Published online before print December 14, 2006, doi: 10.1161/01.ATV.0000255307.65939.59
© 2007 American Heart Association, Inc.
Thrombosis |
A Mechanistic Model for Paradoxical Platelet Activation by Ligand-Mimetic 
IIbß3 (GPIIb/IIIa) Antagonists
From the Centre for Thrombosis & Myocardial Infarction (N.B., C.E.H., S.U.E., K.P.), Baker Heart Research Institute, Melbourne, Australia; the Department of Cardiology (C.L., M.S., I.A., C.B.), Albert-Ludwigs-University, Freiburg, Germany; and the Australian Centre for Blood Diseases (P.M., Y.Y., S.P.J.), Monash University, Melbourne, Australia.
Correspondence to Karlheinz Peter, MD, Baker Heart Research Institute, PO Box 6492 St Kilda Road Central, Melbourne, Victoria 8008, Australia. E-mail karlheinz.peter{at}baker.edu.au
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Abstract |
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Objective— Integrins are attractive therapeutic targets. Inhibition of integrin
IIbß3 effectively blocks platelet aggregation. However, limitations with intravenous IIbß3 antagonists and failure of oral IIbß3 antagonists prompted doubts on the current concept of ligand-mimetic integrin blockade.
Methods and Results— Evaluating P-selectin expression on platelets by flow cytometry, we report a mechanism of paradoxical platelet activation by ligand-mimetic IIbß3 antagonists and define three requirements: (1) Induction of ligand-bound conformation of IIbß3, (2) receptor clustering, (3) prestimulation of platelets. Conformational change is inducible by clinically used ligand-mimetic IIbß3 antagonists, RGD-peptides, and anti-LIBS antibodies. In a mechanistic experimental model, clustering is achieved by crosslinking integrins via antibodies, and preactivation is induced by low-dose ADP. Finally, we demonstrate that platelet adhesion on collagen represents an in vivo correlate of platelet prestimulation and receptor clustering, in which the presence of ligand-mimetic IIbß3 antagonists results in platelet activation as detected by P-selectin, CD63, and CD40L expression as well as by measuring Ca2+-signaling. Blockade of the ADP receptor P2Y12 by AR-C69931MX and clopidogrel inhibits IIbß3 antagonist-induced platelet activation.
Conclusion— These findings can explain limitations of ligand-mimetic anti-IIbß3 therapy. They describe potential benefits of concomitant ADP receptor blockade and support a shift in drug development from ligand-mimetic toward allosteric or activation-specific integrin antagonists.
Limitations with intravenous and failure of oral IIbß3 antagonists question the current concept of ligand-mimetic IIbß3 blockade. We provide a model explaining paradoxical platelet activation as consequence of IIbß3 antagonist-induced conformational change of IIbß3. Concomitant blockade of the ADP-receptor P2Y12, activation-specific and allosteric blockade are described/discussed as alternative IIbß3-blocking strategies.
Key Words: IIbß3 • GPIIb/IIIa • IIbß3 • antagonists • integrin • antiplatelet therapy
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Introduction |
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Integrins are a family of cellular adhesion molecules, which mediate cell–cell and cell–extracellular matrix interactions and therefore are broadly involved in biological processes.1–3 Congruously, integrins are a major target for the development of new therapeutics.4 The best studied and already extensively clinically used integrin therapeutics are
IIbß3 (platelet glycoprotein [GP] IIb/IIIa) antagonists.5 However, the intravenous use of IIbß3 antagonists has revealed side effects and limitations in efficacy.5 Moreover, in large-scale clinical trials, the use of oral GPIIb/IIIa antagonists was associated with a significant increase in mortality.5–7 The mechanism of these unexpected problems of IIbß3 antagonists has not yet been understood.6,7
Clinically used IIbß3 antagonists imitate or directly resemble the peptide sequence RGD, which is one of the binding sites within fibrinogen that is directly involved in binding to IIbß3.5 The antibody-derived IIbß3 antagonist abciximab inhibits IIbß3 via an alternative mechanism, targeting an epitope close to the fibrinogen-binding pocket in IIbß3.8 Thus, the currently clinically used IIbß3 antagonists are to be considered as ligand-mimetics.9,10 Because ligand binding causes a conformational change of IIbß3 and can result in receptor outside-in signaling,10,11 we systematically investigated whether the ligand-mimetic property of IIbß3 antagonists can cause paradoxical platelet activation.
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Materials and Methods |
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Fibrillar Horm-type collagen from equine tendon (Horm type I) was purchased from Nycomed. The anti–P-selectin (anti-CD62P, clone AC1.2) mAb was obtained from Becton Dickinson, the FITC-conjugated goat anti-mouse IgG+M, and the Fc-fragment specific FITC-conjugated goat anti-mouse IgG were obtained from Dianova. The anti-
IIbß3 2G12 mAb was a generous gift of Dr Ginsberg (Scripps Research Institute), the ß3-specific Ab15 mAb was a generous gift of Dr Loftus (Mayo Clinic, Scottsdale). The anti-IIbß3 P2 mAb, the IIb-specific mAb SZ22, the ß3-specific mAb SZ21, the anti–CD40L-FITC mAb (clone TRAP-1), and the anti–CD63-FITC mAb (clone CBL-gran12) were purchased from Immol/Lunotech. Bovine serum albumin (BSA, fraction V) was from Calbiochem. Eptifibatide was obtained from Essex Pharma, tirofiban from MSD, and abciximab from Elli Lilly. The P2Y12 receptor blocker AR-C69931MX was a generous gift of AstraZeneca. Clopidogrel was obtained from Bristol-Myers Squibb. ADP was purchased from Moelab, the RGD-peptide (GRGDSP) was produced by Peptide Specialty Laboratories GmbH. Unspecific mouse IgG was purchased from Chemicon. Production and characterization of the anti–LIBS-145 IgG antibody were performed as previously described.10 Fab fragments of the anti–LIBS-145 IgG were produced using the Immol/LunoPure IgG Fab preparation kit from Pierce. The single chain version (scFv) of the anti-LIBS antibody was cloned in pHOG-21 and produced in E coli TG1 similar as described for an anti-fibrin scFv.9 Binding characteristics of the anti-LIBS-145-scFv for the LIBS epitope on IIbß3 was identical to the anti–LIBS-145-IgG.
Blood Collection
Blood was collected by venipuncture with a 21-gauge butterfly needle from healthy volunteers taking no medications, and blood was anticoagulated with one-tenth volume of citric acid (Monovette, Sarstedt). Platelet-rich plasma (PRP) was obtained by centrifugation at 250g for 10 minutes. Washed platelets were prepared from citrated PRP using a sepharose CL-2B column (Sigma) on a polypropylene column (Pierce). After elution with modified Tyrode buffer (150 mmol/L NaCl, 2.5 mmol/L KCl, 12 mmol/L NaHCO3, 2 mmol/L MgCl2, 2 mmol/L CaCl2, 1mg/ml BSA, 1mg/ml dextrose; pH 7.4) platelets were further diluted 1:10 in modified Tyrode buffer.
Platelets in Suspension Incubated With IIbß3 Antagonists and Antibody Crosslinking
Whole blood was diluted in modified Tyrode buffer and was incubated for 20 minutes with either P2 or 2G12 antibody at final concentrations of 10 µg/ml or with no addition. Samples were incubated with eptifibatide (10 µg/ml), tirofiban (100 ng/ml), or RGD-peptides (2 mmol/L) or no addition and in parallel either with ADP at a final concentration of 1 µmol/L or 20 µmol/L, or no ADP for 15 minutes at room temperature. In experiments evaluating potential concentration-dependent effects the following concentrations (1-9) were used. Eptifibatide: 0, 0.1, 0.25, 0.5, 1, 5, 10, 20, 50 µg/ml. Tirofiban: 0, 0.1, 10, 20, 40, 50, 100, 200, 500 ng/ml. RGD-peptides: 0, 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 10 mmol/L. For the measurement of platelet activation, phycoerythrin-labeled anti–P-selectin antibody was added in saturating concentrations. After incubation for 20 minutes, samples were fixed with CellFix, and the mean fluorescence of platelets was determined by flow cytometry on a FACSCalibur with CellQuest software (all Becton Dickinson).
Anti-LIBS Antibody Experiments
For experiments with anti-LIBS antibodies, samples of diluted whole blood were treated with or without anti-LIBS antibodies at a final concentration of 10 µg/ml for 20 minutes. Unspecific mouse IgG at two concentrations (10 and 200 µg/ml) was used as negative control and to exclude Fc-receptor–dependent effects. Platelet preactivation was achieved with ADP at a final concentration of 1 µmol/L. Platelet activation was determined with an anti–P-selectin antibody as described above.
Immol/Lunofluorescence Staining of Platelets Adhering to Collagen
Glass cover slips (12 mmol/L circular, No 1; Fisher Scientific) were incubated with collagen (50 µg/ml) at 4°C overnight. After washing with modified Tyrode buffer, the cover slips were blocked with 1% BSA and washed twice again with modified Tyrode buffer. The washed platelets (preparation described above) were incubated with eptifibatide (10 µg/ml), tirofiban (100 ng/ml), RGD-peptides (2 mmol/L), abciximab (10 µg/ml), ADP (1 µmol/L or 20 µmol/L), AR-C69931MX (10 µmol/L), or no addition for 15 minutes at room temperature. Then, platelets were allowed to adhere on the coated cover slips for 30 minutes at 37°C and washed twice with modified Tyrode buffer. For microscopy, platelets were stained with an anti-CD62P for 30 minutes, then washed twice again and stained with a FITC-labeled goat anti-mouse IgG+M antibody for 30 minutes. In the case of experiments with abciximab, a FITC-labeled Fc-specific goat anti-mouse IgG antibody was used. After another two washes with Tyrode buffer, platelets were fixed with CellFix (Becton Dickinson) and the cover slips were mounted using Vectashield (Vector). Photographs were taken on an Axioplan 2 microscope with a plan NeoFluar 100x oil immol/Lersion objective (Zeiss). For flow cytometry, platelets were fixed with 1x Cellfix, then scraped off carefully, stained with phycoerythrin-labeled anti–P-selectin antibody, and analyzed with flow cytometry.
Ca2+ Signaling in Adhering Platelets
Cover slips and PRP were prepared as described above. Platelets were purified with a sepharose B (Sigma) column und were eluted with PWB (platelet washing buffer; 43 mmol/L KH2PO4, 43 mmol/L Na2PO4, 243 mmol/L NaH2PO4, 1.13 M NaCl, 55 mmol/L D-Glucose, 100 mmol/L theophylline, 10 % BSA). 3x108 platelets/ml were stained with 1.25 µmol/L FuraRed AM (Molecular Probes) for 30 minutes at 37°C and then with 1µmol/L Oregon Green 488 BAPTA-1 AM (Molecular Probes) for 30 minutes at 37°C. Stained platelets were pelleted at 2000g for one minute and resuspended with modified Tyrode buffer. Samples were preincubated with ADP (20 µmol/L), eptifibatide (10 µg/ml) for 15 minutes at 37°C, then visualized on confocal microscope (Leica) after 10 minutes. For analysis, platelets of 4 representative fields were counted. As a positive control we used dye-loaded platelets with ionophore A23187 and as negative control dye-loaded platelets with DM-BAPTA-AM and EGTA. Platelet calcium was calculated for the individual platelet from ratiometric fluorescence measurements and converted to intracellular calcium concentrations as described previously.13
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Results |
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Description of a Trial Required for Paradoxical Platelet Activation: Ligand-Bound Conformation of
IIbß3, Receptor Crosslinking, and Platelet PreactivationPlatelet activation can be determined in flow cytometry by measuring P-selectin (CD62P) expression on platelets. The sole incubation with a ligand-mimetic
IIbß3 antagonist (RGD-peptide, eptifibatide, or tirofiban) at various concentrations does not result in platelet activation as determined by an anti–P-selectin mAb (Figure 1A and 1B). The addition of ADP induces a concentration-dependent P-selectin expression, which is not influenced by the presence of IIbß3 antagonists (Figure 1A). Because clustering of receptors has been described to participate in integrin outside-in signaling,12 we introduced receptor clustering by addition of crosslinking anti-IIbß3 mAbs. Receptor clustering and receptor occupancy by ligand-mimetic IIbß3 antagonists alone does not cause platelet activation (Figure 1C and 1E). However, if platelets are preactivated by ADP, the combination with IIbß3 crosslinking and receptor occupancy by ligand-mimetic blockers results in strong platelet activation (Figure 1C, 1D, and 1E). This effect could be demonstrated for the IIbß3 complex-specific mAbs P2 and 2G12 and the ß3-specific mAb15, but not for the IIb-specific mAb SZ22 or the ß3-specific SZ21 (results shown for P2 and 2G12), which may be explained by certain steric requirements for crosslinking antibodies to be able to mimic clustering by the native ligand fibrinogen. If the crosslinking ability of the antibody is destroyed by digestion to Fab' fragments, then platelet activation is not induced by IIbß3 antagonists (not shown). P-selectin expression increases with increasing concentrations of IIbß3 antagonists, reaching saturation at higher concentrations (Figure 1D). This finding is consistent with an activating effect mediated by a receptor-ligand interaction in form of antagonist binding to IIbß3.
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5.
Anti-LIBS Antibodies Imitate the Binding of IIbß3 Antagonists
To demonstrate that the ligand-mimetic function of IIbß3 antagonists is essential for paradoxical platelet activation and furthermore to exclude other non–IIbß3-associated effects, we used an anti-LIBS (Ligand-induced Binding Site) antibody (mAb145) to imitate the conformational change of IIbß3 induced by the binding of IIbß3 antagonists.10 Indeed, the original IgG anti-LIBS antibody, imitating the ligand-mimetic blocker occupancy and at the same time crosslinking of IIbß3, causes strong P-selectin expression on prestimulated platelets (Figure 1F). However, if platelets are incubated with the Fab' fragment or a single-chain antibody version of mAb145 or unspecific IgG, platelet activation could not be detected, arguing for the importance of receptor clustering (Figure 1F).
Platelet Adhesion Constitutes Preactivation and Crosslinking Resulting in Paradoxical Platelet Activation by IIbß3 Antagonists
Platelet adhesion on collagen can be considered as an in vivo correlate of the above-described experimental model. Adhesion itself represents a stimulus for platelet preactivation and the adhesive cytoskeleton provides "crosslinking" of adhesion receptors. Indeed, the incubation of adhering platelets with ligand-mimetic IIbß3 antagonists such as eptifibatide, RGD-peptide, tirofiban, and abciximab results in strong platelet activation as demonstrated by staining with anti–P-selectin, anti-CD63, and anti-CD40L mAbs (shown for P-selectin in immunofluorescence microscopy in Figure 2A and 2C and supplemental Figure I, as well as in flow cytometry in Figure 2B).
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Changes in Ca2+ Levels as Measure of Paradoxical Platelet Activation
To further substantiate our previous findings, Ca2+-sensitive probes FuraRed AM and Oregon-Green 488 BAPTA-1 AM were used as previously described13 to directly evaluate Ca2+ response in platelets adhering on collagen and exposed to eptifibatide. As seen in the ratiometrically calculated Ca2+ level of individual platelets (Figure 3) as well as in the overall increased number of platelets with high Ca2+ levels (Figure 3), the ligand-mimetic IIbß3 antagonist eptifibatide indeed causes additional platelet activation.
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Paradoxical Activation of Platelets Is Dependent on the ADP Receptor P2Y12
Platelet activation by platelet adhesion on collagen involves a positive feedback signaling by ADP release from platelet granules and by activation of the P2Y12 receptor.14 We first used the P2Y12 ADP receptor blocker AR-C69931MX to investigate the role of ADP-signaling in ligand-mimetic IIbß3 antagonist-induced paradoxical platelet activation. P-selectin expression of platelets adhering to collagen and incubated with ligand-mimetic IIbß3 antagonists can be blocked by AR-C69931MX (Figure 2C). To address the potential clinical relevance of this finding we evaluated platelets from 5 healthy volunteers, who took 75 mg clopidogrel for 3 days. We could see an inhibitory effect of clopidogrel on eptifibatide-induced platelet activation (Figure 2C). This finding suggests that the P2Y12 receptor is involved in IIbß3 antagonist-induced paradoxical platelet activation.
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Discussion |
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The findings of our study can be summarized in a schematic drawing (Figure 4). Binding of ligand-mimetic antagonists to
IIbß3 causes a conformational change of this integrin resulting in outside-in signaling. However, this outside-in signaling on its own is not sufficient to cause platelet activation. We hypothesized that two other signaling events have to accompany this outside-in signaling of IIbß3 to culminate in strong platelet activation. One is receptor clustering, the other is platelet preactivation mediated by the P2Y12 ADP receptor. To prove our hypothesis, we first defined these two signaling components in a model system using platelets in suspension (Figure 4A). We then demonstrated that adhesion of platelets to a collagen matrix provides both components, receptor clustering and P2Y12-mediated preactivation, sufficient to cause strong platelet activation on exposure to ligand-mimetic IIbß3 antagonists (Figure 4B). This model describes a potential pathomechanism of IIbß3 antagonist-induced paradoxical platelet activation and indeed may explain major limitations and failure seen during clinical use or clinical testing of IIbß3 antagonists.
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Ligand-mimetic binding of RGD-peptides has first been described to cause conformational changes of IIbß3 by Du et al.15 Using anti-LIBS antibodies, conformational changes of IIbß3 have been previously described for the three currently clinically used IIbß3 antagonists, abciximab, eptifibatide, and tirofiban.10,16 LIBS induction on IIbß3 by eptifibatide, tirofiban, and RGD-peptide has been demonstrated to be dependent on the IIbß3 antagonist concentration,16 which is consistent with our finding that the platelet-activating effect of IIbß3 antagonists is concentration-dependent. Based on crystal structures, Xiao et al11 were recently able to directly define the binding sites of ligand-mimetic antagonists within the IIbß3 headpiece. IIbß3 antagonist binding causes an allosteric conformational change that results in an open, extended high affinity conformation for ligand binding and in signal transduction (outside-in signal) along the integrin legs finally resulting in the separation of the transmembrane domain of the and ß subunit.11 The latter is hypothesized to initiate intracellular signaling events.11 For both agonist-induced ligand binding as well as for agonist-induced outside-in signaling, supportive data has been reported. We previously reported induction of fibrinogen binding by IIbß3 antagonists, in particular at low drug concentrations.9 Similar findings with the induction of vitronectin binding to Vß3 integrin at low antagonist concentrations have been published.17 Also, platelet activation induced by IIbß3 antagonist has been directly demonstrated by flow cytometry in patients treated with IIbß3 antagonists.18–22 Furthermore, direct proaggregatory effects, which correlate with the induction of LIBS epitopes, have been demonstrated for IIbß3 antagonists.16
The initial adhesion step of platelets to collagen involves GPVI, which mediates platelet stimulation and release of platelet agonists such as ADP,14 which is one of the proposed requirements for IIbß3 antagonist-induced platelet activation (Figure 4B). Receptor crosslinking, as the second component of the proposed mechanistic model of IIbß3 antagonist-induced paradoxical platelet activation, may involve several receptors. Based on published data, 3 receptors may generate a crosslinking-induced outside-in signal: (1) Stable platelet adhesion on collagen is mediated by the 2ß1 receptor, which is known to develop integrin-typical receptor clustering on engagement with multimeric ligands such as collagen (depicted as an example for receptor clustering in Figure 4B).14,23 Indeed, outside-in signaling via 2ß1 is typically induced with immobilized collagen but not with soluble collagen, arguing for a role of receptor clustering in 2ß1 outside-in signal generation.24 (2) Recent data also suggests that GPVI signaling in response to its multimeric ligands such as collagen involves clustering of GPVI itself.14,25 (3) IIbß3 may be crosslinked during platelet adhesion via release of fibrinogen and von Willebrand factor (vWF) from platelet granules.26 Further evidence for receptor crosslinking on a collagen matrix has been demonstrated in megakaryocytes, via stress fiber and adhesion plaque staining.27 In our experimental setting, we could also demonstrate stress fiber formation and focal adhesion plaque assembly in platelets adhering to collagen (data not shown). Therefore, there are several findings indicative of receptor crosslinking in platelets adhering on collagen. Nevertheless, further systematic evaluation beyond the current mechanistic model may be able to dissect this process and to identify the receptor or the combination of receptors involved in crosslinking.
The finding that P2Y12 ADP receptor blockers inhibit IIbß3 antagonist-induced paradoxical platelet activation fits well to the model proposed by Nieswandt & Watson, which describes ADP as major mediator of GPVI-induced platelet activation.14 Our finding that the clinically used P2Y12 ADP receptor blocker clopidogrel inhibits IIbß3 antagonist-induced platelet activation may be of major clinical importance. Intravenous application of IIbß3 antagonist in nowadays clinical practice is typically accompanied by clopidogrel administration, whereas orally administered IIbß3 antagonists, which failed in clinical trials, were not given together with clopidogrel.7 This raises the interesting question whether IIbß3 antagonists should be accompanied by ADP receptor blockers.
Two alternatives to competitive integrin inhibition by ligand-mimetic antagonists are conceivable: allosteric and activation-specific integrin inhibition. For the leukocyte integrin LFA-1, allosteric inhibition of ligand binding has been demonstrated.28 It is proposed that for all integrins, allosteric inhibitors can be developed.4 Activation-specific inhibition is not associated with changes from the low to the high affinity integrin conformation, because as shown for activation-specific human anti-IIbß3 single-chain antibodies no binding to the nonactivated IIbß3 is seen.29,30 These alternative strategies of integrin antagonism promise to circumvent the reported paradoxical platelet activation and thereby to provide benefits to patients which may be beyond the currently employed ligand-mimetic antagonism. Because platelet inhibition has been proven to result in major cardiovascular risk reduction, effective and safe anti-platelet drugs are highly sought after.4–6 The described new anti-IIbß3 agents are well suited to compete with other selective antiplatelet approaches.4,31
In summary, we present an experimental model that can explain ligand-mimetic IIbß3 antagonist-induced paradoxical platelet activation. Binding of ligand-mimetic IIbß3 antagonists to IIbß3 causes integrin outside-in signaling, which under certain conditions results in paradoxical platelet activation. We could define these conditions and demonstrate their presence in platelets adhering on collagen. Moreover, we could show that ADP receptor blockade prevents paradoxical platelet activation. These findings strongly support the strategy to move beyond the initial approach of ligand-mimetic integrin antagonists toward the development of allosteric or activation-specific integrin antagonists.
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Acknowledgments |
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Sources of Funding
This work was supported by the National Health and Medical Research Council and the National Heart Foundation of Australia.
Disclosures
None.
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Footnotes |
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Original received June 11, 2006; final version accepted November 13, 2006.
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