Ability of anti-glycoprotein IIb/IIIa agents to dissolve platelet thrombi formed on a collagen surface under blood flow conditions
Shinya Goto, MD, FACC*,*,
Noriko Tamura, BS* and
Hideyuki Ishida, PhD
* Department of Medicine, Tokai University School of Medicine, Kanagawa, Japan
Department of Physiology, Division of Cardiology, Tokai University School of Medicine, Kanagawa, Japan
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Figure 1 Experimental protocol. Fifteen ml of blood anticoagulated either by Argatroban or heparin containing platelets rendered fluorescent by the addition of mepacrine was perfused in a parallel-plate flow chamber composed of two glass plates, one of which was covered by immobilized type I collagen. A perfusion of an additional 15 ml of blood obtained from the same donors and treated by the same procedure, containing or not containing one of the anti-glycoprotein (GP) IIb/IIIa agents, was immediately started to perfuse on the same collagen surface for the same length of time (B). The two-dimensional and three-dimensional structures of the platelet thrombi formed on the collagen surface were continually assessed by fluorescence microscopy or by a laser confocal microscope controlled by a piezo-electric motor control system (A). In a laser confocal imaging system, the platelet thrombi were scanned from the bottom to the top (a') at a constant speed by controlling the position of objective lens (a) by piezo-motor control unit. Then, the scanning confocal images were projected from the top to the bottom at every 5° to obtain three-dimensional projection images, including projections from the top, 45° position from the horizontal axis and the side of the platelet thrombi, which are shown in Figures 3 and 4. The maximum height of the platelet thrombi (h) was calculated based on the projection image from the side of the platelet thrombi.
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Figure 2 Effects of anti-glycoprotein (GP) IIb/IIIa agents on platelet thrombus formation on the collagen surface under blood flow conditions. Fifteen ml of blood containing fluorescent platelets were perfused on the collagen surface for 6 min, either in the presence or absence of the anti-GP IIb/IIIa agents abciximab, eptifibatide, and tirofiban at the final concentrations shown in the figure. The results shown represent the mean and standard deviation of the eight sets of replicated experiments. The * and ** indicate values significantly lower than those in the absence of the anti-GP IIb/IIIa agents with the p value <0.05 and <0.01, respectively.
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Figure 3 Effects of anti-glycoprotein (GP) IIb/IIIa agents on three-dimensional platelet thrombus growth on the collagen surface. Experiments were performed as described in the legend for Figure 2, but the platelet thrombi were visualized by laser confocal microscopy. The projection images from the top (A), 45° position from the horizontal axis (B), and the side (C) are shown. Three-dimensional platelet thrombus growth occurred in the control group (left panel), whereas only a single layer of attached platelets was seen in the presence of any of the anti-GP IIb/IIIa agents tested (10 µg/ml abciximab, 0.5 µM eptifibatide, and tirofiban). The results shown are representative of the results of eight replicated experiments. The results with eptifibatide were similar to those obtained with abciximab and tirofiban, but are not shown because of space limitation. There were no differences in the results when the blood was anticoagulated by heparin. For the accompanying videos corresponding to Figure 3 (Videos 1, 2, and 3), please see the July 21 issue of JACC at www.cardiosource.com/jacc.html.
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Figure 4 Effect of anti-glycoprotein (GP) IIb/IIIa agents on the dissolution of platelet thrombi formed on the collagen surface. The experiments were performed as described in the legend for Figure 1B. Blood anticoagulated with Argatroban either containing or not containing anti-GP IIb/IIIa (abciximab: 10 µg/ml, eptifibatide: 0.5 µM, tirofiban 0.5 µM) was perfused on the collagen surface on which platelet thrombi had already formed as a result of previous perfusion of control blood. The three-dimensional projection images of the platelet thrombi (the projection images from the top [A], 45° position from the x axis [B], and the side [C]) before and after the second perfusion of blood containing (II) or not containing one of the anti-GP IIb/IIIa agents of abciximab (I) are shown in the left and right panel of I and II. The results are representative of those of eight replicated experiments. No differences in effects were observed among the three anti-GP IIb/IIIa agents tested. No differences in the effects of anti-GP IIb/IIIa agents were observed when blood anticoagulated with heparin was used. For the accompanying videos corresponding to Figure 4 (Videos 4, 5, 6, and 7), please see the July 21 issue of JACC at www.cardiosource.com/jacc.html.
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Figure 5 Effect of anti-glycoprotein (GP) IIb/IIIa agents on platelet thrombi formed on the collagen surface. The experiments were performed in a manner similar to that described in the legend for Figures 1 and 4. The three-dimensional structure of the platelet thrombi formed on the collagen surface after 6-min perfusion of blood on the collagen surface at 1,500 s–1 was quantified by calculating the cross-sectional areas of the platelet thrombi using the National Institutes of Health image software at every 3 µm from the collagen surface. The results are expressed as a percentage of the largest cross-sectional area obtained at the collagen surface level. The results shown are the mean and standard error of eight replicated experiments. Similar results were obtained in the condition when blood was anticoagulated by heparin.
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Figure 6 Speculative mechanism explaining the thrombus-dissolving effects of anti-glycoprotein (GP) IIb/IIIa agents. This figure summarizes the possible mechanism by which anti-GP IIb/IIIa agents dissolve platelet thrombi formed on a collagen surface, although it is not clear whether von Willebrand factor (VWF), in addition to fibrinogen, also plays some role in stabilizing platelet thrombi. When blood containing one of the anti-GP IIb/IIIa agents began to be perfused, ligand bound with activated GP IIb/IIIa was replaced by the anti-GP IIb/IIIa agent. Parts of the platelet thrombi started to become detached when a certain number of ligands were replaced by anti-GP IIb/IIIa and the strength of the GP IIb/IIIa ligation was no longer sufficient to support the integrity of the thrombi. Details are explained in the "Discussion" section.
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