Inhibition of von Willebrand Factor Binding to Platelet GP Ib by a Fractionated Aurintricarboxylic Acid Prevents Restenosis After Vascular Injury in Hamster Carotid Artery
Background Aurintricarboxylic acid (ATA) prevents von Willebrand factor binding to platelet glycoprotein (GP) Ib, with higher-molecular-weight ATA more effective than the lower-molecular-weight compound. We investigated the effects of high-molecular-weight ATA (Mr=7500), obtained by fractionating commercial ATA, in the injured hamster carotid artery.
Methods and Results Platelet aggregation was induced in vitro with ADP (2.5 μmol/L) or botrocetin (5 μg/mL) in hamster platelet-rich plasma. IC50 values were 348.6±22.4 and 8.2±3.2 μg/mL, respectively. The endothelium of hamster carotid artery was denuded with a modified catheter. Continuous administration of high-molecular-weight ATA (10, 30, and 100 μg·kg−1·h−1) with an infusion pump produced antithrombotic effects in a dose-dependent manner, as evaluated by prolongation of time to occlusion. Neointima formation was observed 2 weeks after catheterization, and proliferating smooth muscle cells (SMCs) were identified by the thymidine analogue 5-bromo-2-deoxy-uridine (BrdU). Continuous treatment with the compound (100 μg·kg−1·h−1) with a 2ML1 Alzet infusion pump resulted in a reduction of neointimal area by 38.0±8.8% and decreased the BrdU index on days 1 and 7 significantly. DNA synthesis in DDT1MF2 hamster SMCs was also decreased by the compound in a dose-dependent manner. In histological observation, the process of endothelial healing was improved by this treatment with the compound.
Conclusions Inhibition of platelet adhesion by von Willebrand factor binding to platelet GP Ib by high-molecular-weight ATA results in the prevention of thrombus formation and the suppression of neointima lesion. In addition, high-molecular-weight ATA has an inhibitory effect on SMC proliferation. This inhibition of both platelet adhesion and SMC proliferation markedly reduced vascular stenosis.
The process of restenosis after vascular injury begins with platelet adhesion to the vessel wall, followed by thrombus formation, secretion of growth factors, and finally SMC migration and proliferation. vWF, a multimeric glycoprotein, mediates the adhesion of circulating platelets to exposed subendothelial structures of the injured vessel wall. vWF binds to components of the subendothelium through multiple interactive sites1 2 3 and at the same time bears binding domains for platelet membrane GP Ib and GP IIb/IIIa.4
Several in vitro experiments have shown the involvement of the vWF–GP Ib axis in the early phase of thrombus formation,5 6 and the involvement of vWF has also been reported in in vivo studies.7 These studies clearly indicate the importance of vWF in platelet-dependent thrombosis in stenosed arteries. ATA, a triphenylmethyl dye compound, inhibits platelet adhesion by interfering with the initiation of vWF binding to platelet GP Ib8 and preventing thrombus formation in vivo.9 Although a number of antagonists have been shown to improve restenosis after vascular injury by the inhibition of GP IIb/IIIa,10 11 12 no studies have been reported on chronic treatment with ATA.
In the present study, we investigated the inhibitory effect of high-molecular-weight ATA fractionated from a commercially obtained ATA on thrombus and neointima formation in injured carotid artery from hamsters, on the assumption that high-molecular-weight ATA is more effective than lower-molecular-weight compounds.13 14
Male hamsters (Gold, SLC, Japan) weighing 100 to 110 g were selected and fed a standard chow (RC4, Oriental Yeast Co, Ltd). Hamsters were anesthetized by injection of 50 mg/kg sodium pentobarbital IP. All experiments were performed in accordance with institutional guidelines.
Fractionated ATA was obtained from a commercially available ATA product (Aldrich) by gel permeation chromatography as described previously.15 Average molecular weight was estimated by comparison with those of standard polystyrene polymers. High-molecular-weight ATA (average molecular weight, 7500; index of dispersion, 1.5) was selected and used for experiments. ADP and botrocetin were obtained from Sigma Chemical Co. [Methyl-3H]thymidine (22 Ci/mmol) was purchased from Amersham Japan.
Platelet Aggregation In Vitro
Blood was collected by heart puncture with added sodium citrate (0.011 mL/L final concentration) and centrifuged for 10 minutes at 155g to obtain platelet-rich plasma. Platelet aggregation was induced by 2.5 μmol/L ADP or 5.0 μg/mL botrocetin and measured with an aggregometer (Aggrecorder II, DA-3220, Kyodaiichi-Chemical) at 37°C with a stirring speed of 800 rpm. Aggregation is expressed as a percentage of maximum light transmission obtained in the absence of drugs. All counts were done in duplicate.
Production of Arterial Injury
The experimental procedure used to induce injury in the hamster common carotid artery has been described in detail elsewhere.16 In brief, the distal right common carotid artery and region of bifurcation were exposed. A 2F 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 seconds and then rotated completely three times. The catheter was then slowly and carefully withdrawn, and the external carotid artery was ligated. By these means, ECs were completely denuded and several parts of the elastic lamina were ruptured. Platelet-rich thrombus was observed in the injured area immediately after the initiation of injury, and adhesive platelet formation was seen in the injured area for several days. Neointima formation was observed 14 days after injury.
Infusion Regimen to Prevent In Vivo Thrombus Formation
High-molecular-weight ATA was administered by continuous intravenous infusion with an infusion pump (TERMO STC-523). Infusion was started 30 minutes before vascular denudation and continued for 60 minutes. Hamsters were divided into a control group (n=12) and groups treated with high-molecular-weight ATA at doses of 10 (n=6), 30 (n=6), and 100 (n=12) μg·kg−1·h−1. Blood flow was continuously monitored during each experiment as follows: A Doppler flow probe (model PDV-20, Crystal Biotech Co) was left in position proximal to the vascular injury area. An occlusive thrombus was considered to have formed when blood flow was decreased to the baseline flow before injury.17
Template Bleeding Time
Template bleeding time was measured at the end of antithrombotic experiments in vivo in each group. A template bleeding device (Simplate; Organon Teknika Co) was placed on a carefully shaved area of the abdominal region. The region of incision was carefully shaved before performance of the first bleeding time. Blood flowing from the incision was wiped away with filter paper every 20 seconds.
Quantification of Neointima Formation
The compound was administered by continuous intravenous infusion with an implanted osmotic pump (Alzet 2ML), starting 30 minutes before injury and continuing for 14 days. Animals were divided into a control group (n=12) and groups treated with high-molecular-weight ATA at doses of 10 (n=8), 30 (n=8), and 100 (n=12) μg·kg−1·h−1. Fourteen days after vascular injury, hamsters in each group were anesthetized, and the common carotid artery was excised, rinsed with saline, and frozen. The frozen sections were divided into several cross sections, then stained with hematoxylin and eosin (Sigma) after immersion fixation. The total IELA and LA were measured with a computerized image graphic analysis system. For this analysis, three consecutive carotid artery cross sections (4 μm thick) were prepared at 100-μm intervals in the most stenotic area, and the intimal area (IA=IELA−LA) was expressed in proportion to the IELA by averaging three measurements for each of the three cross sections. At the end of the experiment, blood samples were taken in each group and platelet aggregation was measured ex vivo as described above.
Hamsters were divided into four groups. Each group was treated with high-molecular-weight ATA (100 μg·kg−1·h−1) for 1, 3 (n=6 each), 7, or 14 days (n=12 each) with an implanted osmotic pump. Neointimal area was also determined 14 days after injury.
Proliferation Index of SMCs In Vivo
Proliferating SMCs were identified by the thymidine analogue BrdU labeling technique.18 19 The proliferation index was measured in a control group (n=6) and a group treated with high-molecular-weight ATA at a dose of 100 μg·kg−1·h−1 (n=6). BrdU (50 mg/kg SC) was injected 1, 8, 16, and 24 hours before removal of the carotid artery at 1 or 5 days after catheterization. Frozen cross sections were then prepared from these arteries. BrdU-positive cells were stained with a murine monoclonal antibody (Sigma), followed by goat anti-mouse Ig antibodies conjugated to peroxidase and detected with DAB. Sections were also stained with hematoxylin for detection of nonproliferating cells. The positive and negative nuclei were counted in the media and newly formed intima. The BrdU labeling index was calculated by the following formula: (positive nuclei stained by DAB)/(total nuclei stained by hematoxylin)×100.
DDT1MF2 hamster SMCs (ATCC-1701) were maintained in DMEM containing 10% FCS at 37°C in a humidified atmosphere of 5% CO2/95% air. The cells (15×104) were seeded into 35-mm-diameter dishes in 2 mL DMEM containing 10% FCS. After 24 hours, the medium was exchanged for 2 mL DMEM. The cells were then used for experiments after 48 hours.
Measurement of DNA Synthesis
The cultured cells were pretreated with various doses of high-molecular-weight ATA for 20 minutes, then stimulated with 5% FCS in 1 mL DMEM at 37°C for 24 hours. Six hours before harvest, the cells were pulsed with [methyl-3H]thymidine (0.5 μCi/dish). Incubation was terminated by addition of 1 mL 10% trichloroacetic acid, and radioactivity in the acid-insoluble materials was determined. The radioactivity of [methyl-3H]thymidine samples was determined with a Beckman LS-6000IC liquid scintillation spectrometer.
Electron Microscopic Observations
Damaged vasculatures were removed for electron microscopic observation of their luminal surfaces on days 3 and 14 in controls and on day 3 of treatment with high-molecular-weight ATA (100 μg·kg−1·h−1) after injury. These segments were prepared without rinsing to leave any formed platelets intact and were fixed in 2.0% glutaraldehyde in 50 mmol/L sodium phosphate buffer for 30 minutes. Each segment was cut open longitudinally to allow visual inspection by scanning electron microscopy as previously described.20
Immunochemical Staining of vWF
Frozen injured carotid arteries taken 7 days after catheterization were cut into sections 4 mm thick. These sections were incubated with preimmune serum for 45 minutes, washed, and then incubated with an anti-vWF antiserum conjugated to peroxidase (DAKO, dilution 1/100) for >4 hours. Finally, they were developed with DAB for 5 minutes in Tris-HCl buffer (pH 7.6) and counterstained with hematoxylin.16
All data are expressed as mean±SEM. Results were compared with the control by ANOVA followed by the Student-Newman-Keuls test or Wilcoxon’s test for time to occlusion in vivo.
High-molecular-weight ATA inhibited platelet aggregation induced by botrocetin and ADP in a dose-dependent manner. IC50 values for aggregation induced by 5 μg/mL botrocetin and 2.5 μmol/L ADP were 8.2±3.2 and 348.6±22.4 μg/mL, respectively. The time to occlusion in the injured arteries is shown in Fig 1⇓. Arterial occlusion in the control group occurred 3.8±1.8 minutes after induction of vascular injury. Time to occlusion with the lowest dose of high-molecular-weight ATA (10 μg·kg−1·h−1) was not significantly changed (3.6±2.2 minutes). In contrast, time to occlusion was prolonged on treatment with the middle dose (30 μg·kg−1·h−1) and significantly prolonged at the highest dose (100 μg·kg−1·h−1). However, all arteries were occluded during the observation period.
Template Bleeding Time
Baseline template bleeding time in hamsters was 45.3±5.8 seconds (n=12). At the end of infusion, this variable was prolonged in a dose-dependent manner (Table⇓). This prolongation was significant at the middle and highest doses of high-molecular-weight ATA, up to 3.8 times longer than that of control.
Reduction of Neointima Formation
The fact that ex vivo botrocetin-induced platelet aggregation was still dose-dependently inhibited at the end of the observation period on day 14 showed that effective delivery of high-molecular-weight ATA by implanted osmotic pumps was maintained throughout the experimental period (Fig 2a⇓). Treatment with the compound dose-dependently decreased neointima formation in hamsters (Fig 2b⇓), with an estimated IC50 of 90 μg·kg−1·h−1. We also performed time-dependent experiments (Fig 3⇓). The shorter treatment period was no longer effective, indicating that inhibition of platelet adhesion was still required even after the initial period in which thrombus formation is believed to occur. Photomicrographs of typical neointima formation under treatment with high-molecular-weight ATA at a dose of 100 μg·kg−1·h−1 and in nontreated controls are shown in Fig 4A⇓ and 4B⇓, respectively.
Proliferation Index of SMCs
Fig 5⇓ shows the ratio of proliferating SMCs in media on day 1 and in newly formed intima on day 7 after vascular injury. Treatment of hamsters with high-molecular-weight ATA caused a significant decrease in medial SMC proliferation and effectively inhibited the early proliferation of neointimal SMCs, measured on day 1 as a 55.8±10.3% decrease in proliferation index. To further characterize the antiproliferative activity of high-molecular-weight ATA, the direct effect on SMCs was investigated in the hamster SMC line DDT1MF2. The compound inhibited DNA synthesis in this cell line in a dose-dependent manner (Fig 6⇓).
Electron microscopic observation showed many activated platelets in the injured area (Fig 7B⇓). In contrast, there were no platelets on day 14 (Fig 7A⇓), and the number of adherent platelets was markedly decreased on treatment with high-molecular-weight ATA, even when a nonrepaired EC surface was observed (Fig 7C⇓). On vWF immunochemical staining, endothelial layers were clearly observed in noninjured vessels (Fig 8A⇓), and repaired endothelial layers were slightly observed on day 7 (Fig 8B⇓). In contrast, repaired endothelial layers were also clearly observed 7 days after injury when high-molecular-weight ATA was given continuously (Fig 8C⇓).
This study demonstrated that a platelet GP Ib antagonist, high-molecular-weight ATA fractionated from commercially obtained ATA,13 14 prevented thrombus formation and reduced neointima formation in an experimental stenosis model in hamster common carotid artery.
Upon binding to an injured vessel wall, platelets play an important role in both thrombus formation and excessive SMC proliferation that ultimately leads to the development of neointima formation. Indeed, recent evidence suggests that potent antiplatelet agents, ie, antagonists of GP IIb/IIIa, the major platelet receptor for fibrinogen, are able not only to decrease thrombosis but also to suppress neointima formation in animals12 and humans.10 However, GP IIb/IIIa antagonists interfere with neither the primary adhesion of platelets to adequate substrates in the injured vessel wall, such as collagen, nor the release of platelet contents, such as platelet-derived growth factor21 and basic fibroblast growth factor.22
Commercially obtained ATA is a potent inhibitor of the vWF–GP Ib axis8 and also inhibits fibrinogen binding to GP IIb/IIIa.23 Although ATA consists of a heterogeneous mixture of polymers of various molecular sizes (Mr=200 to >6000), the high-molecular-weight compounds inhibit vWF-mediated platelet activation more effectively than the lower-molecular-weight compounds.13 14 In hamsters, the IC50 of ATA (Mr=435) on in vitro platelet aggregation induced by botrocetin was 230 μg/mL, whereas thrombus produced by a catheter injury in vivo was prevented by infusion at a dose of 3.0 mg·kg−1·h−1 (data not shown). However, this dose produced marked bleeding in the surgical area and could not be used in long-term studies. We therefore separated commercial ATA and used the high-molecular-weight component in the present experiments because of greater selectivity and sensitivity to GP Ib.13
Our in vitro studies showed that high-molecular-weight ATA inhibited the platelet aggregation induced by botrocetin or ADP. However, the IC50 value with botrocetin was much lower than that with ADP. These results indicated that one could expect to find the inhibitory effect on GP Ib by use of an adequate dose in an in vivo study. Indeed, platelet aggregation induced by botrocetin ex vivo at the end of treatment with high-molecular-weight ATA (100 μg·kg−1·h−1) was inhibited, whereas that by ADP was not.
The results of ex vivo platelet aggregation indicated that the prolongation of time to occlusion in the injured hamster carotid artery represents the inhibitory effect by high-molecular-weight ATA (100 μg·kg−1·h−1) due to the prevention of vWF binding to platelet GP Ib on thrombus formation in vivo. When we administered the compound at a dose of 3.0 mg·kg−1·h−1, blood flow after injury continued for >30 minutes (data not shown), but bleeding time during infusion was markedly prolonged (10 times greater than the control value). Indeed, high-molecular-weight ATA at this dose inhibited both GP Ib and GP IIb/IIIa, because platelet aggregation ex vivo induced by both ADP and botrocetin was completely inhibited at the end of infusion (data not shown). These antithrombotic and hemorrhagic effects are of course closely related. In addition, our previous data showed that a selective GP IIb/IIIa antagonist prevented the development of thrombus formation in this model without extreme prolongation of bleeding time.24 These results support the possibility that the inhibition of GP IIb/IIIa is more effective than that of GP Ib in decreasing in vivo thrombus formation and that antagonism of both GP Ib and GP IIb/IIIa together induced marked hemorrhage.
Neointima formation, as evaluated 14 days after vascular injury in hamster carotid artery, was significantly reduced by continuous infusion of high-molecular-weight ATA (100 μg·kg−1·h−1). The neointimal suppression by the treatment for 7 days was somewhat similar to that by the treatment for 14 days. These results indicate that activated platelets adhering to injured vascular wall play an important role in the early phase of the vascular healing process; indeed, the proliferation index of SMCs was markedly decreased in the injured media by the treatment with high-molecular-weight ATA. The presence of activated platelets was closely related to the stimulation of SMC proliferation and migration, because selective GP IIb/IIIa antagonists also decreased the index of SMC proliferation 1 day after injury in media.19 Newly formed intima could be observed from day 5 onward, and thrombus formation associated with many activated platelets had almost disappeared by day 5 in this model.16 Continuous treatment with high-molecular-weight ATA decreased the SMC proliferation index in neointima on day 7 despite the absence of many adherent platelets. To further investigate this phenomenon, we measured DNA synthesis in cultured hamster SMCs. As a result, high-molecular-weight ATA showed a dose-dependent inhibition of DNA synthesis. The inhibitory effect on neointima formation in the late phase after vascular injury by the compound was therefore mainly the result of direct prevention of SMC proliferation. Indeed, the polymeric compound was reported to show an antiproliferative activity.25
Histological observations that high-molecular-weight ATA scavenged adherent platelets on nonrepaired endothelial surface supported our hypothesis. ECs started to recover quickly after vascular injury in this model,16 but repaired surfaces were not complete and smooth, resulting in residual shear stress in the injured area. This shear stress induced platelet adhesion, and the adherent platelets provoked further shear stress by the change in blood flow around the injured surface. Under these conditions, SMCs under the injured area are exposed to and stimulated by several kinds of growth factors. Moreover, treatment with the compound resulted in the enhancement of EC healing, because the staining of vWF was clearly observed on day 7. The rapid healing of ECs may present the end point of development of neointima formation.
Our findings indicate that high-molecular-weight ATA induces not only selective and strong inhibition of platelet adhesion in vivo and in vitro by the prevention of vWF binding to platelet GP Ib but also a decrease in neointima formation due to the inhibition of both platelet activation and SMC proliferation. These dual beneficial effects in the prevention of vascular stenosis open a new area of therapeutic research.
Selected Abbreviations and Acronyms
|IELA||=||internal elastic lamina area|
|SMC||=||smooth muscle cell|
|vWF||=||von Willebrand factor|
This work was supported by a research grant (SKK-74) from Yamanouchi Pharmaceutical Co, Ltd. We thank Drs Tomihisa Kawasaki and Osamu Inagaki of Yamanouchi Pharmaceutical Co and Dr Guy Harris for their assistance in the preparation of the manuscript.
- Received December 18, 1996.
- Revision received February 25, 1997.
- Accepted February 28, 1997.
- Copyright © 1997 by American Heart Association
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