© 1997 American Heart Association, Inc.
Articles |
Relative Involvement of GPIb/IX-vWF Axis and GPIIb/IIIa in Thrombus Growth at High Shear Rates in the Guinea Pig
From the INSERM U353 (P.A., L.O.D.); the Institut des Vaisseaux et du Sang (P.A., P.H., C.B. dit S., J.P.C., L.O.D.), Paris, France; and the Biotechnology General Ltd, Rehovot, Israel (L.I.G.).
Correspondence to Patrick André, Institut des Vaisseaux et du Sang, Hôpital Lariboisière, 8 rue Guy Patin, 75475 Paris Cedex 10, France.
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Abstract The relative involvement of the glycoprotein (GP) Ib/IX–von Willebrand factor (vWF) axis and GPIIb/IIIa in thrombus growth at high shear rates was assessed and compared by testing the pharmacological effects of VCL, a recombinant GPIb-binding fragment of vWF (residues 504-728), aurintricarboxylic acid (ATA), which binds to the 509-695 disulfide loop of vWF, and lamifiban, a specific synthetic GPIIb/IIIa antagonist. In vivo, their effects were evaluated in guinea pig mesenteric arteries, in a model of a laser-induced cyclic thrombotic process, and ex vivo, at a shear rate of 1800 s-1, in a capillary perfusion chamber model, in which collagen-adherent platelets are exposed to nonanticoagulated guinea pig blood. In vivo, VCL, ATA, and lamifiban administered 2 minutes after intimal injuries stopped thrombus growth, prevented the cyclic thrombotic process, and induced gradual thrombus dissolution. Ex vivo, at 1800 s-1, collagen exposure to untreated blood for 2 minutes, 4 minutes, or two consecutive periods of 2 minutes each resulted in similar platelet adhesion, 56%, 59%, and 61%, respectively, with an average thrombus volume of 6, 19, and 17.5 µm3/µm2, respectively, without any fibrin formation. This indicated that the two consecutive perfusions did not affect the dynamic process of thrombus growth. When collagen-adherent platelets deposited after the first 2-minute perfusion were perfused for 2 minutes with VCL-, ATA-, or lamifiban-treated blood, thrombus growth was prevented and platelet adhesion remained unchanged, but fibrin formation increased on and around the predeposited platelets. These results suggest that both the GPIb/IX-vWF axis and GPIIb/IIIa are involved in in vivo platelet-to-platelet interactions at high shear rates in the guinea pig.
Key Words: thrombus • von Willebrand factor • GPIb/IX • GPIIb/IIIa
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It is generally admitted that initial platelet adhesion and thrombus formation occurring at sites of damaged vessel wall at high shear rates are mainly mediated by von Willebrand factor (vWF), a high-molecular-weight multimeric glycoprotein present in plasma, endothelial cells, subendothelium, and platelets.1 Although it has been shown that at high shear rates, platelet adhesion to the subendothelium is mediated by vWF binding to the platelet membrane glycoprotein (GP) Ib/IX complex2 3 4 5 6 7 and that subsequent platelet spreading and aggregation is mediated by vWF binding to GPIIb/IIIa,8 9 10 11 12 13 14 15 some experiments have also suggested that in cone and plate viscometer models,16 17 18 the platelet aggregation induced by shear rates of 1500 to 2000 s-1 is initiated by shear-modified vWF binding to the GPIb/IX complex. This binding initiates a calcium influx and then activation of platelets that express functionally activated GPIIb/IIIa, which binds to vWF and leads to platelet aggregation.19 20
To establish whether at high shear rates vWF binding to GPIb
is involved in the secondary phase of thrombus growth (ie, the interactions between circulating platelets and activated deposited platelets), VCL (a recombinant peptide of the A1 domain of the vWF subunit that prevents vWF/GPIb binding and inhibits platelet adhesion and thrombus formation21 22 23 24 ) and aurintricarboxylic acid (ATA, which binds to the 509-695 loop of vWF25 and inhibits in vivo arterial thrombosis26 27 ) were tested and compared with lamifiban. The latter is a specific GPIIb/IIIa antagonist that has been shown to inhibit initial platelet adhesion and thrombus formation at high shear rates.28 29
Preventive and curative antithrombotic effects of VCL and lamifiban were sequentially tested in vivo in a model of laser-induced vessel wall damage to guinea pig mesenteric arteries30 and ex vivo in a capillary perfusion chamber, in which collagen or collagen-adherent platelets were exposed to nonanticoagulated guinea pig blood at 1800 s-1.28
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Recombinant VCL fragment (Biotechnology General Ltd) covering residues 504-728, which reproduced the A1 loop conformation of vWF, was prepared as previously described,21 and reconstituted in distilled water. Unfractionated commercial ammonium salt of ATA (Sigma Chimie) was dissolved in PBS, pH 7.4, and filtered through a 22-µm filter (Millipore).26 Lamifiban (Ro44-9883), kindly provided by Dr S. Roux (F. Hoffmann-La Roche, Basel, Switzerland), is a synthetic nonpeptidic GPIIb/IIIa inhibitor with an IC50 of 450 nmol/L in guinea pig platelet-rich plasma.31 The three molecules have been found to be effective in previous experimental thrombosis studies in the guinea pig.24 26 28 29
Animal Selection and Anesthetic Administration
Experiments were conducted in accordance with the legislation of the French Ministry of Agriculture, the guidelines of INSERM (Institut National de la Santé et de la Recherche Médicale), the institutional policies of INRA (Institut National de la Recherche Agronomique), and the international laws and policies stated by Giles32 in the Guidelines for the Use of Animals in Biomedical Research. Male Hartley strain guinea pigs (Saint Antoine, Pleudaniel, France) weighing 450 to 550 g were anesthetized with 50 mg/kg sodium thiopental (Nesdonal, Specia Rhône-Poulenc). Forty guinea pigs served for in vivo thrombosis study (20 for the preventive study, and 20 for the curative study) and another 65 were used for the ex vivo perfusion chamber study.
Laser-Induced Cyclic Thrombotic Process
In vivo thrombosis was induced by a pulsed nitrogen laser (Sopra) pumping a dye laser tuned to 427 nm, the wavelength of the first absorption peak of hemoglobin, as previously described.30 Briefly, guinea pigs were anesthetized with sodium thiopental, and a 3-cm incision was made through the abdominal wall to expose the mesenteric arteries. The dye laser beam was then focused on a 100-µm-diameter section on the adventitial side of the mesenteric arterial vessel wall, and 30 pulses at a repetition rate of 1.5 Hz, each delivering an energy of 30 µJ, were applied on each mesenteric artery studied. The arterial (shear rate >1000 s-130 ) thrombotic response to the vessel wall lesion was observed in real time and followed for 15 minutes. The total number of recurrent thrombi formed and the time of presence, ie, the time between appearance and disappearance, of the first thrombus during the 15-minute period of observation were the thrombotic parameters recorded, to compare the antithrombotic effects of VCL and lamifiban. Boluses of saline, VCL, ATA, and lamifiban were injected intravenously 10 minutes before laser injury (n=20) or 2 minutes thereafter (n=20), allowing a normal initial thrombotic process to occur.
Capillary Perfusion Chamber Studies
Ex vivo perfusions were performed with the previously described capillary perfusion chamber model used in our laboratory.28 Blood flow was fixed at 2.65 mL/min to reconstitute a shear rate of 1800 s-1 in glass capillaries with a radius of 0.315 mm. Human type III collagen (Sigma Chimie) was purified and allowed to polymerize and form fibrils, as previously described.33 The internal surface of the capillaries was prepared for coating as previously indicated, to reach a final density of 
2.6 µg/cm2.28 Perfusions were carried out at 37°C using an occlusive roller pump (2120 Varioperpex(R)II Pump, LKB Bromma), which was positioned distally to the chamber and drew nonanticoagulated blood directly from the abdominal aorta. The aorta was punctured with a 19-gauge butterfly infusion set (Venisystems, Abbott Lab). This set was connected through a T-piece shunt with silicone elastomer tubing previously filled with rinsing buffer (130 mmol/L NaCl, 2 mmol/L KCl, 12 mmol/L NaHCO3, 2.5 mmol/L CaCl2, 2H2O, 0.9 mmol/L MgCl2, 6H2O, and 5 mmol/L glucose, pH 7.4) at 37°C to avoid any contact with air. Chambers were perfused at 1800 s-1 with nonanticoagulated blood drawn from control untreated guinea pigs either with a single perfusion for 2 minutes (n=5) or 4 minutes (n=5) or with two consecutive perfusions of 2 minutes each (n=10). Chambers were also perfused for 4 minutes with blood from guinea pigs injected 10 minutes previously with either 4 mg/kg VCL (n=5), 10 mg/kg ATA (n=5), or 3 mg/kg lamifiban (n=5). For the experiments with two different animals, collagen-adherent platelets deposited in the capillary chambers after a 2-minute perfusion of untreated blood were rinsed but not fixed (n=15) and were immediately exposed for 2 minutes to blood drawn from guinea pigs that were previously injected with either 4 mg/kg VCL (n=5), 10 mg/kg ATA (n=5), or 3 mg/kg lamifiban (n=5). At the end of the experiments, capillaries were rinsed, fixed, postfixed, and embedded in epoxy resin as previously described.28
To study platelet-capillary surface interactions, 1-µm semithin cross-sections were cut for light microscopic computer-assisted morphometry at an axial position of 5 mm downstream from the flow inlet at the capillary and perpendicular to the direction of blood flow. Computer-assisted morphometry with a PC 486DX33 computer (Elonex) and a LUCIE program (Microvision, Evry, France) was used to quantify the percentage of surface coverage with platelets (platelet adhesion) and the average thrombus volume according to the method of Sakariassen et al.34
Statistical Analysis
Differences in the number of thrombi and the time of presence of the first thrombus were analyzed using ANOVA and the Dunnett tests. Differences in the percentage of capillary surface covered with platelets and average thrombus volume were analyzed using multifactor ANOVA. A value of P<.05 was considered significant. Data were expressed as mean±SEM.
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In Vivo Study
In the in vivo model, injection of 4 mg/kg VCL, 10 mg/kg ATA, or 3 mg/kg lamifiban 10 minutes before injury prevented the cyclic thrombotic process by reducing the number of thrombi from 3.3±0.2 for the saline-treated group to 1, 1, and 0, respectively. VCL and ATA treatment increased the time of appearance and time of presence of the only thrombus formed. This thrombus did not reach occlusive size, gradually dissolved, and never reformed.
IV injection of saline 2 minutes after injury did not modify the cyclic thrombotic process. For VCL, ATA, and lamifiban injection 2 minutes after injury, the cyclic thrombotic phenomenon was prevented (Fig 1A
), and the times of presence of the single preformed thrombus increased, 275±30 seconds for control, 585±35 seconds (P<.001) for 4 mg/kg VCL, 690±50 seconds (P<.001) for 10 mg/kg ATA, and 457±31 seconds (P<.001) for 3 mg/kg lamifiban (Fig 1B). The time of presence was slightly longer in the VCL- and ATA-treated groups than in the lamifiban-treated group, because after VCL and ATA injections, the thrombus continued to grow for 2 and 3 minutes, whereas its growth was quickly stopped by lamifiban (Fig 2). Each treatment enhanced similar gradual dissolution and disappearance of the nonocclusive thrombus within 10 minutes (Fig 2).
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Ex Vivo Study
Ex vivo, perfusing native untreated blood for 2 or 4 minutes through a collagen-coated capillary at 1800 s-1 induced similar platelet adhesion but different thrombus volume values (Fig 3A and 3B). Two consecutive native untreated blood perfusions of 2 minutes each and one continuous perfusion of untreated native blood for 4 minutes induced similar platelet adhesion and thrombus volume values (Fig 3A and 3B).
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VCL-, ATA-, and lamifiban-treated blood perfused for 4 minutes through collagen-coated capillaries exhibited a significant reduction of platelet adhesion (29±5%, 25±4%, and 22±7%, respectively, versus 59±6% for control) and average thrombus volume (7±2.5, 6±2, and 6±1.5 µm3/µm2, respectively, versus 19±3.5 µm3/µm2 for control).
When VCL-, ATA-, or lamifiban-treated blood was perfused for 2 minutes through a collagen-coated capillary previously perfused for 2 minutes with untreated blood, there were no changes in platelet adhesion, but thrombus growth was prevented (Fig 3A
and 3B).
Minor fibrin formation was found around thrombotic deposits either after one perfusion of 2 or 4 minutes of untreated blood or two consecutive perfusions of 2 minutes each of untreated blood. When the second perfusion was performed with VCL-, ATA-, or lamifiban-treated blood, a thin fibrin meshwork was observed on and around the platelets adhering to the collagen-coated capillary surface (Fig 4A through 4E).
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Discussion |
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GPIIb/IIIa inhibitors and GPIb/IX-vWF axis inhibitors were recently shown to prevent the process of thrombotic occlusion in in vivo arterial thrombosis models.35 36 37 38 39 The antithrombotic activities of these compounds were explained by the interdependence of GPIb
and GPIIb/IIIa in platelet adhesion and aggregation.40 This interdependence has been shown in cone-and-plate viscometer models in which aggregation induced by high shear rates (1500 to 2000 s-1) was inhibited by the blockade of both GPIb/IX and GPIIb/IIIa.16 17 18 vWF binding to GPIb initiates a transmembrane flux of calcium ions that leads to platelet activation and irreversible binding to platelets via GPIIb/IIIa.19 20 41 42 43 Here we studied the inhibition of the GPIb/IX-vWF axis and GPIIb/IIIa separately with VCL, ATA, and lamifiban, in an in vivo and an ex vivo thrombosis model. In vivo, the thrombogenic reactive surface was either a damaged arterial wall or an active growing thrombus initiated by laser-induced intimal injuries of the guinea pig mesenteric artery and ex vivo, either collagen or collagen-adherent platelets, in a capillary perfusion chamber model. The three compounds exhibited potent antithrombotic effects when they were injected before the intimal injuries in the in vivo model. Whatever the treatment in the ex vivo model, the perfusion of nonanticoagulated treated blood produced similar quantitative antithrombotic effects but different qualitative effects (3 mg/kg lamifiban reduced platelet adhesion but increased the contact/spread ratio in platelets previously shown28 ).
When VCL and ATA were injected 2 minutes after injury in the in vivo model, it prevented the cyclic thrombotic phenomenon but did not block thrombus growth immediately, as slight growth continued for another 2 and 3 minutes, in contrast with the immediate antithrombotic effect of lamifiban. Each compound induced a similar gradual dissolution of the thrombus, indicating that thrombus formation results from a permanent balance between growth and dissolution. Transmission electron microscopy studies have revealed that the luminal thrombus formed in this model in <5 minutes was a platelet-rich thrombus with a thin fibrin meshwork, which might explain the rapid thrombus lysis observed (unpublished data). This balance between growth and dissolution might be due to normal hemostasis, which acts to limit the intraluminal extension of an arterial parietal thrombus formed at the site of vascular injury.44 In our ex vivo model, we showed that two consecutive blood perfusions of 2 minutes each or one continuous perfusion of 4 minutes through a collagen-coated capillary led to similar platelet adhesion and thrombus volume values, with limited fibrin formation, indicating that the two consecutive perfusions did not affect the dynamic process of thrombus growth. At a shear rate corresponding to moderately stenotic arteries (1800 s-1), both glycoproteins appeared to be necessary to mediate the interactions between circulating platelets and collagen-adherent platelets in native nonanticoagulated blood. One hypothesis might be that high shear forces are present at the apex of an arterial thrombus and that the thrombotic process is similar to the one generated in the cone-and-plate viscometer model. Thus, a fixed thrombogenic support might be associated with the mechanism of shear-induced vWF binding to platelets as described by Goto et al.43
The absence of thrombus lysis in our ex vivo model might be due to the higher shear rates, the short period of exposure to treated blood (2 minutes), and the fact that the thrombogenic surface is not a vessel wall, which plays a key role in the thrombotic and dethrombotic processes.
We have shown that blocking vWF binding to GPIb did not abolish platelet adhesion and first thrombus formation in the preventive study. We have also shown that when the thrombus disappeared, the thrombotic process did not reappear in the next 3 hours, whereas normal thrombotic responsiveness is restored 1.5 hours after the bolus injection (unpublished data). This might indicate that the luminal thrombotic surface exposed to arterial blood flow after the thrombus dissolution is probably less thrombogenic than the original damaged vessel wall. The weak thrombogenic reactivity of the luminal surface of the platelets implicated in the parietal thrombus might explain this apparent nonthrombogenicity,44 but several plasmatic components of circulating blood have also been suspected of interfering in the thrombogenic reactivity of the exposed surface.45 46 47 In this study, 3 hours after the injuries, optical microscopy observation of the damaged area has revealed a parietal thrombus that was not extended by a luminal thrombus. Transmission electron microscopy analysis of the luminal surface of the parietal thrombus revealed nonactivated platelets implicated in a moderate fibrin meshwork (unpublished data). In our ex vivo study, it was interesting to observe that perfusing treated blood after untreated blood at high shear rates induced the formation of a thin fibrin meshwork on the collagen surface and collagen-adherent platelet surface. This phenomenon was also observed, but at low shear rates, by others48 and was explained by the procoagulant activity of the platelet phospholipid surface exposed to the blood flow.49 Experiments are currently in progress to evaluate the possible part played by fibrin deposition in reducing the thrombogenicity of a highly thrombogenic surface under high shear rates.
All our observations lead us to conclude that inhibition of both GPIIb/IIIa and the GPIb/IX-vWF axis is a major goal in antithrombotic therapy, because it prevents occlusive thrombus formation, inhibits both platelet adhesion and platelet-to-platelet interactions at high shear rates, and also restrains early rethrombosis at arterial shear rates.
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Acknowledgments |
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During this study, Patrick André was the recipient of a scholarship from the Sanofi Thrombose Foundation, Paris, France. We thank Drs Matthew Konneh and Sébastien Roux for their expert assistance.
Received April 25, 1996; accepted July 26, 1996.
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