Platelets play a key role in acute coronary syndromes. The underlying pathophysiologic mechanism is the formation of a platelet-rich thrombus at the site of a preexisting atherosclerotic lesion resulting in partial or complete occlusion of the artery (1). Improvement of platelet-inhibiting therapy has been shown to be crucial in the attempt of reopening the occluded vessel and thus minimizing myocardial damage. A new class of platelet aggregation inhibitors has been developed in the past years (2). These substances block the GPIIb/IIIa receptor—the common final pathway of platelet aggregation independent of the mechanisms of platelet activation. The first GPIIb/IIIa antagonist that has been widely tested in humans is abciximab (Reopro; Lilly Germany Company, Bad Homburg, Germany) (3,4,5). It represents the Fab fragment of a human–mouse chimeric monoclonal antibody against GPIIb/IIIa. Eptifibatide (Integrilin; Essex Pharma GmbH, München, Germany) (6) and tirofiban (Aggrastat; Merck Sharp and Dohme, Haar, Germany) are two low molecular weight synthetic GPIIb/IIIa antagonists. Eptifibatide has been shown to reduce death or myocardial infarction 30 days after an acute coronary syndrome (7). Tirofiban was proven to reduce the short-term rate of cardiac events after coronary angioplasty for unstable angina pectoris (8). All three GPIIb/IIIa antagonists are approved for clinical use and are beneficial in situations associated with platelet activation such as high-risk percutaneous transluminal coronary angioplasty (PTCA) (9) and thrombolysis for acute myocardial infarction (10). Some investigators have reported that abciximab administered as a high-dose bolus alone was able to reinstall coronary blood flow even without additional thrombolytic therapy or mechanical manipulation (11–14). For other GPIIb/IIIa antagonists, these effects have not been reported so far. In the present work, we investigated for the first time whether three GPIIb/IIIa antagonists that are often applied in the setting of acute coronary syndromes possess the potency to dissolve existing platelet aggregates and the potential mechanisms involved.
Unless otherwise mentioned, all chemicals were purchased from Sigma Chemicals Company (St. Louis, MO, U.S.A.).
For the determination of platelet aggregation, peripheral blood samples were collected from an antecubital vein of healthy volunteers into sodium citrate (final concentration, 0.011 M). Blood samples for the measurement of ex vivo platelet aggregation were centrifuged at 160 g for 10 minutes. The supernatant, platelet-rich plasma (PRP), was removed, and the remaining blood underwent a second centrifugation step at 2500 g for 10 minutes to obtain platelet-poor plasma. Platelet-rich plasma was diluted with autologous platelet-poor plasma to adjust platelet count to 250/nl. All procedures with PRP were performed at room temperature to avoid premature platelet activation.
Platelet aggregation was determined by light transmission in a four-channel aggregometer (PAP-4; Bio/Data Corporation, Horsham, PA, U.S.A.) as previously described (15). In brief, the aggregometer was adjusted before each test with light transmission of PRP corresponding to 0% and that of platelet-poor plasma corresponding to 100%. Platelets in 200 μl PRP at 37°C were stimulated by the addition of ADP (final concentration, 1 μM) and stirred at 900 g. Each aggregation curve was registered for a minimum of 5 minutes. Aggregation was quantified by increase in light transmission compared with initial levels (adjusted to 0). First, maximal aggregation was determined for each volunteer. In a next sample, GPIIb/IIIa antagonists in the described concentrations or NaCl as a control were added when the aggregation curve reached half of the expected maximal level. This was typically the case between 30 and 60 seconds after initiation of aggregation. Disaggregation potency (DP) was calculated as follows: DP = 100 − (final aggregation after addition of GPIIb/IIIa antagonist/maximum aggregation) × 100. Each experiment was performed in triplicates.
Plasma Clot Assay
The assay was performed as described elsewhere (16,17). In brief, fresh frozen plasma obtained from the blood bank was mixed with iodine I 125 fibrinogen (125I fibrinogen), 0.5 M CaCl2, and 8 U/ml thrombin in a silastic tubing. Standardized clots with the same 125I activity were prepared and, after washing, placed in 1.9 ml fresh plasma each. The GPIIb/IIIa antagonists were added to the plasma. NaCl and reteplase (Actilyse; Boehringer Ingelheim Pharma, Ingelheim, Germany), a tissue-plasminogen activator deletion mutant, were used as negative and positive controls, respectively. Radioactivity was measured in a gamma-counter before and at 3 hours and 24 hours after addition of agonists.
Cell Adhesion Assay
As a model for GPIIb/IIIa-related aggregation, Chinese hamster ovary (CHO) cells expressing the human GPIIb/IIIa receptor (CHO-GPIIb/IIIa) locked in the activated form were used. A stable cell line was created by cotransfection of CHO cells with a plasmid containing the wild-type 3 subunit and a plasmid containing a deletion of five amino acids of the intracellular tail of the alpha IIb subunit. These cells were shown previously to express the GPIIb/IIIa receptor in an activated state compared with CHO cells transfected with both wild-type subunits (18). Mutated GPIIb/IIIa receptors are disconnected from the cytoskeleton impairing inside-out and outside-in signaling. In this model, the interactions between fibrinogen and its receptor can be studied without interferences from signaling events or other platelet molecules. Cells were grown to 80% confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and 1% penicillin and streptomycin (15140122; Gibco, Invitrogen GmbH, Karlsruhe, Germany). Before each experiment, the existence and activity of the GPIIb/IIIa receptor was proven by flow cytometry.
Tissue culture dishes (Maxisorb; Nunc GmbH, Wiesbaden, Germany) were coated with human fibrinogen and blocked with bovine serum albumin (BSA) (1%). 1 × 105 cells were added to each well. Cells were incubated for 15 minutes on a tilting rocker at medium speed. Then wells were washed six times with phosphate-buffered saline to reject unbound cells. Adherent cells were counted and documented by photography. GPIIb/IIIa antagonists dissolved in phosphate-buffered saline as well as phosphate-buffered saline alone as a control were added and incubated for another 30 minutes under continuous rocking. After the second incubation period, adherent cells were recounted. Cell disaggregation potency (CDP) was calculated as follows: CDP = 100 − (cell counts after GPIIb/IIIa antagonist incubation/cell count before GPIIb/IIIa antagonist incubation) × 100. Experiments were performed in triplicates and in five individual sets of experiments.
Flow cytometry analysis was performed as described previously (19). In brief, CHO cells were detached and washed two times in modified Tyrode's buffer. Three hundred thousand cells per 50 μl Tyrode buffer were incubated with the appropriate antibody labeled to a fluorochrome for 20 minutes at room temperature in the dark. Analysis was performed using a FACScan with Lysis II software (both Becton Dickinson, Mountain View, CA, U.S.A.). The presence of the activated GPIIb/IIIa complex was shown with a fluorescein isothiocyanate (FITC)-conjugated activation-specific antibody (PAC-1). For the determination of the alpha IIb subunit (CD41) and the beta-3 subunit (CD 61), phycoerythrin (PE)-conjugated anti CD 41 and anti CD 61 antibodies were used, respectively. All antibodies were purchased from Becton Dickinson.
When appropriate, data were expressed as mean ± standard error of mean. For descriptive statistics and correlation analysis (Pearson product moment correlation coefficient), computing software (SigmaStat for Windows 1.0, Jandel Scientific, SPSS Science, Erkrath, Germany) was applied.
Platelet aggregation experiments were performed to investigate the potencies of three GPIIb/IIIa antagonists to dissolve platelet aggregates. Addition of 1 μM ADP to plasma of five healthy volunteers who had not taken any medication 14 days before the experiment resulted in maximal platelet aggregation within the first 4 minutes. GPIIb/IIIa antagonists were added after induction of platelet aggregation by ADP. After addition of GPIIb/IIIa antagonists, dissociation of the platelet aggregates occurred as measured by optical density of the sample that returned toward baseline (Fig. 1). The dosages of GPIIb/IIIa antagonists were chosen to cover a range that is reported to be achieved in clinical use of the respective drug (20–22).
The addition of increasing amounts of the respective GPIIb/IIIa antagonist after initiation of platelet aggregation resulted in increasing platelet disaggregation (Fig. 2). Tirofiban was used in a dose range from 0.0625 μg/ml to 0.5 μg/ml (0.126 μM–1.01 μM, all concentrations are final concentrations) and resulted in significant platelet disaggregation compared with controls (NaCl, 0.9%), ranging from 21.4% to 72.4% (P < 0.05 at medium and high concentrations). The same pattern could be observed after addition of eptifibatide in dosages ranging from 0.1875 μg/ml to 3.75 μg/ml (0.225 μM–4.51 μM), which led to disaggregation by 27.9% to 91.5%, respectively (P < 0.05 compared with controls at the highest concentration). The addition of abciximab in dosages ranging from 10 μg/ml to 50 μg/ml (0.21 μM–1.05 μM) was followed by platelet disaggregation of 14.8% to 48.4%, with the highest dose reaching significant difference compared with controls (P < 0.05). The highest dose of eptifibatide resulted in a significantly higher platelet disaggregation (92%) than the highest dose of abciximab (48.4%;P < 0.05).
To exclude proteolytic activity of the substances under investigation, we repeated the disaggregation experiments in the presence of aprotinin. Aprotinin alone had no effect on platelet aggregation induced by ADP. The addition of aprotinin before the induction of platelet aggregation did not change the platelet disaggregation results with or without GPIIb/IIIa antagonists (data not shown).
To further exclude intrinsic fibrinolytic activity of the investigated GPIIb/IIIa inhibitors, plasma clot assays were performed. Radiolabeled fibrin-rich plasma clots were created from fresh frozen plasma supplied with 125I-labeled fibrinogen by the addition of thrombin. After clot formation, clots of equal radioactivity were placed in fresh plasma with or without GPIIb/IIIa antagonists. The tissue plasminogen activator deletion mutant reteplase was used as a positive control. Radioactivity was measured in aliquots of the clot supernatant plasma taken before, at 3 hours after, and at 24 hours after placement of the clots in the plasma. All three GPIIb/IIIa antagonists as well as the negative control (NaCl) did not result in significant clot lysis, as indicated by low count numbers in the clot supernatant at all time points, whereas the positive control reteplase resulted in a significant increase in clot lysis (P < 0.05;Fig. 3). These experiments support the hypothesis that GPIIb/IIIa antagonists do not possess intrinsic fibrinolytic activity.
To confirm platelet aggregation results, we investigated GPIIb/IIIa-related aggregation in CHO cells expressing the activated GPIIb/IIIa receptor (CHO-GPIIb/IIIa). This model also allows for investigation of the interaction of fibrinogen with its receptor independent from signaling events that usually occur in platelets. Before each experiment, cells were monitored by flow cytometry for the presence and activity of GPIIb/IIIa. In Figure 4, a representative histogram of control CHO cells and CHO cells expressing the GPIIb/IIIa receptor in the activated form is depicted, as detected by the activation-specific antibody PAC-1. Compared with control cells, CHO-GPIIb/IIIa cells were able to bind to fibrinogen-coated tissue culture dishes. This binding of CHO-GPIIb/IIIa to the wells could be reversed significantly by all three GPIIb/IIIa antagonists (P < 0.05 compared with controls;Fig. 5).
The main goal in modern therapy for acute myocardial infarction is to reinstall coronary blood flow in the occluded coronary artery as soon as possible. Enzymatic fibrinolysis or interventional procedures such as balloon angioplasty or stent implantation are currently used to achieve this goal. It appears as if enzymatic fibrinolysis alone does not result in sufficient recanalization compared with interventional invasive procedures (23). Therefore, recent reports are significant in that they indicate a potential of the platelet GPIIb/IIIa antagonist abciximab to dissolve thrombi in vivo (11–14,24). In these observations in animals and humans with coronary thrombosis, the administration of abciximab alone resulted in restoration of blood flow of the occluded infarct-related artery. We tested the hypothesis that GPIIb/IIIa antagonists have the potency to dissolve platelet-rich arterial blood clots and we compared their efficacy to do so. All three GPIIb/IIIa antagonists analyzed in this study are currently approved for clinical use in patients: tirofiban, eptifibatide, and abciximab. Platelet aggregates were created by the addition of ADP to platelet-rich plasma in an aggregometer. Early addition of GPIIb/IIIa antagonists in clinically relevant doses not only prevented further platelet aggregation but also disaggregated freshly created platelet aggregates in a dose-dependent manner, ranging from no disaggregation at low concentrations up to significant disaggregation at high concentrations. The results presented here are supported by previous findings with the experimental GPIIb/IIIa antagonist SR 121566, which reverses platelet aggregation in a dose-dependent fashion when added early after platelet activation (25).
Maximal disaggregating effects were highest using eptifibatide, with a significant difference to abciximab at each of the highest concentrations. Several reasons for this difference may apply. First, the molar concentration of the highest dose of eptifibatide (4.51 μM) is higher compared with tirofiban (1.01 μM) and abciximab (1.05 μM). However, the dosages of the GPIIb/IIIa antagonists studied in our experiments were chosen according to therapeutic concentrations determined in vitro and in vivo, thus representing clinical practice (20–22,26). Second, the compounds used have different molecular weights, with tirofiban and eptifibatide being low molecular weight antagonists (MW 495 Da and 832 Da, respectively), whereas the antibody abciximab has a molecular weight of 47.600 Da. The GPIIb/IIIa receptors within preexisting platelet aggregates that are surrounded by a forming fibrin–fibrinogen network may be more accessible to smaller than to larger molecules, resulting in a higher disaggregation potency for the smaller agonists. Third, the differences in dissociation capacity may also be explained by the differences in receptor affinity. Abciximab binds with high affinity to the IIb/IIIa-receptor (KD = 5 n M), whereas the affinity of tirofiban and eptifibatide is substantially lower (KD = 15 n M and KD = 120 n M, respectively) (27). Although for the prevention of platelet aggregation the highest affinity may be advantageous, for disaggregating activities, a lower affinity could enable a compound to dissociate more molecules in a given time, as seen with eptifibatide. Fourth, interactions of the compounds other than with the IIb/IIIa receptor may account for the differences seen in our experiments. The interaction between fibrinogen and platelets (adhesion, aggregation, clot retraction) appears to involve receptors other than IIb/IIIa (28). Abciximab itself binds to several integrin receptors in addition to IIb/IIIa. However, whether this difference in receptor specificity accounts for the effects seen here remains speculative.
Because it was expected that disaggregation occurs by competitive inhibition at the extracellular fibrinogen binding domains of GPIIb/IIIa, a special emphasis was put on the exclusion of proteolytic activity in this setting. Fibrinolytic cleavage of cross-linking fibrinogen molecules would represent a different mechanism of action. The addition of the protease inhibitor aprotinin to the samples before induction of platelet aggregation had no influence on the platelet disaggregating activities of the tested GPIIb/IIIa antagonists. In addition, plasma clot assays were performed to rule out intrinsic fibrinolytic activity of the tested substances. As expected, GPIIb/IIIa antagonists did not result in lysis of a fibrin rich and platelet poor clot, whereas the control fibrinolytic reteplase did result in significant clot lysis. These results are in accordance to experiments by Collet et al. (28), in which perfusion of cross-linked clots with abciximab had no significant influence on clot size but facilitated clot lysis by a plasminogen activator.
Given the in vitro experimental setup of this study, additional effects of GPIIb/IIIa inhibitors, such as activation of endothelium-based fibrinolysis and reduction of thrombin generation, could be excluded (29). We also used a model of the activated GPIIb/IIIa receptor expressed in a stable CHO cell line to confirm further the results found in platelets. In this model, the GPIIb/IIIa complex is locked in the activated state by a deletion of the alpha subunit of the receptor. The receptor is detached from inside-out signaling, allowing for independent investigation of extracellular ligands or inhibitors. Using this model, all tested GPIIb/IIIa antagonists had the capability to detach cells attached to fibrinogen-coated surfaces. This set of experiments provides additional data, suggesting that the GPIIb/IIIa receptor complex is involved in the disaggregating activity of the agonists. They also indicate that extracellular GPIIb/IIIa antagonists have the potential to reverse fibrinogen binding to the GPIIb/IIIa receptor. Signaling events or changes of the receptor activity level do not appear to be involved in this process.
The capacity of GPIIb/IIIa antagonists to disaggregate fresh platelet aggregates but not older clots that are covered and organized by a fibrin network would provide characteristics that are ideal for the therapy of acute thrombotic diseases involving platelet-rich thrombi. At the site of a fresh platelet-rich clot, GPIIb/IIIa antagonists would be most active in dissolving the thrombus, but older fibrin-rich clots, for example, in the cerebral circulation, would not be affected. Thus, the thrombus-dissolving potency of GPIIb/IIIa antagonists is targeted to fresh platelet-rich thrombi and the risk–benefit ratio of a thrombus-dissolving therapy may be improved. These effects may explain why the intracerebral bleeding rate created by the therapy with GPIIb/IIIa antagonists alone is relatively low (9).
Our results indicate for the first time that all three GPIIb/IIIa antagonists investigated in this study have the potential to dissolve freshly created platelet aggregates. It appears as if the small molecular-weight compounds eptifibatide and tirofiban may have a higher disaggregation potential than the high molecular-weight compound abciximab. More studies are needed to elucidate further this potential difference between the substances. We could prove that the GPIIb/IIIa antagonists under investigation had no proteolytic or fibrinolytic activities. These results are consistent with the clinical observation that platelet disaggregation occurred only after early addition of GPIIb/IIIa antagonists, most likely because increasing fibrin cross-linking prevents disaggregation at later time points. This would be an additional reason for early therapy with this group of drugs.
We conclude from these experiments that the GPIIb/IIIa antagonists do not exhibit fibrinolytic activities, nor do they induce endogenous fibrinolysis. Direct competition between fibrinogen and GPIIb/IIIa antagonists at the receptor level is the most likely underlying mechanism.
Here we report for the first time that all three clinically applied GPIIb/IIIa antagonists are capable of dissolving platelet aggregates. This gives an explanation for clinical observations of early clot dissolution by abciximab alone and provides new information about the mechanisms of action of GPIIb/IIIa antagonists. The smaller size of eptifibatide and tirofiban compared with abciximab may even result in better penetration of platelet aggregates, resulting in increased disaggregation capacity. Lacking fibrinolytic activity, the platelet inhibitors most likely induce platelet disaggregation by direct competition with fibrinogen on the GPIIb/IIIa receptor level.
Given the low intracerebral bleeding risk of the administration of GPIIb/IIIa antagonists alone and their ability to dissolve fresh platelet aggregates, the possibility may raise that GPIIb/IIIa antagonists alone or in combination with heparin and aspirin could become a therapeutic option for early preclinical therapy of patients with acute coronary syndromes.
The authors thank Dr. Cam Patterson (Chapel Hill, NC, U.S.A.) for critical review of the manuscript and helpful comments, and Iris Dockhorn for excellent technical assistance with the experiments.
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