sisted throughout the 3 hour period investigated. At 3 hrs after l-B1 injection, ex vivo TXB2 production was still inhibited 74 + 13% (Table 2). However, the platelet aggregation response to arachidonic acid varied greatly (from 0 to 50% inhibited) in the 5 animals tested. Since l-B1 was effective in the inhibition of rabbit platelet TXB2 production, the in vivo effect of 10 and 30 mg/kg of l-B1 on platelet-vessel wall interaction was assessed using three groups of rabbits. All three groups were similar in body weight, sex, radioactivity received and dry weight of their aortic tissue (Table 3). Three hours after acute injury, the rabbits were sacrificed and the injured and uninjured segments of aorta removed. The radioactivity and dry weight of these tissues were determined. The radioactivity and dry weight of the blood sample drawn prior to sacrifice were also determined. The radioactive counts as well as percentage of injected radioactivity per gm of dry weight of blood were approximately the same in the control and the l-B1 treated groups, indicating that the number of adhering platelets was insufficient to affect the total blood volume. In the control animals the number of platelets (i.e. cpm lllIn) adhering to the lesion site was 16.7 times greater than the number that bound non-specifically to the uninjured tissue. In the presence of 10 mg/kg of l-B1 the number of counts adhering to the injured aortic segment was significantly decreased (P
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TABLE 2 Decredse in Thromboxane B2 Production hy Platelets 30 min and 3 hr after l-Benzylimidazole Injection

Sample

ng TXB2/108 platelets

Pre-injection*

283 + 30t

% Inhibition

0.0

30 min post*

51 ? 115

82 + 4

3 hrs post

73 r 36

74 i:13

* Rabbits were given an IV injection of 30 mg/kg 1-benzylimidazole. f Values represent the mean f S.D. for four experiments. !JP CO.05 when compared to the control group.

TABLE 3 Comparison of Experimental Parameters among the Groups of Rabbits

Control

No of Rabbits Weight (kg) Sex Total 11'In injected (cpm x 106)

10

l-B1 10 mglkg

l-B1 30 mglkg

5

5

2.45 ?r0.12

2.35 ? 0.95

2.33 i:0.25

M

M

$1

268 f.44

295 + 57

266 5 44

Dry weight of injured tissue (mg)

70.0 f 21.3

63.5 ? 21.5

69.7 i.7.3

Dry weight of uninjured tissue (mg)

60.5 + 28.0

54.4 f 22.8

52.5 2 24.6

5

l-B1 (30 m&kg)

0.44 0.16 0.09

48 ?:3" 24 i:2"

0.03

%+

117 f 21(

8

x lo4 cpm/gm*

16.7

0.03 0.03 0.03

724 9?1 Et1

"P CO.01 when compared to the vehicle group.

lIValuesrepresent mean f S.D.

§Animals received a sham operation and were killed 3 hrs afterward. Since only two animals were used, average values only are given.

3.0"

5.3"

1.1

0.02

7

x lo4 cpmlgm

Injured/Intact

Ratio of cpm

%

Radioactivity of Intact Aorta

tPercentage of injected radioactivity per gm of dry weight of tissue.

* cpm per gm dry weight of tissue.

5

l-B1 (10 mg/kg)

10

2

Shams

l-B1 vehicle

Rabbits

Groups

Radioactivity of Injured Aorta

Effect of 1-Benzylimidazole on Lesion Radioactivity

TABLE 4

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DISCUSSION The present study was undertaken to investigate the effect of l-BI, a in vitro, selective thromboxane synthetase inhibitor, on rabbit platelets -ex vivo and -in vivo. l-B1 is an effective inhibitor of rabbit platelet -thromboxane synthetase. In addition, l-B1 inhibited platelet aggregation both _in vitro and _ex vivo. There is some disagreement in the literature whether or not selective thromboxane synthetase inhibitors are sufficient to inhibit platelet aggregation. Grimm et al (22) have shown that 7-(l-imidazolyl) heptanoic acid (7-IHA) is a more effective inhibitor of TXB2 synthesis (150 = 1.9 uM) than platelet aggregation (I50 = 1.35 mM) in humans. Their conclusion was that the formation of the endoperoxides or other cyclooxygenase products may be sufficient to mediate aggregation induced by arachidonic acid even in the presence of thromboxane synthetase inhibitors. Our data show that 0.9 mM l-B1 will inhibit platelet aggregation 50% and TXB2 production by 84% in rabbit platelets. Although TXB2 synthesis was more sensitive to l-B1 than platelet aggregation, we did not find as large a difference in sensitivity as seen by Grimm et al. There may be at least two reasons for this 1) that l-B1 is not as selective for thromboxane synthetase as 7-IHA and/or 2) that the response of human and rabbit platelets to arachidonic acid and its metabolites is different. Although the selectivity of l-B1 has not been investigated in detail, several other imidazole derivatives have been reported to selectively inhibit thromboxane synthetase and not cyclooxygenase, prostacyclin synthetase or endoperoxide isomerase (9-11, 22). On the other hand, there are differences in the response of rabbit and human platelets to arachidonic acid. Although rabbits are more sensitive to arachidonic acid induced aggregation, they are less sensitive to U46619, an agonist of the platelet thromboxane/endoperoxide receptor (23,24). The minimal concentration of U46619 needed to induce maximal aggregation in humans was 1.6 + 0.5 (n=8> and rabbits was 6.8 + 3.8 nM (n=8). Therefore, human platelets would be challenged with more arachidonic acid and would be more sensitive to the endoperoxides accumuIn vivo, however, lated in the presence of thromboxane synthetase inhibitors. -one would not expect to see an accumulation of endoperoxides, as they should be rapidly metabolized. We have also investigated the effect of l-B1 -in vivo using the acute injury model with lllIn-labeled platelets as described by Wu et al (25). The thrombi formed are primarily composed of platelets with minimal fibrin formation. Thus, this model is of particular value for studying -in vivo platelet-subendothelial interaction and platelet aggregate formation. It is currently thought that the balance between PG12 and TXA2 plays an important role in modulating platelet aggregate formation in acute vascular injury. The selective inhibition of thromboxane synthetase by l-B1 would not only limit the production of TXA2, thereby increasing the effectiveness of any available prostacyclin, but it would also lead to an increase in endoperoxide concentration and release. The release of endoperoxides from the platelets may then be utilized by the vasculature for the production of PG12 (26,27). Our data suggest that l-B1 can effectively inhibit platelet deposition -in vivo after acute vascular injury. Both doses of l-B1 tested were effective in reducing platelet thrombus formation at the deendothelialized surface of the aorta, although as might be expected, the higher dose was the more effective of the two. Since the 10 mg/ kg dose of l-B1 was effective in our acute injury model, it is likely that a much lower dose would be sufficient in less extreme cases.

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Wu et al (25) employed the same acute injury model to determine the doserelated effects of aspirin on platelet thrombus formation. They found that 30 mgfkg of aspirin significantly reduced platelet thrombus formation at the deendothelialized surface of the aorta, yet it did not inhibit the adhesion of a single layer of platelets to the subendothelium. This observation corresponds to those of other investigators, that platelet adhesion is more resistant to PG12 than platelet aggregation (28,29). The corollary is probably also true, i.e. that platelet aggregation is more dependent on TXA2 than is platelet adhesion. Therefore, the observed effectiveness of l-B1 in inhibiting platelet thrombus formation in our study may not be a result of limiting platelet adherence to the damaged vessel wall but instead representative of its ability to inhibit platelet aggregation and thus subsequent thrombus formation. ACKNOWLEDGEMENTS The authors would like to thank Michael McNeal and Debbie Matayoshi for their technical assistance and Therese Molyneux and Rosa Ocasio for their preparation of the manuscript. This work was supported in part by the American Heart Association (#77-1039) and by a Rush University Research Fund (847898). REFERENCES 1.

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TAI, H.H. and YUAN, B. Development of radioimmunoassay for thromboxane B2. Anal. Biochem. 87, 343-349, 1978.

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DWANA, E., CAZENAVE, J.P., GROVES, H.M., KINLOUGH-RATHBONE, R.L., RICHARDSON, M., PACKHAM, M.A. and MUSTARD, J.F. The effect of aspirin inhibition of PG12 production on platelet adherence to normal and damaged rabbit aortae. Thromb. Res. 17, 453-464, 1980.