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Effect of GR144053, a Fibrinogen-Receptor Antagonist, on Thrombus Formation and Vascular Patency After Thrombolysis by tPA in the Injured Carotid Artery of the Hamster

Matsuno, Hiroyuki; Kozawa, Osamu; Niwa, Masayuki; Ito, Takeshi; Tanabe, Kumiko; Nishida, Motoi; Hayashi, Hidehiko*; Uematsu, Toshihiko

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Journal of Cardiovascular Pharmacology: August 1998 - Volume 32 - Issue 2 - p 191-197
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Abstract

Platelets play important roles both in thrombus formation after vascular injury and in subsequent reocclusion alter successful thrombolytic therapy (1-3). The current standard conjunctive treatment with thrombolysis is aspirin and heparin (4,5). However, aspirin is a relatively weak platelet inhibitor, which mainly interrupts the thromboxane pathway (6). Inhibition of thrombus formation has been successfully performed in experimental models by using different types of antiplatelet agents (7-9). However, most of these drugs again act on a single platelet-activation pathway. The final common mechanism in platelet aggregation is the cross-linking of adjacent activated platelets by the binding of fibrinogen to the glycoprotein (GP) complex IIb/IIIa (10). In fact, monoclonal anti-GPIIb/IIIa antibodies have been demonstrated to be potent inhibitors of platelet aggregation and of platelet-dependent thrombus formation in experimental models (11,12) and patients (13). Very recently the incidence of reperfusion was enhanced when a potent GPIIb/IIIa antagonist was combined with tissue plasminogen activator (tPA) in patients (14). In addition, platelets contribute to the development of restenosis because they provide both cornice (15) and progression factors (16) leading to smooth-muscle cell (SMC) migration and proliferation.

With these considerations in mind, we developed an experimental model in hamsters suitable for studying arterial thrombus formation and neointima formation (17) and reported that an RGD (Arg-Gly-Asp)-containing peptide (a nonselective GPIIb/IIIa antagonist) reduced both thrombus and neointima formation in this hamster model (18). In this study, we evaluated the antithrombotic, thrombolysis-assisting, and antistenotic effects of GR144053 because this compound was shown to be a potent and selective inhibitor of fibrinogen binding to GPIIb/IIIa (19).

MATERIALS AND METHODS

Animals

Male hamsters (Gold, SLC, Sizuoka, Japan) weighing between 100 and 120 g were selected. All experiments were performed in accordance with institutional guidelines. The hamsters were anesthetized by intraperitoneal injection of 50 mg/kg sodium pentobarbital.

Reagents

GR144053, 4-[4-{4-(aminoiminomethyl)phenyl}-1-piperazinyl]-1-piperidineacetic acid hydrochloride trihydrate, was synthesized by Glaxo Wellcome U.K. GR144053 and tPA were kind gifts from Glaxo Wellcome U.K. and Toyobo Co., Tokyo, Japan., respectively. ADP and fibronectin were obtained from Sigma Chemical Co. and ICN Biomedicals, respectively.

Platelet aggregation

Blood was collected by heart puncture in tubes containing 1:10 (vol/vol) sodium citrate (3.15%) and then centrifuged for 10 min at 155 g to obtain platelet-rich plasma (PRP). Platelet aggregation was induced by adding 2.5 μM adenosine diphosphate (ADP) and followed in an aggregometer (Aggrecorder II, DA-3220; Kyoto Daiichi-Chemical, Kyoto, Japan) at 37°C with 800 r/min stirring speed. All measurements were done in duplicate.

Production of vascular injury in the carotid artery

The experimental procedure used to induce endothelial denudation was described previously (17). In brief, the distal right common carotid artery and the region of bifurcation were care-fully exposed. A 2FG catheter (Portex) with a roughened tip was inserted through the external carotid artery and advanced into the thoracic aorta. The catheter was left in position for 30 s and rotated completely 3 times. The external carotid artery was ligated after the catheter was slowly and carefully with-drawn. Endothelial cells in the injured area were completely stripped, and the elastic lamina was broken on several places. Blood flow in the carotid artery was continuously monitored by using a Doppler flow probe (model PDV-20; Crystal Biotech Co. Ltd., Tokyo, Japan) positioned proximal to the injured area of the carotid artery. The probe was removed after the first observation (day 0) and replaced on each observation day (days 1, 3, and 5). Occlusive thrombus formation was judged to have occurred when blood flow was reduced to zero.

Antithrombotic effect of GR144053 in vivo

GR144053 was administered by a continuous intravenous infusion via an infusion pump (STC-523; Termo Inc., Tokyo, Japan). The infusion of GR144053 (0, 0.1, 0.3, or 1.0 mg/kg/h; n = 8 each) was started 30 min before the infliction of endothelial injury. Blood flow was continuously monitored from the start of the infusion until 30 min after the injury.

Template bleeding time determination

Template bleeding times (20,21) was determined in each group. For this purpose, a template bleeding device (Simplate; Organon Teknika Co., Durham, NC, U.S.A.) was placed on the abdominal region, which was carefully shaved before the first bleeding time was measured. Blood flowing from the incision was gently wiped away with litter paper every 20 s.

Antistenotic effects of GR144053 after reperfusion by tPA

In separate experiments, the thrombus was allowed to age for 30 min before treatment with drugs was started. Ten percent of the total dose of tPA (0.52 mg/kg) was administered by an intravenous bolus injection, and the remaining was infused intravenously over 30 min by using an infusion pump (STC-523; Termo). GR144053 (0, 0.1, 0.3, and 1.0 mg/kg/h) was infused intravenously through the left jugular vein by way of a catheter (ID, 0.5 mm; OD, 0.8 mm;polyethylene sp3; Natsume Co. Ltd., Japan) connected to an implanted osmotic pump (2ML1; Alzet, Palo Alto, CA, U.S.A.). The osmotic pump was incubated for 30 min in saline (37°C) before the compound was injected by a exclusive syringe, and it was subcutaneously buried on the back of anesthetized hamsters. The start of infusion was at 30 min after thrombus had formed (together with the start of the infusion of tPA) and continued for the next 14 days.

Blood flow was monitored continuously from 5 min before until 90 min after the vascular injury and was measured additionally for 15 min at 1, 3, and 5 days after the injury. Finally, hamsters of each treated group (n = 8 each) were anesthetized 14 days after the injury, and the common carotid artery was immediately excised. The excised arterial segments were rinsed with saline, frozen in liquid nitrogen, and stored at −80°C until analyzed. Several cross sections were obtained from the frozen specimen and stained with hematoxylin and eosin (Sigma) after immersion fixation. The total areas circumscribed by the internal elastic lamina (IELA) and luminal surface of the artery (LA) were measured by using a computerized graphic image analyzer system. For this purpose, three consecutive cross sections of 4-μm thickness were prepared at 100-μm intervals in the vascular segment where stenosis was most prominent. The intimal area (IA = IELA - LA) was then calculated and expressed as a percentage of IELA by averaging three measurements taken from each of the three cross sections.

In vitro effects of GR144053 on SMC growth

SST1MF2 hamster SMCs (18) were grown to confluence in 75 cm2 culture flasks in Dulbecco's modified Eagle's medium (Gibco BRL, Rockville, MD, U.S.A.; 5% fetal calf serum, 100 μg/ml streptomycin, 100 U/ml penicillin, 4 μM glutamine) at 37°C under humidified 5% CO2/95% air. Cells were detached with 0.25% trypsin in phosphate-buffered saline (pH 7.4), washed, and counted. Isolated cells (8 × 104) were cultured as previously described in the absence or presence of various concentrations of GR144053 for 8 days in 16-mm Falcon multiple-well dishes previously coated with fibronectin by the following method. Human fibronectin was dissolved at 20 mg/ml in saline and adsorbed onto the dishes at 25°C for 60 min, after which they were rinsed 3 times with medium containing 0.2% bovine serum albumin (BSA) before use. The cell-culture medium containing GR144053 was renewed every 2 days. On day 8, calls were detached with trypsin and then counted.

In vivo effects of GR144053 on SMC proliferation

Proliferating SMCs were identified by the thymidine analog 5-bromo-2-deoxyuridine (BrdU)-labeling technique (22,23). After the infliction of carotid artery injury, the hamsters were divided into four groups: a control group (n = 4), one treated with tPA (0.52 mg/kg/day, n = 4), one with a continuous infusion of GR144053 (0.3 mg/kg/h, n = 4) and one with the combination of both drugs (n = 5). BrdU (50 mg/kg) was injected subcutaneously 1, 8, 16, and 24 h before removal of the carotid artery. After removal, 1 day after the catheterization, frozen cross sections were prepared from these arteries. BrdU-positive cells were stained with a murine monoclonal antibody (Sigma, St. Louis, Mo.), followed by goat antimouse immunoglobulin (Ig) antibodies conjugated to horseradish peroxidase and detection with diaminobenzidine (DAB). Sections were also stained for background with hematoxylin. The number of positive and negative nuclei were counted in the media and newly formed intima. The BrdU labeling index was calculated by using the following formula: (positive nuclei stained by DAB)/(total nuclei stained by hematoxylin) × 100 (10).

Statistics

All data are presented as mean ± SEM. The significance of drug effects (p < 0.05 and 0.01) versus the control was determined by analysis of variance (ANOVA) followed by the Student-Newman-Keuls test or Wilcoxon's test for the time to occlusion in vivo.

RESULTS

Inhibition of platelet aggregation in vitro and ex vivo

The inhibitory effect of GR144053 on platelet aggregation induced by ADP was evaluated in PRP collected from normal hamsters. An IC50 of 2.2 ± 0.4 × 10−5M (n = 8) was found. In ex vivo experiments, blood samples were collected from each hamster at the end of a GR144053 infusion at 0 (saline), 0.1, 0.3, and 1.0 mg/kg/h, and platelet aggregation induced by ADP was examined. Maximal aggregations in the four dosage groups (n = 8, each) were 69.43 ± 4.4, 68.3 ± 10.2, 19.3 ± 11.3, and 4.8 ± 4.1%, respectively, and the differences with the control value (infusion of saline) were statistically significant when using doses of 0.3 and 1.0 mg/kg/h.

Antithrombotic effect of GR144053 in vivo

Thrombotic occlusion of the carotid artery, in control animals (infusion of saline, n = 8), occurred at 4.4 ± 2.3 min after the induction of vascular injury (Fig. 1). When animals were treated with 0.1 mg/kg/h of GR144053, the time required to occlude the carotid artery (3.4 ± 2.0 min; n = 8) was not significantly changed. In contrast, it was significantly prolonged to 16.4 ± 6.1 min with 0.3 mg/kg/h of GR144053 (n = 8), whereas two of eight arteries examined were not occluded within 30 min with the highest dose (1.0 mg/kg/h), although the blood flow still was decreased.

FIG. 1
FIG. 1

Thrombolytic effect of tPA coadministered with GR144053

The reperfusion results obtained in the damaged carotid artery on thrombolysis with tPA plus GR144053 are summarized in Table 1. Blood flow was initially restored by tPA alone to ∼90% of the control value measured before the vascular injury but gradually decreased to 40 to 30% from 90 min until 5 days after reperfusion. On the contrary, the combined treatment with both drugs maintained arterial reflow >70% until 5 days after injury. Continuous i.v. infusion of GR144053 enhanced the thrombolysis with tPA, improving the vascular patency after reperfusion (Table 2). The alteration of schematic blood flow in each hamster is shown in Fig. 2.

TABLE 1
TABLE 1
TABLE 2
TABLE 2:
Vascular patency status after reperfusion by tPA (0.52 mg/kg) in the absence (0) or presence (0.3 and 1.0) of GR144053
FIG. 2
FIG. 2:
A: Treatment with tPA alone. B, C: GR144053 was administered i.v. by using an implanted osmotic pump at 0.3 and 1.0 mg/kg/h, respectively.

Template bleeding times

Baseline template bleeding time in hamsters is shown in Table 3. At the end of GR144053 infusions, bleeding times were dose-dependently prolonged. When the highest dose of GR144053 was coadministered with tPA, bleeding time was markedly prolonged (i.e., ≤9 times longer than that of control). However, by the end of the observation period (60 min after the end of infusion), bleeding time was shortened.

TABLE 3
TABLE 3:
Template bleeding time

Inhibitory effect of GR144053 on neointima formation

Figure 3a illustrates the development of neointima in the injured carotid artery evaluated 14 days after the induction of injury. Neointimal area in the injured artery, treated with continuous i.v. infusion of GR144053 (0.3 mg/kg/h) for 14 days after vascular reperfusion with tPA, was significantly reduced as compared with the nontreated group. In animals treated with either GR144053 (0.3 mg/kg/h) or tPA alone, neointima formation was not significantly reduced.

FIG. 3
FIG. 3:
Left panel (a) shows neointima formation. Open bars, control (Cont); treatment with tissue-type plasminogen activator alone (tPA); GR144053 alone (GR); and with the combination of both drugs (tPA + GR). GR144053 (0.3 mg/kg/h) was continuously administered i.v. for 14 days by using an implanted osmotic pump. tPA (0.52 mg/kg) was coadministered i.v. for the first 30 min. Hamsters were killed 14 days after the injury, and the neointimal area was determined on the cross sections of the excised carotid arteries. Internal elastic luminal areas of all arteries were not significantly changed. Right panel (b) shows smooth-muscle cell (SMC) proliferation measured as the bromodeoxyuridine (BrdU) proliferation index in the injured area 24 h after the catheterization. Treatment of the different groups was as in a. *p < 0.05 and **p < 0.01 versus control. Data are expressed as mean ± SEM.

Proliferation index of SMC in vivo and vitro

Combined treatment with tPA and GR144053 significantly reduced the proliferation index of SMC in vivo (Fig. 3b). When hamsters were treated with GR144053 alone, the index was decreased but not significantly, whereas treatment with tPA alone slightly increased the index. Finally, GR144053 did not inhibit the growth of the DDT1MF2 hamster SMCs on fibronectin (Fig. 4).

FIG. 4
FIG. 4:
Growth of DDT1MF2 hamster smooth-muscle cells on human fibronectin as a function of GR144053 concentration. Dashed line, number of cells grown on fibronectin obtained in control conditions after 8 days of culture. Solid circles, number of cells in growth medium containing GR144053. Each point is the mean of triplicate cultures. Data are expressed as mean ± SEM.

DISCUSSION

This study demonstrated the antithrombotic and antistenotic effects of GR144053, a specific fibrinogen-receptor antagonist in the injured hamster carotid artery. Inhibition of GPIIb/IIIa, required for the final common pathway of platelet aggregation (i.e., fibrinogen binding to platelets), is expected to decrease thrombus formation resulting from multiple proaggregatory stimuli. In our hands, GR144053 strongly inhibited platelet aggregation in vitro and thrombus formation in vivo. Previous studies showed that the addition of a GPIIb/IIIa antagonist to tPA decreased the time required to attain vascular reperfusion and subsequently maintained the arterial blood flow (21). In agreement with this observation, the concomitant administration of GR144053 and tPA in our model decreased the number of cyclic flow reductions (CRs) and increased blood flow after reperfusion, as compared with tPA alone. These findings indicate that platelets are mainly involved in the CRs and that the inhibition of activated platelets by a GPIIb/IIIa antagonist contributes to the maintenance of vascular patency after reperfusion.

A disadvantage of antithrombotic interventions is that treatment may often be complicated by hemorrhagic events, especially when drugs acting on different pharmacologic pathways are combined, so that patients show a bleeding tendency. In our experiment, the highest dose of GR144053 elicited a notable prolongation of the bleeding time when administered in combination with tPA. However, this effect was less pronounced when using the middle dose of GR144053 where the enhancement of the thrombolytic effect of tPA was still similar to the one seen with the highest dose of GR144053. The combination of a not fully inhibitory dose of an antiplatelet drug with a thrombolytic agent therefore may result in a synergistic effect on thrombus dissolution and prevention, without much additional risk for bleeding problems.

Chronic reocclusion due to neointima formation as a consequence of SM proliferation and migration is also an important problem after successful reperfusion has been achieved with the aid of tPA. In this process, vascular SMCs are activated by a variety of stimulating factors, where platelets may contribute by releasing platelet-derived growth factor (PDGF; 5,24) during thrombus formation. GPIIb/IIIa antagonists therefore can be expected partially to prevent the supply of PDGF to the injured vasculature by inhibiting platelet aggregation and secretion. To define the inhibitory effect on chronic reocclusion mainly due to neointima formation, we continuously infused GR144053 in hamsters with a damaged carotid artery. Treatment with GR144053 (0.3 mg/kg/h) alone was unable significantly to prevent neointima formation, even though it was continuously infused. However, when the hamsters were treated by GR144053 in combination with a thrombolytic agent, tPA, the consequent vascular stenosis was reduced significantly.

Further to characterize the inhibitory effect of continuous treatment with GR144053 on vascular late stenosis, we performed BrdU-labeling experiments in vivo and in vitro by using a hamster smooth-muscle cell line. The results indicated that GR144053 did not inhibit proliferation of SMCs directly, even if the same concentration of GR114053 could reduce platelet aggregation induced by ADP. However, GR144053 alone slightly reduced proliferating SMCs in vivo via inhibition of platelet activation. The combined treatment with GR144053 and tPA again strongly inhibited the activation of SMCs after vascular injury. Overall our experiments indicate that after damage, an occlusive thrombus formed because of both activation of platelets and the coagulation cascade. Activated platelets play a role in neointima formation by releasing PDGF, resulting in late restenosis.

Antithrombotic treatment, with either a specific GPIIb/IIIa blocker or with tPA alone, did not result in a significant reduction of neointima formation, whereas the combination was effective. Treatment with tPA alone dissolves the formed thrombus but leaves the initial trigger for platelet activation, and thus PDGF secretion, intact, which is evidenced by the occurrence of cyclic flow reductions. The lack of effect of GR144053 alone at first sight seems to be in contrast with our earlier findings (18) in which an RGD-containing compound, G4120, was found to be effective in preventing neointima formation. However, G4120 not only blocks GPIIb/IIIa, but also αvβ3, a vitronectin/fibronectin receptor also present on SMCs. GR144053 is a specific GPIIb/IIIa antagonist, because no effects were seen on SMC growth on fibronectin.

On the other hand, the dose of GR144053 used by itself was not sufficient fully to maintain vessel patency, thus still allowing platelet activation to occur. However, this dose did not further prolong the bleeding time by tPA, a combination that, despite this, apparently is powerful enough to provoke a synergistic effect on neointima formation.

In conclusion, our experiments indicate that it may be possible to find a combination of a thrombolytic drug and an antiplatelet agent that on the one hand is able to restore and maintain vessel patency and on the other hand to reduce excessive neointima formation significantly, without causing additional bleeding risks.

Acknowledgment: We thank Prof. Hans Deckmyn (K.U. Leuven, Belgium) for the helpful discussion and English.

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Keywords:

Fibrinogen-receptor antagonist; Neointima formation; Thrombolysis; Restenosis

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