VCL, an Antagonist of the Platelet GP1b Receptor, Markedly Inhibits Platelet Adhesion and Intimal Thickening After Balloon Injury in the Rat
Background Arterial injury is immediately followed by platelet adhesion at the site of injury, a process that requires the interaction of subendothelial von Willebrand factor with the platelet GP1b receptor. VCL, a recombinant von Willebrand factor GP1b binding domain, inhibits platelet binding to von Willebrand factor. The aim of this study was to determine whether VCL inhibits platelet adhesion at the site of arterial injury and affects neointimal thickening after injury in rats.
Methods and Results Sprague-Dawley rats were randomized to receive VCL, 4 mg/kg bolus followed by a continuous infusion of 2 mg · kg−1 · h−1 for 72 hours, or an identical volume of saline. Balloon injury of the femoral artery was performed 15 minutes after the initial bolus injection of VCL. Scanning electron microscopy performed 1 and 3 days after injury indicated that VCL-treated rats had >80% reduction in the number of platelets adherent to the vessel wall at the site of injury compared with controls (P<.003). Histological examination at day 14 showed that, compared with controls, VCL-treated rats had a 60% reduction in the intima-media ratio (0.21±0.03 versus 0.53±0.06, P=.001) and a reduced luminal area stenosis (12±3% versus 38±10%, P=.04). At 28 days after injury, there was no rebound of neointimal thickening in VCL-treated rats (intima-media ratio, 0.19±0.04; luminal stenosis, 17±5%). The difference between VCL-treated rats and control rats persisted but was attenuated (intima-media ratio, 0.19±0.04 versus 0.28±.0.1, P=.162; luminal stenosis, 17±5% versus 31±5%, P=.058) as neointimal thickening regressed in untreated rats. With the use of proliferating cell nuclear antigen immunohistochemistry on day 3, VCL had no effect on smooth muscle cell (SMC) proliferation.
Conclusions Antagonism of the platelet GP1b receptor by VCL profoundly decreased platelet deposition at the site of balloon injury in the rat femoral artery. This effect was associated with a persistent reduction in neointimal thickening. The lack of effect of VCL on SMC proliferation suggests that the decrease in neointimal thickening may have been mediated through inhibition of SMC migration and/or modulation of the extracellular matrix.
Platelets adhere to the injured arterial wall immediately after balloon injury.1 2 Platelet adherence and aggregation are followed by degranulation and the release of various mitogens, which may contribute to the proliferation and migration of smooth muscle cells (SMCs), characteristic of the vascular response to injury.3 4
The interaction of von Willebrand factor with the platelet GP1b receptor is essential for platelet adhesion, the necessary first phase in platelet activation.5 6 7 Pigs that are congenitally deficient in von Willebrand factor have reduced magnitude of thrombus formation at sites of arterial injury8 and less neointimal thickening after balloon injury.9 VCL is a recombinant monomeric fragment of von Willebrand factor containing the GP1b binding domain (Leu-504–Lys-728, one disulfide bond), produced in Escherichia coli. It inhibits botrocetin- and ristocetin-induced platelet aggregation,10 inhibits platelet adhesion to the damaged arterial wall, and reduces platelet deposition in a rat model of arterial thrombosis.11 VCL has also been shown to abolish cyclic flow variations in stenosed coronary arteries of nonhuman primates12 and to facilitate sustained reperfusion in a canine model of tissue-type plasminogen activator–induced thrombolysis.13
We studied the effect of VCL on platelet deposition at the site of arterial injury and on the vascular response to balloon injury in the femoral artery of the rat.
This study conformed to the guiding principles of the American Physiological Society and was approved by the Institutional Animal Care and Use Committee.
Animals and Surgical Preparation
Adult male Sprague-Dawley rats, weighing 400 to 500 g, were anesthetized with subcutaneous sodium pentobarbital (40 mg/kg). A PE-50 catheter was inserted into the left jugular vein for drug administration and exteriorized in the interscapular region. After intravenous administration of heparin (10 U/kg), a 2F balloon catheter was advanced through a left carotid cutdown into either femoral artery. The femoral artery was exposed, and the catheter was advanced just proximal to the first branch of the femoral artery, inflated for 1 minute, and withdrawn into the aorta. The inflation was repeated three times, and the rats were allowed to recover.
At the time of tissue harvesting, 14 and 28 days after injury, rats were anesthetized and killed by exsanguination, followed by pressure fixation with 1% glutaraldehyde at physiological pressure. The injured femoral artery was removed and placed in 2% glutaraldehyde. All arteries were cut at 2-mm intervals within 6 hours and stored in 70% alcohol until they were embedded in paraffin and sectioned for histological evaluation.
Rats were randomized to either VCL (4 mg/kg bolus 15 minutes before balloon inflation, followed by a continuous infusion of 2 mg · kg−1 · h−1 for 72 hours by an infusion pump) or an equivalent volume of saline. Four series of experiments were conducted.
Group 1 consisted of 14 rats, 6 given VCL and 8 given saline, that were killed on day 14 after injury. Cross sections of the injured arterial segments were stained with hematoxylin and eosin, and computer-assisted planimetry was used to measure luminal, intimal, and medial areas. Neointimal thickness was expressed as the ratio of the intimal and medial areas.
Group 2 consisted of 11 rats, 5 given VCL and 6 given saline, that were killed 28 days after injury. Injured arteries were processed as for group 1.
Group 3 consisted of 8 rats, 4 given VCL and 4 given saline. Two rats from each treatment group were killed either 1 or 3 days after injury. Platelet adhesion to the injured arterial wall was assessed in this group by scanning electron microscopy (SEM).
Group 4 consisted of 9 rats, 3 given VCL and 6 given saline. These rats were killed 3 days after injury, and immunohistochemistry for proliferating cell nuclear antigen (PCNA) was performed in the histological cross sections. This was done to determine whether an observed effect on neointimal thickening reflected a change in SMC proliferation.
PCNA immunohistochemistry14 was performed with mouse monoclonal anti-PCNA antibody as the primary antibody. The secondary antibody was horseradish peroxidase–conjugated rabbit anti-mouse immunoglobulin; the tertiary antibody was horseradish peroxidase–conjugated swine anti-rabbit immunoglobulin. The negative control was mouse anti–human factor VIII antibody. Rat small intestine served as positive control. All antibodies were obtained from Dako and were used in 1:50 dilution. Slides were counterstained with hematoxylin. Positive cells were counted in all sections of the distal femoral artery, and the number of positive cells was expressed as the average for each rat and for treated or control groups.
Scanning Electron Microscopy
The femoral artery was opened longitudinally and cut into three or four segments. The segments were fixed in 2.5% glutaraldehyde, rinsed in phosphate buffer, and dehydrated in ethanol. Samples were then immersed in Hexamethyldisilazane, mounted, and coated with gold-palladium for scanning in an S405 Hitachi scanning electron microscope under a magnification of ×1500. A “blinded” investigator examined multiple fields, and the number of adherent platelets per field was expressed for each vessel as the average of 10 fields.
These experiments were conducted to exclude a direct effect of VCL on SMC.
Isolation and Culture of SMCs
SMCs were isolated from normal aortic media obtained from a cardiac transplantation donor. Tissue specimens were carefully dissected, cut into small pieces, and allowed to attach to the surface of six-well multiplates by drying for 15 minutes. The explants were then cultivated in Ham’s F-12 medium containing 10% FCS and 50 μg/mL gentamycin at 37°C in an atmosphere of 5% CO2 in air. Cells began migrating out from the explants within 1 to 2 weeks and reached confluence within another 2 weeks. Secondary cultures were established by trypsinization and seeding of the cells in 75-cm2 culture flasks. The purity of the SMC populations was determined by the presence of muscle-specific α-actin immunoreactivity by use of the HHF35 antibody.15
Analysis of DNA Synthesis
The growth of subconfluent cultures of SMCs in passage 9 grown in 22×22-mm glass coverslips in six-well plates was arrested by transfer to serum-free medium (Ham’s F-12 containing 0.1% BSA) for 48 hours. The cells were then exposed to 2 μCi/mL 3H-thymidine in Ham’s F-12 medium containing 0.1% BSA, 1% FCS, or 10% FCS for another 48 hours with or without the addition of 150 μg/mL VCL. This concentration of VCL was chosen on the basis of the calculated serum concentration achieved in the average rat after bolus intravenous injection of 4 mg/kg. Cells were then fixed in 1% buffered paraformaldehyde, dehydrated in ethanol, and mounted on glass slides. The slides were dipped in Kodak NTB2 emulsion, air-dried, and exposed at 4°C for 3 days. The fraction of labeled nuclei (labeling index) was determined by counting in triplicate at least 300 randomly selected cells in each coverslip.
An unpaired Student’s t test was used to compare results in VCL and saline-treated rats. Data are expressed as mean±SEM, and a value of P<.05 was considered significant.
Scanning electron microscopy 1 day after balloon injury showed that the number of adhering platelets per field in control and VCL-treated rats was 81±1 versus 14±3, respectively (83% reduction, P<.002). Three days after injury, the number of adhering platelets was 126±5 versus 15±3, respectively (88% reduction, P<.003; Fig 1⇓).
Morphometric analysis of injured vessels 14 days after balloon injury revealed a 60% reduction in the intima-media ratio (0.21±0.03 versus 0.53±0.06, respectively; P=.001) and less luminal stenosis (12±3% versus 38±10%, respectively; P=.04) in VCL-treated rats compared with control rats (Fig 2⇓). There was no late increase in intima-media ratio (0.19±0.04) or luminal stenosis (17±5%) at 28 days in rats treated with VCL. In untreated rats killed 28 days after injury, the intima-media ratio and luminal stenosis were 52% and 18% less, respectively, compared with those in untreated rats killed 14 days after injury (Fig 3⇓).
VCL was tolerated well by the rats, and no bleeding complications were encountered.
The number of PCNA-positive cells on day 3 after injury was similar in control and VCL-treated rats (27±2 versus 21±9, respectively; P=.5).
Effect of VCL on DNA Synthesis and Viability of Cultured Arterial SMCs
In serum-starved SMCs incubated with serum-free medium containing 0.1% BSA and 3H-thymidine for 48 hours, the labeling index was 6±1%. Exposure of cells to medium containing 1% and 10% FCS increased the labeling index to 23±2% and 90±1%, respectively (P<.001 BSA versus 1% FCS and 1% FCS versus 10% FCS). As Fig 4⇓ shows, the addition of VCL did not change the labeling index significantly with either 1% or 10% FCS (28±1% and 91±2%, respectively; P=.5 VCL-treated rats versus control). VCL did not have an adverse effect on SMC viability as determined by trypan blue exclusion.
This study demonstrates that VCL, a platelet GP1b receptor antagonist, profoundly inhibits both early platelet adhesion and late intimal thickening in the balloon-injured rat femoral artery. Because SMC proliferation was unaffected by VCL treatment, inhibition of cell migration or matrix deposition may have been the mechanism involved.
Von Willebrand factor is essential for platelet–vessel wall interaction.16 VCL was previously shown to inhibit platelet adhesion to the injured vessel wall and to human umbilical artery subendothelium ex vivo, particularly at high shear rates.11 17 18 Our data indicate that this effect persists even under the low shear rate conditions (<500 second−1) characteristic of large arteries.8 This observation may be explained, at least in part, by the findings of Badimon et al,8 who showed that von Willebrand–deficient pigs have a defect in platelet–vessel wall interaction even at low shear rates when given heparin. In our study, the rats were treated with heparin. These observations may become relevant where aggressive heparinization is used, as in angioplasty, conditions of high shear rate, or vessels narrowed by atherosclerosis.16
The reduction in platelet deposition by VCL was accompanied by a marked decrease of neointimal thickening. Because inhibition of platelet deposition was not complete in our study, it is possible that higher doses of VCL would inhibit platelet adhesion completely and reduce neointimal formation even further. Our data, however, do suggest that complete inhibition of platelet deposition is not a prerequisite for a marked inhibition of neointimal thickening after arterial injury. In dogs treated with dual thromboxane A2 synthetase inhibitor and receptor antagonist and serotonin S2 receptor antagonist, Willerson et al19 showed a correlation between the frequency and severity of cyclic flow variations and the severity of coronary neointimal thickening. Animals without cyclic coronary flow variations during the first 7 days after instrumentation had the most pronounced reduction of neointimal thickening at 21 days.
Thrombocytopenia is known to inhibit intimal thickening after balloon injury.20 21 22 Fingerle et al22 used polyclonal antiplatelet antibody to produce thrombocytopenia in rats for 24 to 72 hours. Although treated rats demonstrated reduced neointimal formation 4 and 7 days after injury, the degree of SMC proliferation was not different in thrombocytopenic and control rats. These results, like ours, suggest that the inhibition of SMC proliferation is not the mechanism of suppression. Potential alternative mechanisms by which VCL could inhibit neointimal thickening include suppression of platelet-derived growth factor–stimulated SMC migration23 24 and modulation of the extracellular matrix, which is important in regulating cell migration.25
In a study by Fingerle et al,22 the effect on neointimal thickening was no longer evident on day 14 after injury. In contrast, we observed persistent reduction of neointimal formation on days 14 and 28 after injury. This difference may be explained by the longer duration of treatment in our study. Indeed, in the study of Fingerle et al, platelet adhesion to the injured arterial wall was markedly reduced at 24 hours, but by 48 hours, as the platelet count recovered, platelet adhesion was similar to that in controls. In our study, platelet adhesion was suppressed at least for the duration of the 3-day course of VCL administration.
At 28 days after injury, the difference in neointimal thickening between control and VCL-treated rats was not significant because of the decrease in neointimal thickening in control rats. The decrease of neointimal thickening between days 14 and 28 after injury was previously shown to occur in injured arterial segments with complete endothelial regrowth.26 Indeed, in our model, reendothelialization was complete in rats killed 14 days after injury (data not shown).
Although various antiplatelet agents27 have been shown to inhibit neointimal thickening after experimental balloon injury, clinical studies have been negative.28 29 30 These studies may have been limited by insufficient drug doses. A recent study with an antibody against the IIb/IIa receptor reported a reduction of restenosis.31 Because platelets are activated immediately after injury, prevention of adhesion, the initial step of activation, may prove more useful than interventions aimed at blocking platelet activation or degranulation.
The absence of any bleeding complications during the 3-day infusion of VCL is similar to the experience of McGhie et al,12 who also found no bleeding in nonhuman primates treated with VCL. The reported prolongation of bleeding time and reduction of platelet count in their study may become of significance when VCL is used in conjunction with other anticoagulants or thrombolytic agents.
In conclusion, VCL, the platelet GP1b receptor antagonist, profoundly reduced both platelet adhesion and late intimal thickening after balloon injury of the rat femoral artery.
This work was supported in part by the Grand Sweepstakes Foundation. Dr Zahger was the William Ganz Research Fellow of the Save A Heart Foundation. We are indebted to Federico Calara, Peter Shintaku, PhD, and Loyda Nolasco for their excellent technical assistance.
Reprint requests to Bojan Cercek, MD, Division of Cardiology, Room 5314, Cedars-Sinai Medical Center, 8700 Beverly Blvd, Los Angeles, CA 90048.
- Received February 1, 1995.
- Revision received February 28, 1995.
- Accepted February 28, 1995.
- Copyright © 1995 by American Heart Association
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