Published online before print April 16, 2007, doi: 10.1161/CIRCULATIONAHA.107.691279
(Circulation. 2007;115:2323-2330.)
© 2007 American Heart Association, Inc.
Stroke |
Targeting Platelets in Acute Experimental Stroke
Impact of Glycoprotein Ib, VI, and IIb/IIIa Blockade on Infarct Size, Functional Outcome, and Intracranial Bleeding
From the Departments of Neurology (C.K., G.S.), Clinical Biochemistry and Pathophysiology (M. Pozgajova, B.N.), and Neuroradiology (M. Pham, M.B.) and the Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine (M. Pozgajova, B.N.), University of Würzburg, Würzburg, Germany.
Correspondence to Guido Stoll, MD, Department of Neurology, Bayerische Julius-Maximilians-Universität Würzburg, Josef-Schneider-Str 11, D-97080 Würzburg, Germany. E-mail stoll_g{at}klinik.uni-wuerzburg.de
Received January 18, 2007; accepted March 9, 2007.
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Abstract |
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Background— Ischemic stroke is a frequent and serious disease with limited treatment options. Platelets can adhere to hypoxic cerebral endothelial cells by binding of their glycoprotein (GP) Ib receptor to von Willebrand factor. Exposure of subendothelial matrix proteins further facilitates firm attachment of platelets to the vessel wall by binding of collagen to their GPVI receptor. In the present study, we addressed the pathogenic role of GPIb, GPVI, and the aggregation receptor GPIIb/IIIa in experimental stroke in mice.
Methods and Results— Complete blockade of GPIb
was achieved by intravenous injection of 100 µg Fab fragments of the monoclonal antibody p0p/B to mice undergoing 1 hour of transient middle cerebral artery occlusion. At 24 hours after transient middle cerebral artery occlusion, cerebral infarct volumes were assessed by 2,3,5-triphenyltetrazolium chloride staining. In mice treated with anti-GPIb Fab 1 hour before middle cerebral artery occlusion, ischemic lesions were reduced to 
40% compared with controls (28.5±12.7 versus 73.9±17.4 mm3, respectively; P<0.001). Application of anti-GPIb Fab 1 hour after middle cerebral artery occlusion likewise reduced brain infarct volumes (24.5±7.7 mm3; P<0.001) and improved the neurological status. Similarly, depletion of GPVI significantly diminished the infarct volume but to a lesser extent (49.4±19.1 mm3; P<0.05). Importantly, the disruption of early steps of platelet activation was not accompanied by an increase in bleeding complications as revealed by serial magnetic resonance imaging. In contrast, blockade of the final common pathway of platelet aggregation with anti-GPIIb/IIIa F(ab)2 fragments had no positive effect on stroke size and functional outcome but increased the incidence of intracerebral hemorrhage and mortality after transient middle cerebral artery occlusion in a dose-dependent manner.
Conclusions— Our data indicate that the selective blockade of key signaling pathways of platelet adhesion and aggregation has a different impact on stroke outcome and bleeding complications. Inhibition of early steps of platelet adhesion to the ischemic endothelium and the subendothelial matrix may offer a novel and safe treatment strategy in acute stroke.
Key Words: cerebrovascular disorders • drug therapy • glycoproteins • intracerebral hemorrhage • magnetic resonance imaging • platelets • stroke
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Introduction |
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Ischemic stroke is the third-leading cause of death and permanent disability in industrialized countries.1 Although the beneficial role of anticoagulation and the platelet aggregation inhibitors acetylsalicylic acid, clopidogrel, and dipyridamole in stroke prevention is well established, their use in the acute phase of cerebral ischemia is a matter of debate. A moderate benefit on stroke progression and recurrence often is outweighed by a significant increase in the rate of intracerebral hemorrhage (ICH).2–5
Clinical Perspective p 2330
Recently, major progress has been made in the functional analysis of platelet activation and platelet-dependent thrombus formation. Platelets can adhere to hypoxic endothelial cells by binding of their glycoprotein (GP) Ib receptor to von Willebrand factor (vWF) on the endothelial surface.6 Exposure of subendothelial matrix proteins during ischemia further facilitates firm attachment of platelets to the vessel wall by binding of collagens to their GPVI receptor.7,8 These processes lead to activation of platelet GPIIb/IIIa and platelet aggregation.9 Although it is well established that endothelial cells undergo activation10 and that microvascular integrity is disturbed during cerebral ischemia,11 less is known about the signaling cascades that lead to intravascular thrombus formation in the brain. We recently demonstrated that coagulation factor XII plays a decisive role in thrombus formation after transient focal cerebral ischemia.12 In the present study, we show that targeting platelet GPIb or GPVI receptors protects mice from ischemic brain injury in an experimental stroke model without an increase in bleeding complications. In contrast, blockade of the final common pathway of platelet aggregation with anti-GPIIb/IIIa antibodies had no positive effect on stroke outcome and dose-dependently raised the incidence of ICH and mortality.
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Methods |
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Anti-Platelet Antibodies
All monoclonal antibodies (mAbs) were produced, characterized, and modified in our laboratories as described previously in detail. The following mAbs were used: mAbs against mouse GPIIb/IIIa (JON/A),13 GPIb
(p0p/B),14 and GPVI (JAQ1).15 Fab and F(ab)2 fragments were prepared as described.16 Briefly, antibodies were incubated for 6 to 8 hours with immobilized papain or for 24 hours with immobilized pepsin according to the manufacturer’s instructions (Pierce Biotechnology, Inc, Rockford, Ill), and the preparations were then applied to an immobilized protein A column, followed by an immobilized protein G column (Pharmacia, Freiburg, Germany) to remove Fc fragments and undigested IgG. Purity of Fab or F(ab)2 fragments was tested by SDS-PAGE. For control experiments, purified rat IgG2a (Serotec, Darmstadt, Germany) and nonimmune control rat IgG Fab were used.
Antibody Administration
To inhibit GPIb, mice received 100 µg p0p/B Fab IV 1 hour before or 1 hour after transient middle cerebral artery occlusion (tMCAO). GPIIb/IIIa receptors were blocked by injection of 100 µg (>95% receptor blockade; see the Table), 20 µg (78.4% receptor blockade), or 10 µg (67.8% receptor blockade) JON/A F(ab)2 IV 1 hour before the start of the experiment. To inhibit GPVI function, mice received 100 µg JAQ1 IP 5 days before infarct induction. Mice had at that time point no detectable GPVI in platelets for at least 5 more days.17 Two different groups of control animals received either 100 µg purified rat IgG2a or 100 µg rat IgG Fab.
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Bleeding Time Experiments
To determine bleeding times, mice were anesthetized, and a 3-mm segment of the tail tip was amputated with a scalpel. The tail was then blotted with filter paper every 15 seconds until the paper was no longer blood stained.18 When necessary, bleeding was manually stopped after 20 minutes to prevent death.
Animal Studies
Animal studies were approved by the Regierung von Unterfranken and conducted according to the recently published recommendations for research in mechanism-driven basic stroke studies.19 Adult male C57/BL6 mice (20 to 25 g) were purchased from Charles River (Sulzfeld, Germany). The tMCAO model was used to induce focal cerebral ischemia as described in detail elsewhere.12,20 Briefly, mice were anesthetized with 2% isoflurane in a 70% N2O/30% O2 mixture. A servo-controlled heating blanket was used to maintain core body temperature close to 37°C throughout surgery. After a midline neck incision was made, a standardized silicon rubber–coated 6.0 nylon monofilament (60–1720RE, Doccol, Redlands, Calif) was inserted into the right common carotid artery and advanced via the internal carotid artery to occlude the origin of the MCA. After 1 hour, mice were reanesthetized, and the occluding filament was removed to allow reperfusion. All animals were operated on by the same operator (C.K.) to reduce infarct variability; operation time per animal did not exceed 15 minutes.
After recovery from anesthesia and again after 24 hours, neurological function was assessed by 2 blinded investigators. Global neurological status was scored according to Bederson et al.21 Motor function and coordination were graded with the grip test.22
Laser Doppler flowmetry (Moor Instruments, Devon, UK) was used to monitor regional cerebral blood flow in the MCA territory in antibody-treated animals and controls before surgery (baseline), immediately after MCA occlusion, and again 5 minutes after removal of the occluding monofilament (reperfusion).12,23 After the thread was advanced, regional cerebral blood was reduced by 95±3% and recovered to 68±5% of baseline (100%) after removal of the filament, indicating sufficient occlusion and reperfusion of the vessel beds. Values did not differ statistically between the groups at any time point (not shown).
After the right femoral artery was punctured, blood gases (PO2, PCO2, pH) were analyzed during the operation in 3 animals per group (antibody-treated mice or controls). The measured values were within the physiological range and showed no significant differences (not shown).
Determination of Infarct Size and ICH
Mice were killed 24 hours after tMCAO. Brains were quickly removed and cut into 2-mm-thick coronal sections using a mouse brain slice matrix. The slices were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich, Seelze, Germany) in PBS to visualize the infarctions. Planimetric measurements (ImageJ software, National Institutes of Health, Bethesda, Md) were performed by researchers blinded to the treatment group and were used to calculate lesion volumes, which were corrected for brain edema as described.24
The occurrence of ICH was macroscopically assessed on whole brains and again after the 2-mm-thick coronal brain slices were cut (see above) before TTC staining. Brains showing ICH were excluded from the assessment of infarct volumes.
Stroke Assessment by Magnetic Resonance Imaging
To investigate the frequency of ICH over time after tMCAO, magnetic resonance imaging (MRI) was performed repeatedly at an early stage (24 hours) and at 7 days after stroke on a 1.5-T MR unit (Vision, Siemens, Berlin, Germany) under inhalation anesthesia as described previously.12 For all measurements, a custom-made dual-channel surface coil designed for the examination of mice head was used (A063HACG, Rapid Biomedical, Würzburg, Germany). The image protocol comprised a coronal T2-weighted sequence (slice thickness, 2 mm) and a coronal 3D T2-weighted gradient-echo constructed interference in steady state (slice thickness, 1 mm) sequence. MRIs were visually assessed by researchers blinded to the prior treatment with respect to infarct morphology and, in particular, the occurrence of ICH.
Statistical Analysis
Results are presented as mean±SD. The frequency of ICH and the mortality rate at day 1 were compared between groups with the 
2 test. Infarct volumes and functional data were tested for gaussian distribution with the D‘Agostino and Pearson omnibus normality test and then analyzed by Bonferroni-corrected 1-way ANOVA. For statistical analysis, PrismGraph 4.0 software (GraphPad Software, San Diego, Calif) was used. Values of P<0.05 were considered statistically significant.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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GPIb and GPVI Blockade Improves Stroke Outcome After Transient Cerebral Ischemia
To study the involvement of platelets in the development of ischemic stroke, we inhibited the function of platelet membrane receptors involved in primary adhesion and activation. Complete blockade of GPIb
was achieved by intravenous injection of 100 µg Fab fragments of the monoclonal antibody p0p/B. In contrast to the intact IgG,16 the Fab is not cytotoxic and therefore had no significant influence on platelet counts. After 1 hour, receptor occupancy was >95% as shown by flow cytometric analysis of p0p/BFITC binding (the Table). Tail bleeding times in anti-GPIb–treated mice were markedly prolonged, with 6 of 14 mice being unable to stop bleeding within the 20-minute observation period and the other 8 mice showing a strong prolongation compared with control IgG- or control Fab-treated animals. In parallel, mice were treated with the anti-GPVI antibody JAQ1 (100 µg IP) and analyzed on day 5. As previously described,17 these animals lacked detectable GPVI in circulating platelets but displayed normal platelet counts and only very moderately increased tail bleeding times (the Table).
Mice treated with anti-GPIb Fab 1 hour before or anti-GPVI mAbs 5 days before challenge were subjected to transient (60 minutes) cerebral ischemia and analyzed after 24 hours. At that time point, infarct volumes were reduced dramatically in anti-GPIb–treated mice to 40% compared with controls (28.5±12.7 versus 73.9±17.4 mm3; P<0.001; Figure 1A). Similarly, depletion of GPVI significantly diminished the infarct volume but to a lesser extent (49.4±19.1 mm3; P<0.05; Figure 1A). Reduction in infarct size after anti-GPIb treatment was functionally relevant in that the Bederson score assessing global neurological function and the grip test that specifically measures motor function and coordination were significantly better than in controls (Figure 1B) (Bederson score, 3.7±0.6 versus 2.2±0.6, respectively; P<0.001; grip test, 1.7±0.9 versus 3.4±0.7, respectively; P<0.01). Anti-GPVI–treated mice tended to develop less severe neurological deficits compared with controls, but the differences did not reach statistical significance (Figure 1B). To test whether GPIb blockade also is beneficial in the acute phase after focal cerebral ischemia, anti-GPIb Fab was applied 1 hour after the induction of tMCAO. Indeed, this therapeutic approach was as effective as prophylactic anti-GPIb infusions before tMCAO because brain infarct volumes were reduced to a comparable extent (24.5±7.7 mm3; P<0.001; Figure 1A) and the neurological status again was improved (Bederson score, 1.9±0.7; P<0.001; grip test, 3.4±1.1; P<0.01; Figure 1B). Taken together, these results indicate that the platelet receptors GPIb and GPVI may contribute critically to stroke development after tMCAO.
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We next analyzed the impact of platelet inhibition on the occurrence of intracerebral bleeding complications after experimental cerebral ischemia. Mice receiving anti-GPIb Fab or anti-GPVI mAbs did not show hemorrhagic transformation of infarcted brain regions as assessed by morphological analysis (not shown). Accordingly, mortality rates were not increased compared with IgG or Fab controls (not shown). This important notion could be confirmed by serial MRI studies. In animals treated with anti-GPIb Fab, ischemic infarcts always appeared hyperintense on T2-weighted MRI. There were no additional hypointense areas indicating hemorrhage on days 1 and 7 after tMCAO when blood-sensitive T2-weighted gradient-echo MR sequences were used. These MRI findings exclude the occurrence of ICH (Figure 2).
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GPIIb/IIIa Blockade Is Ineffective in tMCAO and Dose-Dependently Increases the Risk of ICH
We next asked whether blockade of the final common pathway of platelet aggregation via GPIIb/IIIa would effectively reduce infarct volumes even more after tMCAO. Unexpectedly, 4 of the 7 animals that had received 100 µg anti-GPIIb/IIIa F(ab)2 leading to a virtually complete receptor blockade (see the Table) died as a result of ICH (P<0.05; Figure 3A and 3B), and the 3 surviving animals exhibited infarct volumes of the same extension as controls (65.7±3.4 mm3; P>0.05; Figure 4). The high incidence of ICH was very similar to that in mice in which platelets had been depleted by >98% by injection of cytotoxic anti-GPIb antibodies (not shown).16 To analyze whether the risk of ICH after GPIIb/IIIa blockade is dose dependent and to further evaluate the efficacy of anti-GPIIb/IIIa F(ab)2 treatment in experimental stroke, additional groups of mice received 20 or 10 µg anti-GPIIb/IIIa F(ab)2, which led to a 78.4% and 67.8% receptor blockade, respectively (see the Table). In contrast to complete GPIIb/IIIa inhibition, only 1 of 15 animals developed ICH and died (Figure 3B), but both concentrations failed to influence infarct volumes (58.8±6.3 and 61.6±12.4 mm3; P>0.05; Figure 4) or neurological outcome (Bederson score, 3.0±0.6 and 3.2±0.9; P>0.05; grip test, 2.0±0.8 and 2.3±1.0; P>0.05).
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Discussion |
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As a principal finding, we demonstrate here that selective blockade of key pathways mediating platelet adhesion and aggregation has different impacts on stroke outcome. Our study shows for the first time that interfering with early steps of platelet–vessel wall interactions mediated by GPIb and GPVI reduces stroke severity after tMCAO. Anti-GPIb
Fab treatment had a very strong protective effect when performed both before and after tMCAO. This suggests a central role of this receptor in the pathogenesis of ischemic stroke, whereas GPVI may have a significant but less prominent function. This is in agreement with the model that GPIb is mandatory for the initial attachment of platelets to the vessel wall under conditions of elevated shear, whereas GPVI serves mainly as an activating receptor, a function that also can be fulfilled by alternative pathways, most notably G protein–coupled receptors.25,26 Importantly, stroke protection after anti-GPIb Fab or anti-GPVI mAb treatment was not accompanied by intracranial bleeding complications. In contrast, application of F(ab)2 targeting the GPIIb/IIIa receptor had no positive effect on stroke outcome but significantly increased the rate of intracerebral bleedings and mortality in a dose-dependent manner.
The cerebral microvasculature rapidly responds to brain ischemia.10 Endothelial cells upregulate cell adhesion molecules, and endothelial denudation of vessels exposes subendothelial matrix proteins such as collagen to the bloodstream. Recently, 2 important pathways have been described that facilitate early adhesion of platelets to vessel walls: binding of the platelet surface receptor GPIb to endothelial vWF6 and adhesion of platelets to collagen via their GPVI receptor.27 In accordance with our findings in experimental stroke, inhibition of either platelet GPIb or vWF reversed flow reductions after experimental femoral artery stenosis.28–31 Importantly, reduced stroke volumes after GPIb inhibition in our study were accompanied by a significant reduction in neurological deficits even when anti-GPIb Fab was injected with a delay of 1 hour after the induction of tMCAO. This underlines the functional significance of this novel therapeutic approach and indicates its potential suitability for clinical application during the acute phase of ischemic stroke in humans, in whom treatment options are very limited. Interestingly, polymorphisms of platelet GPIb exist, and variants that lead to enhanced vWF/GPIb interactions are associated with an increased risk of ischemic stroke in humans.32,33 Similarly, increased serum levels of vWF represent an independent stroke risk factor.34 Although tail bleeding times are strongly elevated after treatment of mice with anti-GPIb Fab fragments, no increase in ICH, which represents the main obstacle for antithrombotic therapy during the acute stroke phase in clinical practice, was detected. This surprising finding suggests that the mechanisms by which platelets prevent intracranial bleeding are different from those involved in the sealing of a tail bleeding wound. Both processes are clearly platelet dependent because platelet depletion or virtually complete inhibition of GPIIb/IIIa results in both markedly prolonged tail bleeding times and intracranial bleeding after tMCAO (Figure 3A). Thus, our results strongly confirm the previous finding that no direct correlation exists between bleeding time and bleeding risk.35
The initial loose adhesion of platelets to the damaged endothelium is followed by firm attachment, which is mediated through the platelet collagen receptors.6 Among the numerous collagen receptors expressed in platelets, GPVI is of central importance for cellular activation and subsequent firm arrest.36 Treatment of mice with anti-GPVI antibodies specifically and persistently depletes GPVI from platelets.17,37 Several reports have demonstrated a profound antithrombotic effect of GPVI inhibition after artificial arterial wall injury and collagen-induced thromboembolism.14,17,38 We now extend these previous findings by showing that treatment of mice with the anti-GPVI antibody JAQ1 significantly reduced the brain infarct volumes at day 1 after tMCAO. This indicates that platelet/collagen interactions via GPVI also may be involved in stroke development. GPVI depletion was less effective than GPIb blockade and did not significantly affect clinical outcome variables. However, it is well established that significant reductions in stroke volumes on histological examination after MCAO often do not translate into measurable clinical improvement.39 Although tail bleeding times were slightly increased by JAQ1, intracerebral bleeding frequency and mortality after 24 hour were not altered, indicating a favorable safety profile. The different extent of stroke protection in favor of GPIb blockade may be due to a more general role of GPIb in stroke development. GPIb, but not GPVI, additionally mediates leukocyte adhesion to attached platelets by a Mac-1–dependent pathway.6,40 Furthermore, resting platelets can bind to the activated endothelium via GPIb interaction with P-selectin.41 Both, leukocyte adhesion and P-selectin upregulation have been shown to contribute to stroke development, probably by impairing reperfusion of the cerebral microvasculature.10,42
GPIIb/IIIa antagonists inhibit the final common pathway of platelet aggregation regardless of the agonist that stimulates platelet activation.6 In agreement with previous reports,43 antibody-mediated blockade of the GPIIb/IIIa receptor had no significant effect on peripheral platelet counts but completely inhibited ex vivo platelet aggregation in response to different stimuli and resulted in tail bleeding times consistently >20 minutes in our study (the Table). Impaired hemostasis after >95% GPIIb/IIIa blockade could explain the high frequency of ICH and mortality after tMCAO in our study. A substantial risk of ICH has previously been reported after tMCAO in mice treated with the GPIIb/IIIa inhibitor SDZGPI562.44 The unexpected high rate of bleeding complications is consistent with similar observations during a recent phase III, double-blind, placebo-controlled, multicenter study testing the safety and efficacy of abciximab in ischemic stroke. This clinical study was stopped prematurely because of significantly increased ICH and mortality, as well as lack of efficacy.45 In the preceding phase II trial, treatment with abciximab had shown a nonsignificant shift in favorable outcomes only.46 Several experimental studies reported a beneficial effect of GPIIb/IIIa antagonists on stroke size and functional outcome.44,47,48 GPIIb/IIIa antagonists also have been successfully used in experimental and clinical settings in conjunction with recombinant tissue-type plasminogen activator thrombolysis therapy without major complications reported.49,50 This is in contrast to our study in which no effect on stroke volume or functional deficit was observed, regardless of the anti-GPIIb/IIIa F(ab)2 dosage used. Increasing evidence exists, however, that the extent of GPIIb/IIIa inhibition may be critical, which could explain the divergent experimental and clinical observations. At peak concentrations, GPIIb/IIIa inhibitors may effectively act as platelet antagonists accompanied by increased bleeding complications, whereas subthreshold GPIIb/IIIa antagonism may lead to platelet activation and thrombus formation.51 In line with this, a 67.8% or 78.4% GPIIb/IIIa receptor blockade was safe (but ineffective) in our experimental stroke model, whereas complete inhibition substantially increased ICH and mortality. Taken together, it appears that GPIIb/IIIa antagonists have a very narrow therapeutic window, limiting their clinical use at least in cerebral ischemia,51,52 in contrast to their proven utility in percutaneous coronary artery interventions, which, however, is age dependent.53,54 Currently, not enough evidence exists from randomized controlled trials on the efficacy or safety of GPIIb/IIIa inhibitor therapy in acute stroke or its use combined with thrombolysis.55 The devastating consequences of ICH in patients require a particularly high safety profile for any antiplatelet therapy or anticoagulation during stroke because, depending on location, even small bleedings can cause major neurological deficits. Our present study provides evidence that blocking of the platelet receptor GPIb involved in platelet adhesion can diminish infarct development in mice and, because of a lack of ICH, may open new avenues for acute stroke treatment in humans in the future.
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Acknowledgments |
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We thank Dr S. Bräuninger (Würzburg) for help with the functional tests and Professor K. Toyka (Würzburg) for critical reading of the manuscript and continuous support.
Sources of Funding
This study was supported by the Deutsche Forschungsgemeinschaft, Bonn (SFB 688 grants A1/B1 to Drs Nieswandt, Stoll, and Bendszus). Dr Bendszus holds a professorship for neuroimaging donated by the Schering GmbH, Berlin, Germany, to the Department of Neurology, University of Würzburg, but has no financial interest.
Disclosures
None.
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Footnotes |
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*Drs Kleinschnitz, Nieswandt, and Stoll contributed equally to this work.
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