In Vivo Molecular Imaging of Thrombosis and Thrombolysis Using a Fibrin-Binding Positron Emission Tomographic Probe Ilknur Ay, Francesco Blasi, Tyson A. Rietz, Nicholas J. Rotile, Sreekanth Kura, Anna Liisa Brownell, Helen Day, Bruno L. Oliveira, Richard J. Looby and Peter Caravan Circ Cardiovasc Imaging. 2014;7:697-705; originally published online April 28, 2014; doi: 10.1161/CIRCIMAGING.113.001806 Circulation: Cardiovascular Imaging is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 1941-9651. Online ISSN: 1942-0080
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Molecular Imaging In Vivo Molecular Imaging of Thrombosis and Thrombolysis Using a Fibrin-Binding Positron Emission Tomographic Probe Ilknur Ay, MD, PhD*; Francesco Blasi, PharmD, PhD*; Tyson A. Rietz, BS; Nicholas J. Rotile, BA; Sreekanth Kura, MS; Anna Liisa Brownell, PhD; Helen Day, MS; Bruno L. Oliveira, PhD; Richard J. Looby, BS; Peter Caravan, PhD Background—Fibrin is a major component of arterial and venous thrombi and represents an ideal candidate for molecular imaging of thrombosis. Here, we describe imaging properties and target uptake of a new fibrin-specific positron emission tomographic probe for thrombus detection and therapy monitoring in 2 rat thrombosis models. Methods and Results—The fibrin-binding probe FBP7 was synthesized by conjugation of a known short cyclic peptide to a cross-bridged chelator (CB-TE2A), followed by labeling with copper-64. Adult male Wistar rats (n=26) underwent either carotid crush injury (mural thrombosis model) or embolic stroke (occlusive thrombosis model) followed by recombinant tissue-type plasminogen activator treatment (10 mg/kg, IV). FBP7 detected thrombus location in both animal models with a high positron emission tomographic target-to-background ratio that increased over time (>5-fold at 30–90 minutes, >15-fold at 240–285 minutes). In the carotid crush injury animals, biodistribution analysis confirmed high probe uptake in the thrombotic artery (≈0.5%ID/g; >5-fold greater than blood and other tissues of the head and thorax). Similar results were obtained from ex vivo autoradiography of the ipsilateral versus contralateral carotid arteries. In embolic stroke animals, positron emission tomographic–computed tomographic imaging localized the clot in the internal carotid/middle cerebral artery segment of all rats. Time-dependent reduction of activity at the level of the thrombus was detected in recombinant tissue-type plasminogen activator–treated rats but not in vehicle-injected animals. Brain autoradiography confirmed clot dissolution in recombinant tissue-type plasminogen activator–treated animals, but enduring high thrombus activity in control rats. Conclusions—We demonstrated that FBP7 is suitable for molecular imaging of thrombosis and thrombolysis in vivo and represents a promising candidate for bench-to-bedside translation. (Circ Cardiovasc Imaging. 2014;7:697-705.) Key Words: fibrin ◼ stroke ◼ thrombosis
T
hrombosis, the underlying cause in the majority of cardiovascular disorders, has a high incidence and prevalence in the United States.1 Recent advances in medical imaging have facilitated the diagnosis of thrombotic and thromboembolic disorders; however, some challenges still remain. First, although recurrences occur in most cases, diagnostic techniques including magnetic resonance (MR) imaging and computed tomography (CT) are not efficient to identify the source thrombus, especially when used as a single test.2,3 Identification of source thrombus requires a technique with whole body scanning and high sensitivity. Second, modalities that are used to detect thrombus, including carotid or pelvic ultrasound, transesophageal echocardiogram, CT, and MRI, have some pitfalls with respect to sensitivity, specificity, and feasibility.4–7 Multimodal imaging with a molecular probe designed to detect thrombus offers a potential solution to both of these challenges.8,9 To date, coagulation factors (thrombin,
Factor XIII, fibrinogen, fibrin) and activated platelets have been targeted for molecular thrombus imaging.10 Among them, fibrin represents an ideal target because of its high specificity (present at high concentration in all clots but not in circulating blood) and high sensitivity (present in all thrombi whether arterial or venous, fresh or aged).11
Editorial see p 581 Clinical Perspective on p 705 Positron emission tomography (PET) is a quantitative, high sensitivity technique that offers the possibility for whole body scanning. Hybrid imaging that combines PET with CT, or more recently PET with MR, provides a high resolution anatomic image (CT or MR) onto which the lower resolution molecular PET image can be overlaid. Thus, a sensitive thrombus-specific PET probe may detect even minor thrombotic events and CT or MR can localize this PET activity to a specific location in the
Received December 11, 2013; accepted April 24, 2014. From the Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA. The Data Supplement is available at http://circimaging.ahajournals.org/lookup/suppl/doi:10.1161/CIRCIMAGING.113.001806/-/DC1 *Drs Ay and Blasi contributed equally to this work. Correspondence to Peter Caravan, PhD, Building 149, Room 2301, 13th St, Charlestown, MA 02129. E-mail [email protected] © 2014 American Heart Association, Inc. Circ Cardiovasc Imaging is available at http://circimaging.ahajournals.org
DOI: 10.1161/CIRCIMAGING.113.001806
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698 Circ Cardiovasc Imaging July 2014 vascular tree. An ideal fibrin/thrombus PET probe should have high affinity for fibrin, high specificity for fibrin over fibrinogen and other blood proteins, rapid clearance from the circulation to increase thrombus-to-background ratio, and small size to enable penetration into thrombi. To this end, we have labeled a short cyclic peptide that was previously shown to exhibit high affinity for fibrin and great specificity for fibrin versus fibrinogen (>100-fold) and other serum proteins.12–14 We recently demonstrated preliminary efficacy of PET and bimodal PET–MR probes based on these fibrin-specific binding peptides for molecular imaging of thrombus, although these probes suffered from instability in vivo.15–17 In the present study, we report the efficacy of a newly developed, chemically stable, fibrin-binding PET probe termed FBP7 in 2 different animal models of arterial thrombosis: carotid crush injury as a nonocclusive intramural thrombosis model and embolic stroke as an occlusive thrombosis model. To extend the translational outcome of our work, we also tested whether FBP7 was able to visualize and quantify in vivo thrombolysis after recombinant tissue-type plasminogen activator (rtPA) treatment in embolic stroke.
Methods Synthesis and Characterization of FBP7 FBP7 was synthesized by conjugation of the CB-TE2A chelator to a cyclic, disulfide bridged 11-amino acid fibrin-binding peptide, followed by labeling with copper-64 and high-performance liquid chromatography purification.16 The cyclic peptide was previously shown to be effective for fibrin targeting.14,16 FBP7 was evaluated for in vitro fibrin affinity in the presence and absence of heparin and also for its plasma stability. Additional information about reagents and methods are reported in Methods in the Data Supplement.
Animal Thrombosis Models All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Massachusetts General Hospital. Adult male Wistar rats (330–400 g; Charles River Laboratories) were anesthetized by isoflurane (4% for induction, 2%–2.5% for maintenance in medical air) and were kept under anesthesia throughout the study. Body temperature was maintained at 37°C using a thermoregulated heating pad (Harvard Apparatus). The right femoral vein and artery were catheterized using PE-50 tubing for probe/drug injection and blood sampling, respectively. Intramural thrombus was induced by carotid artery crush injury (n=10). Under isoflurane anesthesia, the right common carotid artery was exposed and crush injury was induced by clamping the vessel with a hemostat for 5 minutes, as previously described.16 Injury was performed 1 to 2 cm proximal to the carotid bifurcation, using the same hemostat and by the same investigator to minimize variability. Occlusive thrombus was produced by embolic stroke (n=16) using a previously published method.18 Briefly, homologous thrombus was prepared 1 day before by retaining freshly collected arterial blood in a PE-50 tube for 2 hours at room temperature and 22 hours at 4°C. The next day, a single 25-mm-long clot was injected into the right internal carotid artery (ICA) via an external carotid stump to occlude the middle cerebral artery (MCA) under isoflurane anesthesia. In a subgroup of animals, the clot was incubated in an Evans blue solution (2%, wt/ vol) to enhance postmortem clot visualization.19 Approximately 1 hour after the clot injection, rats were injected with either rtPA (alteplase, 10 mg/kg, IV, 10% bolus and 90% infusion over 30 minutes; n=9) or vehicle (saline solution, 1 mL; n=7). Because the embolic stroke model is associated with a small but significant mortality and failure rate, rats that died during the study period, had hemorrhage at the base of
the skull, and were unresponsive to rtPA-induced thrombolysis were considered technical failures and excluded from the final analysis.
PET–CT Imaging After the surgical procedures, animals were placed in a dedicated small-animal PET/SPECT/CT scanner (Triumph; TriFoil Imaging), equipped with inhalation anesthesia and heating pad system. Instrument calibration was performed with phantoms containing small known amounts of radioactivity. FBP7 was injected 15 to 30 minutes after the end of the surgical procedures. Each rat was injected with ≈300 μCi probe solution in 0.3 to 0.4 mL, followed by saline flush. The total activity injected was calculated by subtracting the activity in the syringe before injection from the activity remaining in the syringe after injection as measured by a dose calibrator (Capintec CRC-25PET). A CT scan with contrast (iopamidol, Isovue 370, Bracco; 0.3 mL/ min, IV) was performed either before or at the end of the PET acquisition. Isotropic (0.3 mm) CT images were acquired for 6 minutes with 512 projections with 3 frames per projection (exposure time per frame, ≈200 ms; peak tube voltage, 70 kV; tube current, 177 mA). PET and CT images were reconstructed using the LabPET software (TriFoil Imaging) and the CT data were used to provide attenuation correction for the PET reconstructions. The PET data were reconstructed using a maximum-likelihood expectation-maximization algorithm run over 30 iterations to a voxel size of 0.5×0.5×0.6 mm3. For the pharmacokinetic analyses, the PET data were reconstructed in 1-minute (first 10 frames), 3-minute (next 10 frames), and 10-minute (last 5 frames) intervals out to 90-minute postinjection. For the embolic stroke experiments, the PET data were reconstructed in 10-minute intervals. Imaging protocols are described in Figure 1. In crush experiments, rats were imaged for 90 minutes immediately after the injection of the FBP7 or for 45 minutes at 4-hour postinjection. In stroke experiments, animals were imaged for 45 minutes to acquire a baseline image and then for 90 minutes starting at the same time of rtPA or vehicle administration. All animals were euthanized at the end of the imaging session; tissues were harvested and processed for ex vivo analyses.
Biodistribution, Blood Analyses, and Autoradiography To quantify the blood clearance and body distribution of FBP7, radioactivity in the arterial blood samples (collected at 0-, 2-, 5-, 10-, 15-, 30-, 60-, 120-, 240-, and 300-minute postinjection) and tissues obtained after euthanasia were measured.16 Blood samples and urine were analyzed by radio-high-performance liquid chromatography to evaluate the metabolic stability of FBP7.16 We also measured the
Figure 1. Timeline of the carotid crush injury (CRUSH) and embolic stroke (STROKE) experiments. All rats were injected with the positron emission tomographic (PET) probe FBP7 immediately after the surgery. In CRUSH experiments, rats were imaged immediately or 240 minutes after the injury. In STROKE experiments, animals were imaged for 45 minutes, injected with recombinant tissue-type plasminogen activator (rtPA) or vehicle, and imaged for additional 90 minutes. CT indicates computed tomography.
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Ay et al Molecular Imaging of Thrombosis and Thrombolysis 699 fraction of activity in each plasma sample that was capable of binding to fibrin immobilized on a well plate and compared this with pure probe spiked into plasma as a means of assaying for functional probe.16 The right and left common carotid arteries from crush experiments and the brain sections (2-mm-thick, coronal or horizontal) from stroke experiments were analyzed by autoradiography.16 Additional information is reported in Methods in the Data Supplement.
Data Analysis Reconstructed PET–CT data were quantitatively evaluated using PMOD 3.2 (PMOD Technologies Ltd, Zurich, Switzerland) and AMIDE software packages.20 For the MCA occlusion model, volumes of interest in the right and left MCA (0.8 mm3) and extracranial ICA (6.3 mm3) were drawn based on CT landmarks. For the crush injury model, the fused, coregistered CT and PET images were used to localize the hot spot at the site of the injured common carotid artery and a 12.5-mm3 volume of interest was placed at this hot spot and at the same level in the contralateral vessel. Brain, heart, and contralateral arterial regions were used to measure background signal. Results were expressed as percent injected dose per cubic centimeter of tissue (%ID/cc). Autoradiography data were quantified using PerkinElmer OptiQuant 5.0 software. Regions of interest were drawn around the ipsilateral (injured) and contralateral carotid arteries for crush experiments and ipsilateral and contralateral ICA/MCA segments for stroke experiments. Ipsi:contra activity ratios were obtained by dividing matched ipsilateral and contralateral raw values from each animal. Data were expressed as mean±SEM. Differences between groups were compared using Student t test and ANOVA (1-way or repeated measures) followed by Dunnett or Bonferroni post hoc test, whenever appropriate. A P value 70% of intact probe in the blood even after 4-hour postinjection Because of the improved stability, little copper-64 is released to bind to plasma proteins, whereas the intact small probe FBP7 is rapidly eliminated by the kidneys and this results in rapid systemic clearance of the activity. The affinity for fibrin is sufficient such that activity in the thrombus persists over ≥5 hours resulting in high target:background ratios that increase with time. The fast systemic clearance is a major pharmacokinetic advantage for the small FBP7 probe, especially in comparison with other thrombus-targeted imaging approaches (such as antibodies that exhibit long half-lives in blood causing low target-to-background ratios).33,34 Compared with other commonly used PET radionuclides (eg, fluorine-18), copper-64 has a longer half-life (110 minutes versus 12.7 hours, respectively). This means that the probe can be synthesized in advance and be ready to use when needed or even produced remotely and then shipped on demand. The latter feature is useful for institutions that are geographically isolated from a cyclotron. Although a longer half-life can raise radiation exposure concerns, FBP7 showed rapid whole body elimination with little activity retained in the kidneys and liver at the 5-hour time point. However, because of its chemical properties, fast clearance, and the rapid target-specific accumulation, our fibrin-binding peptide could be readily labeled with radionuclides with shorter half-lives (eg, fluorine-18, gallium-68), although we expect blood clearance to be slower in humans compared with rats because of lower cardiac output. The peptide used in FBP7 is a derivative of that used in the MR probe EP-2104R. EP-2104R was evaluated in small phase II trial for the detection of thrombus in the deep veins, the lungs, cardiac chambers, ascending aorta, and carotid arteries.35 Although EP-2104R showed promise in thrombus detection, clots were more conspicuous at 2 hours or later postprobe injection. For MR imaging, where a baseline image is required, this resulted in 2 imaging sessions and is not ideal for patient workflow or for comparing the preprobe and postprobe images. The PET technique does not suffer this limitation as a baseline scan is not required. There is also a much lower safety risk of the PET probe because of the much lower mass dose compared with MR imaging, although this is offset by the radiation exposure with PET. In our study, we found about 2- to 3-fold lower thrombus:background ratios for the intracranial clots compared with the extracranial thrombi when measured by PET imaging. However, ex vivo autoradiography showed comparable ipsi:contra ratios between the intra- and extracranial thrombi. Histology also revealed that the intracranial clots were fibrin rich. This discrepancy between the PET imaging and autoradiography is due to a partial volume effect related to the vessel size (internal diameter in adult rats are >600 μm for extracranial ICA, ≈200–360 μm for intracranial ICA and MCA),36 where the thrombus is smaller than the resolution of the micro-PET camera used for the present study (≈1.2 mm). For smaller thrombi, this partial volume loss is more pronounced and will ultimately limit the size of thrombus that can be detected. However, the high target:background achieved with FBP7 already demonstrates that we can still detect pathology well below the resolution limit of the PET camera (eg,
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704 Circ Cardiovasc Imaging July 2014 the MCA thrombus occupies at most 1/10th of a PET voxel). Combining PET with CT also provided valuable anatomic context that enables confident diagnosis of small thrombi.
Study Limitations There are some limitations in the present study, including the small sample size and the animal models, which do not entirely mimic the clinical setting. We did not perform any specific experiment to assess whether FBP7 is able to visualize clots older than 24 hours, but previous works have shown that fibrin-targeted probes can detect aged and organized thrombi.37 Thrombus aging is a dynamic process and time-dependent changes in thrombus composition (eg, fibrin content) affect clot stability and thus thrombolysis.38 Future studies are needed to assess the time window of efficacy for FBP7, as well as its potential to discriminate between different stages of thrombus formation based on fibrin content.
Conclusions In this study, we demonstrated the feasibility of PET imaging of thrombosis and thrombolysis using a new fibrin-binding probe. FBP7 imaging detected both nonocclusive (mural) and occlusive thrombi; furthermore, FBP7 enabled both visualization and quantification of in vivo thrombolysis after rtPA treatment. Indeed, FBP7 represents a promising candidate for translation in clinical testing.
Acknowledgments Prof. Carolyn J. Anderson of the University of Pittsburgh Medical Center is gratefully acknowledged for a gift of the cross-bridged chelator CB-TE2A used in this study.
Sources of Funding This work was supported by HL109448 (to P. Caravan) from the National Heart, Lung, and Blood Institute, and the micro-PET/ SPECT/CT system was funded by RR029495 (to A.L. Brownell) from the National Center for Research Resources. This research was carried out in whole at the Athinoula A. Martinos Center for Biomedical Imaging at the Massachusetts General Hospital, using resources provided by the Center for Functional Neuroimaging Technologies, P41RR14075, a P41 Regional Resource supported by the Biomedical Technology Program of the National Center for Research Resources, National Institutes of Health.
Disclosures P. Caravan has equity in Factor 1A, LLC, the company holding the patent rights to the peptide used in this work. The other authors report no conflicts.
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Clinical Perspective FBP7 can be used to detect source thrombus in patients admitted to the hospital with cardiovascular or neurological symptoms typical of thrombosis. In these patients, etiologic work-up includes the parallel use of several imaging modalities that may be expensive and time consuming, causing delays in secondary prevention strategies. Instead of performing sequential imaging studies using ultrasound, transesophageal echocardiogram, computed tomography/magnetic resonance imaging, a bimodal positron emission tomographic scan using FBP7 as a molecular contrast agent can help visualizing both the thrombus causing the symptoms (eg, an embolus lodged in the lungs or in the brain), the original thrombus (eg, a thrombus in the deep vein of the leg, carotid artery, or the aortic arch), and tissue ischemia in 1 session. Another possible application of FBP7 is in combination with angiography (eg, positron emission tomographic–magnetic resonance angiography) for patients undergoing thrombolytic therapy. In these cases, in addition to image post–recombinant tissue-type plasminogen activator recanalization with angiography, it would be also possible to detect the presence of nonocclusive residual thrombi in the vascular bed. This can provide additional information to predict the chance of possible recurrent thrombotic episodes.
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SUPPLEMENTAL MATERIAL
In vivo molecular imaging of thrombosis and thrombolysis using a fibrin-binding PET probe
Ilknur Ay*, MD PhD; Francesco Blasi*, PharmD PhD; Tyson A. Rietz, BS; Nicholas J. Rotile, BA; Sreekanth Kura, MS, Anna Liisa Brownell, PhD, Helen Day, MS; Bruno L. Oliveira, PhD; Richard J. Looby, BS; Peter Caravan, PhD
Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital East, 149 13th Street, Charlestown, Massachusetts 02129, USA
1
SUPPLEMENTAL METHODS
Reagents CB-TE2A [C16H30N4O4 (2,2'-(1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diyl)diacetic acid)] was a gift of Prof. Carolyn Anderson, University of Pittsburgh Medical Center. 64CuCl2 was obtained from the University of Wisconsin, Medical Physics Department. All other chemicals were purchased commercially and used without further purification.
High Performance Liquid Chromatography (HPLC) Preparative HPLC purification was performed on a Varian Prostar system with two Prostar 210 pumps and a Prostar 325 UV/Vis detector, using a Phenomenex Luna C18 column (250 x 21.2 mm, 10 μm). Liquid chromatography-electrospray mass spectrometry (LC-MS) was performed using an Agilent 1100 Series apparatus with a Phenomenex Luna C18 column (100 mm × 2 mm, 5 μm). This system is equipped with an LC/MSD trap and a Daly conversion dynode detector. Radio-HPLC analyses were obtained on an analytical Agilent 1100 Series system using a Phenomenex Luna C18 column (150 x 4.6 mm, 5 μm; to check radiochemical yields and purity) or a MetaChem Polaris C18 (150 x 4.6 mm, 5 μm; for analysis of urine and blood samples). Four different HPLC methods were used depending on whether HPLC was being used for preparative purification (Method 1), to assess purity (Method 2), to check radiochemical yields and purity (Method 3) or to analyze urine and blood metabolites (Method 4). For all the methods the mobile phase A was H2O with 0.1% trifluoroacetic acid (TFA) and mobile phase B was CH3CN with 0.1% TFA. Method 1 (flow rate of 15 mL/min): 0 – 3 min, 5% B; 3 – 15 min, 5 to
2
95% B; 15 – 18 min, 95% B; 18 – 19 min, 95 to 5% B; 19 – 22 min, 5% B. Method 2 (flow rate of 0.8 mL/min): 0 – 1 min, 5% B; 1 – 10 min, 5 to 95% B; 10 – 12 min, 95% B; 12 – 12.5 min, 95 to 5% B; 12.5 – 15 min, 5% B. Method 3 (flow rate of 1.0 mL/min): 0 – 12 min, 5 to 80% B; 12 - 12.1 min, 80 to 5% B; 12.1 – 13 min, 5% B. Method 4 (flow rate of 1 mL/min): 0 – 17 min, 0 to 100% B; 17 – 17.1 min, 100 to 0% B; 17.1 – 18 min, 0% B.
Probe Synthesis The cyclic disulfide peptide precursor F.H.C.Hyp.Y(3-CI).D.L.C.H.I.L.PXD (Hyp=L-4hydroxyproline, Y(3-Cl)=L-3-chlorotyrosine, PXD=paraxylenediamine) was prepared with the use of a rink amide MBHA resin, Fmoc coupling strategy, and aqueous DMSO cyclization. This peptide (24 mg), CB-TE2A (17.2 mg, 3eq), and Pybop (3eq) were added to 5 ml DMF while the pH of the reaction (monitored by spotting the reaction on a wet pH stick) was adjusted to ca. 6 with diisopropylethylamine. The reaction was stirred at RT for 16 h. The reaction was concentrated to an oil and purified using Method 1. Lyophilization of fractions containing the desired product provided 10 mg of white powder (theoretical MW for [M + 2H]2+ = 1089.3, observed 1089.0).1 The peptide-(CB2)2 ligand (25 μL, 1.0 mM in 10mM in NH4OAc) was diluted in 400 μL of 40 mM NH4OAc, pH 4.5.
CuCl2 was added (0.5 - 5 mCi) and heated at 95 C for 60 minutes.
64
The reaction was monitored by radio-HPLC, which showed 90 - 95% conversion to the desired product. EDTA (1 mL of 10 mM solution) was added and the mixture stirred an additional 20 min to scavenge any unchelated copper ion. The compound was purified using Method 3, concentrated via rotary evaporation, and reconstituted with 10 mM NH4OAc and saline (1:1). This purified material was again checked by HPLC (Method 3) for final radiochemical purity.
3
In vitro stability assay FBP7 was incubated with citrated rat plasma (Innovative Research Inc) at 37°C. Aliquots (100 μL) were collected after 0, 2 and 16 hours of incubation and mixed with 100 μL ice-cold methanol to precipitate plasma protein. Proteins were then sedimented by centrifugation (10,000 g x 5 min) and the supernatant was injected into HPLC (Method 4).1
DD(E) Binding Assay The affinity of FBP7 to the soluble fibrin fragment DD(E) was assessed in the presence and absence of heparin using a fluorescence polarization assay as previously described.1, 2 Here we used FBP7 labeled with natural abundance 63,65Cu. This assay measures the displacement of a tetramethylrhodamine-labeled peptide (TRITC-Tn6) from DD(E) upon addition of FBP7 by observing the corresponding change in fluorescence anisotropy. The Kd of the TRITC-Tn6 probe binding to DD(E) is 0.92 µM2, and based on this Kd and the observed displacement as a function of FBP7 concentration, an inhibition constant Ki for FBP7 can be determined.2 The experiment was performed at room temperature using a probe concentration of 0.1 μM in the following assay buffer: Tris base (50 mM), NaCl (100 mM), CaCl2 (2 mM), Triton X-100 (0.01%), pH=7.8. For the heparin competition, the assay buffer was mixed in 33 IU sodium heparin blood collection tubes to allow for a 5U/ml final concentration. The anisotropy measurements were made using a TECAN Infinity F200 Pro plate reader equipped with the appropriate filter set for tetramethylrhodamine (excitation 535 nm, emission 590 nm). A series of solutions of FBP7 were prepared through serial dilutions from a stock solution. These solutions were then added to a mixture of the DD(E) protein and TRITC-Tn6 peptide in assay buffer with or without heparin. The final concentrations of DD(E) and fluorescent probe used in these experiments were 2 μM
4
and 0.1 μM, respectively. All measurements were performed in a 384-well plate. The inhibition constant, Ki of FBP7, were then calculated using least-squares regression and the known Kd of the fluorescent probe.
Biodistribution analysis Serial blood samples were collected from the femoral artery at 0, 2, 5, 10, 15, 30, 60, 120, 240, and 300 min post-injection into EDTA blood tubes. Blood was weighed and radioactivity in the blood was measured on a gamma counter (Packard, CobraII Auto gamma) to assess clearance of total activity. To quantify organ and body fluid distribution of copper-64, blood, chest and abdominal organs, brain, left rectus femoris muscle, and left femur were collected from all animals after euthanasia. Additionally, the right common carotid artery containing the intramural thrombus and the contralateral common carotid artery were collected from animals that had carotid crush injury. The tissues were weighed and radioactivity in each tissue was measured using a gamma counter, along with an aliquot of the injected FBP7 solution.1 The radioactivity was reported as percent injected dose per gram (%ID/g) which was calculated by dividing the counts of copper-64 per gram of tissue by the total counts of the injected dose, all decay corrected to the time of injection.
5
Autoradiography Autoradiography of the common carotid arteries and the brain sections was performed using a multipurpose film and a Perkin-Elmer Cyclone Plus Phosphor Imager and PerkinElmer OptiQuant 5.0 software. Exposure time was between 1-10 min.1
Metabolic Stability Assay Blood samples collected at 2, 15, 120 and 240 min were immediately centrifuged for 10 min at 2000 rpm and the supernatant passed through a 0.22-μm Millipore filter and injected onto an analytical HPLC column at a flow rate of 1 mL/min using Method 4. The eluent was collected every 0.5 min and the activity of each fraction was measured by the gamma counter. The HPLC analysis was performed in duplicate or triplicate. Data obtained from the gamma counter were plotted to reconstruct the HPLC chromatograms.1
Functional Fibrin Binding Assay Human fibrinogen (American Diagnostica) was dialyzed against Tris (50 mM, pH 7.4), sodium chloride (150 mM), and sodium citrate (TBS·citrate; 5 mM), and the fibrinogen concentration was adjusted to 5 mg/mL (based on the absorbance at 280 nm and ε280 = 1.512 L/g/cm). After adding CaCl2 (7 mM), 50 μL of this fibrinogen solution was dispensed into a 96-well polystyrene microplate (Immulon-II). Human thrombin solution (50 μL; 2 U/mL in TBS) was added into each well to clot the fibrinogen yielding final fibrin concentration close to 2.5 mg/mL (7.3 μM based on fibrinogen MW = 340 kDa). The plates were incubated at 37°C overnight and evaporated to dryness. Blood plasma that was obtained after centrifugation (2000 rpm for 20 min at 4°C) diluted in TBS (1:1) was incubated in fibrin immobilized wells as well as in empty
6
wells for 2h on a shaker at 300 rpm and room temperature. The counts in the supernatant in both the fibrin-containing and empty wells were measured on a gamma counter and divided by the weight of plasma to determine the concentration of unbound probe and total probe, respectively. The amount of copper-64 containing species bound to fibrin was calculated from [bound] = [total] − [unbound]. The amount of functional probe in the blood at time T was determined by taking the ratio of the % bound to fibrin at time T compared to the % bound at T = 0, and multiplying this ratio by the measured total copper-64 %ID/g in the blood.1
Histopathology Ipsilateral (right) and contralateral (left) common carotid arteries were collected two hours after crush injury (n=2); brains were harvested one hour after embolic stroke induction (n=2). Samples were carefully rinsed in Phosphate Buffer, embedded in OCT mounting media (Tissue-Tek), and snap-frozen in -45°C isopentane. The brains and the arteries were cryosectioned (20 μm thickness) and processed for Hematoxylin and Eosin staining according to the standard protocol. For fibrin immunostaining, adjacent slices were incubated with a mouse anti-fibrin alpha-chain antibody (Abcam, U45). Griffonia Simplicifolia Lectin 1 (Vector Laboratories) was used to stain the vascular endothelium; Hoechst 33342 (Sigma) was used as nuclear marker. Negative control experiments were performed by omitting the primary antibody anti-fibrin U45, resulting in the absence of specific fibrin immunostaining. Images were acquired using a Nikon TE-2000 microscope equipped with epifluorescence illumination.
7
Supplemental Figures and Figure Legends
Figure I. Synthesis and chemical structure of FBP7.
8
Figure II. Displacement of TRITC-Tn6 peptide (0.2 μM) from DD(E) by FBP7 in absence (clear symbols) and presence of heparin (5U/mL, solid symbols). The reduction in fluorescence anisotropy is used to calculate the inhibition constant (Ki). The binding of FBP7 to DD(E) in presence (Ki = 620 nM) or absence (Ki = 660 nM) of heparin was comparable.
9
Figure III. (A) Data showing blood clearance (clear symbols) of FBP7 and result of fibrinbinding assay (solid symbols). (B) Radio-HPLC analysis from serial blood draws showing the percentage of intact probe at 2, 15, 120 and 240 min post-injection. (C) Representative radioHPLC tracing of plasma and urine showing high levels of probe and just few metabolites up to 4 hours post-injection.
10
Figure IV. Ex vivo autoradiography (upper panels) of crushed (RC) and contralateral (LC) common carotid arteries at 2 hours and 5 hours post-injection confirmed high thrombus uptake of FBP7. Lower panels show the photographs of arteries taken before autoradiography.
11
B
A
C
D
Figure V. Macroscopical inspection of the carotid arteries suggests thrombosis at the level of the ipsilateral common carotid artery, but not contralaterally (center panel). Hematoxylin and Eosin staining reveals the presence of arterial thrombosis after carotid crush injury (A, top). High magnification microphotograph shows that mural thrombi are mainly localized at the level of the endothelial cells layer (A, arrows, bottom); this finding is consistent with previously reported models of mural thrombosis3. Fibrin immunostaining performed on adjacent histological sections reveals an immunopositive signal (green) in proximity of the endothelial cells layer (B, arrows), consistent with fibrin deposition. Histopathology did not reveal the presence of mural thrombi (C) or fibrin (D) in the contralateral common carotid artery. Green, fibrin; red, endothelial cells layer; blue, nuclei. Arrows in A, mural thrombi; arrows in B, fibrin deposits. Scale bars: 0.2 (A and C, top), 0.05 (A and C, bottom), and 0.025 mm (B and D).
12
Figure VI. Hematoxylin and Eosin staining reveals an occlusive thrombus in the right middle cerebral artery of rats subjected to embolic stroke (top panel). Insets (middle panels) show high magnification (40X) images of the contralateral (left) and ipsilateral (right) cerebral arteries. The entire lumen of the right middle cerebral artery is occluded by the embolus. Adjacent sections were immunostained for fibrin (bottom panels). An immunopositive signal (green) was present in the lumen of the ipsilateral middle cerebral artery (right), but not contralaterally (left). Green, fibrin; red, endothelial cells layer; blue, nuclei. Scale bars: 0.2 (top) and 0.05 mm (middle and bottom).
13
SUPPLEMENTAL VIDEO LEGENDS
Video I. 3D reconstruction of a representative rat following carotid crush injury and FBP7 injection. Carotid thrombus is clearly visible and distinguishable from background organs and tissues. A superficial hyperintense area is also present at the level of the surgical incision.
14
SUPPLEMENTAL REFERENCES
1.
Ciesienski KL, Yang Y, Ay I, Chonde DB, Loving GS, Rietz TA, Catana C and Caravan
P. Fibrin-targeted PET probes for the detection of thrombi. Mol Pharmaceutics. 2013;10:110010. 2.
Kolodziej AF, Nair SA, Graham P, McMurry TJ, Ladner RC, Wescott C, Sexton DJ and
Caravan P. Fibrin specific peptides derived by phage display: characterization of peptides and conjugates for imaging. Bioconjug Chem. 2012;23:548-56. 3.
Yamashita A, Furukoji E, Marutsuka K, Hatakeyama K, Yamamoto H, Tamura S, Ikeda
Y, Sumiyoshi A and Asada Y. Increased vascular wall thrombogenicity combined with reduced blood flow promotes occlusive thrombus formation in rabbit femoral artery. Arterioscler Thromb Vasc Biol. 2004;24:2420-2424.
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The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circimaging.ahajournals.org/content/7/4/697
Data Supplement (unedited) at: http://circimaging.ahajournals.org/circcvim/suppl/2014/04/28/CIRCIMAGING.113.001806.DC1.html
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Molecular Imaging In Vivo Molecular Imaging of Thrombosis and Thrombolysis Using a Fibrin-Binding Positron Emission Tomographic Probe Ilknur Ay, MD, PhD*; Francesco Blasi, PharmD, PhD*; Tyson A. Rietz, BS; Nicholas J. Rotile, BA; Sreekanth Kura, MS; Anna Liisa Brownell, PhD; Helen Day, MS; Bruno L. Oliveira, PhD; Richard J. Looby, BS; Peter Caravan, PhD Background—Fibrin is a major component of arterial and venous thrombi and represents an ideal candidate for molecular imaging of thrombosis. Here, we describe imaging properties and target uptake of a new fibrin-specific positron emission tomographic probe for thrombus detection and therapy monitoring in 2 rat thrombosis models. Methods and Results—The fibrin-binding probe FBP7 was synthesized by conjugation of a known short cyclic peptide to a cross-bridged chelator (CB-TE2A), followed by labeling with copper-64. Adult male Wistar rats (n=26) underwent either carotid crush injury (mural thrombosis model) or embolic stroke (occlusive thrombosis model) followed by recombinant tissue-type plasminogen activator treatment (10 mg/kg, IV). FBP7 detected thrombus location in both animal models with a high positron emission tomographic target-to-background ratio that increased over time (>5-fold at 30–90 minutes, >15-fold at 240–285 minutes). In the carotid crush injury animals, biodistribution analysis confirmed high probe uptake in the thrombotic artery (≈0.5%ID/g; >5-fold greater than blood and other tissues of the head and thorax). Similar results were obtained from ex vivo autoradiography of the ipsilateral versus contralateral carotid arteries. In embolic stroke animals, positron emission tomographic–computed tomographic imaging localized the clot in the internal carotid/middle cerebral artery segment of all rats. Time-dependent reduction of activity at the level of the thrombus was detected in recombinant tissue-type plasminogen activator–treated rats but not in vehicle-injected animals. Brain autoradiography confirmed clot dissolution in recombinant tissue-type plasminogen activator–treated animals, but enduring high thrombus activity in control rats. Conclusions—We demonstrated that FBP7 is suitable for molecular imaging of thrombosis and thrombolysis in vivo and represents a promising candidate for bench-to-bedside translation. (Circ Cardiovasc Imaging. 2014;7:697-705.) Key Words: fibrin ◼ stroke ◼ thrombosis
T
hrombosis, the underlying cause in the majority of cardiovascular disorders, has a high incidence and prevalence in the United States.1 Recent advances in medical imaging have facilitated the diagnosis of thrombotic and thromboembolic disorders; however, some challenges still remain. First, although recurrences occur in most cases, diagnostic techniques including magnetic resonance (MR) imaging and computed tomography (CT) are not efficient to identify the source thrombus, especially when used as a single test.2,3 Identification of source thrombus requires a technique with whole body scanning and high sensitivity. Second, modalities that are used to detect thrombus, including carotid or pelvic ultrasound, transesophageal echocardiogram, CT, and MRI, have some pitfalls with respect to sensitivity, specificity, and feasibility.4–7 Multimodal imaging with a molecular probe designed to detect thrombus offers a potential solution to both of these challenges.8,9 To date, coagulation factors (thrombin,
Factor XIII, fibrinogen, fibrin) and activated platelets have been targeted for molecular thrombus imaging.10 Among them, fibrin represents an ideal target because of its high specificity (present at high concentration in all clots but not in circulating blood) and high sensitivity (present in all thrombi whether arterial or venous, fresh or aged).11
Editorial see p 581 Clinical Perspective on p 705 Positron emission tomography (PET) is a quantitative, high sensitivity technique that offers the possibility for whole body scanning. Hybrid imaging that combines PET with CT, or more recently PET with MR, provides a high resolution anatomic image (CT or MR) onto which the lower resolution molecular PET image can be overlaid. Thus, a sensitive thrombus-specific PET probe may detect even minor thrombotic events and CT or MR can localize this PET activity to a specific location in the
Received December 11, 2013; accepted April 24, 2014. From the Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA. The Data Supplement is available at http://circimaging.ahajournals.org/lookup/suppl/doi:10.1161/CIRCIMAGING.113.001806/-/DC1 *Drs Ay and Blasi contributed equally to this work. Correspondence to Peter Caravan, PhD, Building 149, Room 2301, 13th St, Charlestown, MA 02129. E-mail [email protected] © 2014 American Heart Association, Inc. Circ Cardiovasc Imaging is available at http://circimaging.ahajournals.org
DOI: 10.1161/CIRCIMAGING.113.001806
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698 Circ Cardiovasc Imaging July 2014 vascular tree. An ideal fibrin/thrombus PET probe should have high affinity for fibrin, high specificity for fibrin over fibrinogen and other blood proteins, rapid clearance from the circulation to increase thrombus-to-background ratio, and small size to enable penetration into thrombi. To this end, we have labeled a short cyclic peptide that was previously shown to exhibit high affinity for fibrin and great specificity for fibrin versus fibrinogen (>100-fold) and other serum proteins.12–14 We recently demonstrated preliminary efficacy of PET and bimodal PET–MR probes based on these fibrin-specific binding peptides for molecular imaging of thrombus, although these probes suffered from instability in vivo.15–17 In the present study, we report the efficacy of a newly developed, chemically stable, fibrin-binding PET probe termed FBP7 in 2 different animal models of arterial thrombosis: carotid crush injury as a nonocclusive intramural thrombosis model and embolic stroke as an occlusive thrombosis model. To extend the translational outcome of our work, we also tested whether FBP7 was able to visualize and quantify in vivo thrombolysis after recombinant tissue-type plasminogen activator (rtPA) treatment in embolic stroke.
Methods Synthesis and Characterization of FBP7 FBP7 was synthesized by conjugation of the CB-TE2A chelator to a cyclic, disulfide bridged 11-amino acid fibrin-binding peptide, followed by labeling with copper-64 and high-performance liquid chromatography purification.16 The cyclic peptide was previously shown to be effective for fibrin targeting.14,16 FBP7 was evaluated for in vitro fibrin affinity in the presence and absence of heparin and also for its plasma stability. Additional information about reagents and methods are reported in Methods in the Data Supplement.
Animal Thrombosis Models All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Massachusetts General Hospital. Adult male Wistar rats (330–400 g; Charles River Laboratories) were anesthetized by isoflurane (4% for induction, 2%–2.5% for maintenance in medical air) and were kept under anesthesia throughout the study. Body temperature was maintained at 37°C using a thermoregulated heating pad (Harvard Apparatus). The right femoral vein and artery were catheterized using PE-50 tubing for probe/drug injection and blood sampling, respectively. Intramural thrombus was induced by carotid artery crush injury (n=10). Under isoflurane anesthesia, the right common carotid artery was exposed and crush injury was induced by clamping the vessel with a hemostat for 5 minutes, as previously described.16 Injury was performed 1 to 2 cm proximal to the carotid bifurcation, using the same hemostat and by the same investigator to minimize variability. Occlusive thrombus was produced by embolic stroke (n=16) using a previously published method.18 Briefly, homologous thrombus was prepared 1 day before by retaining freshly collected arterial blood in a PE-50 tube for 2 hours at room temperature and 22 hours at 4°C. The next day, a single 25-mm-long clot was injected into the right internal carotid artery (ICA) via an external carotid stump to occlude the middle cerebral artery (MCA) under isoflurane anesthesia. In a subgroup of animals, the clot was incubated in an Evans blue solution (2%, wt/ vol) to enhance postmortem clot visualization.19 Approximately 1 hour after the clot injection, rats were injected with either rtPA (alteplase, 10 mg/kg, IV, 10% bolus and 90% infusion over 30 minutes; n=9) or vehicle (saline solution, 1 mL; n=7). Because the embolic stroke model is associated with a small but significant mortality and failure rate, rats that died during the study period, had hemorrhage at the base of
the skull, and were unresponsive to rtPA-induced thrombolysis were considered technical failures and excluded from the final analysis.
PET–CT Imaging After the surgical procedures, animals were placed in a dedicated small-animal PET/SPECT/CT scanner (Triumph; TriFoil Imaging), equipped with inhalation anesthesia and heating pad system. Instrument calibration was performed with phantoms containing small known amounts of radioactivity. FBP7 was injected 15 to 30 minutes after the end of the surgical procedures. Each rat was injected with ≈300 μCi probe solution in 0.3 to 0.4 mL, followed by saline flush. The total activity injected was calculated by subtracting the activity in the syringe before injection from the activity remaining in the syringe after injection as measured by a dose calibrator (Capintec CRC-25PET). A CT scan with contrast (iopamidol, Isovue 370, Bracco; 0.3 mL/ min, IV) was performed either before or at the end of the PET acquisition. Isotropic (0.3 mm) CT images were acquired for 6 minutes with 512 projections with 3 frames per projection (exposure time per frame, ≈200 ms; peak tube voltage, 70 kV; tube current, 177 mA). PET and CT images were reconstructed using the LabPET software (TriFoil Imaging) and the CT data were used to provide attenuation correction for the PET reconstructions. The PET data were reconstructed using a maximum-likelihood expectation-maximization algorithm run over 30 iterations to a voxel size of 0.5×0.5×0.6 mm3. For the pharmacokinetic analyses, the PET data were reconstructed in 1-minute (first 10 frames), 3-minute (next 10 frames), and 10-minute (last 5 frames) intervals out to 90-minute postinjection. For the embolic stroke experiments, the PET data were reconstructed in 10-minute intervals. Imaging protocols are described in Figure 1. In crush experiments, rats were imaged for 90 minutes immediately after the injection of the FBP7 or for 45 minutes at 4-hour postinjection. In stroke experiments, animals were imaged for 45 minutes to acquire a baseline image and then for 90 minutes starting at the same time of rtPA or vehicle administration. All animals were euthanized at the end of the imaging session; tissues were harvested and processed for ex vivo analyses.
Biodistribution, Blood Analyses, and Autoradiography To quantify the blood clearance and body distribution of FBP7, radioactivity in the arterial blood samples (collected at 0-, 2-, 5-, 10-, 15-, 30-, 60-, 120-, 240-, and 300-minute postinjection) and tissues obtained after euthanasia were measured.16 Blood samples and urine were analyzed by radio-high-performance liquid chromatography to evaluate the metabolic stability of FBP7.16 We also measured the
Figure 1. Timeline of the carotid crush injury (CRUSH) and embolic stroke (STROKE) experiments. All rats were injected with the positron emission tomographic (PET) probe FBP7 immediately after the surgery. In CRUSH experiments, rats were imaged immediately or 240 minutes after the injury. In STROKE experiments, animals were imaged for 45 minutes, injected with recombinant tissue-type plasminogen activator (rtPA) or vehicle, and imaged for additional 90 minutes. CT indicates computed tomography.
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Ay et al Molecular Imaging of Thrombosis and Thrombolysis 699 fraction of activity in each plasma sample that was capable of binding to fibrin immobilized on a well plate and compared this with pure probe spiked into plasma as a means of assaying for functional probe.16 The right and left common carotid arteries from crush experiments and the brain sections (2-mm-thick, coronal or horizontal) from stroke experiments were analyzed by autoradiography.16 Additional information is reported in Methods in the Data Supplement.
Data Analysis Reconstructed PET–CT data were quantitatively evaluated using PMOD 3.2 (PMOD Technologies Ltd, Zurich, Switzerland) and AMIDE software packages.20 For the MCA occlusion model, volumes of interest in the right and left MCA (0.8 mm3) and extracranial ICA (6.3 mm3) were drawn based on CT landmarks. For the crush injury model, the fused, coregistered CT and PET images were used to localize the hot spot at the site of the injured common carotid artery and a 12.5-mm3 volume of interest was placed at this hot spot and at the same level in the contralateral vessel. Brain, heart, and contralateral arterial regions were used to measure background signal. Results were expressed as percent injected dose per cubic centimeter of tissue (%ID/cc). Autoradiography data were quantified using PerkinElmer OptiQuant 5.0 software. Regions of interest were drawn around the ipsilateral (injured) and contralateral carotid arteries for crush experiments and ipsilateral and contralateral ICA/MCA segments for stroke experiments. Ipsi:contra activity ratios were obtained by dividing matched ipsilateral and contralateral raw values from each animal. Data were expressed as mean±SEM. Differences between groups were compared using Student t test and ANOVA (1-way or repeated measures) followed by Dunnett or Bonferroni post hoc test, whenever appropriate. A P value 70% of intact probe in the blood even after 4-hour postinjection Because of the improved stability, little copper-64 is released to bind to plasma proteins, whereas the intact small probe FBP7 is rapidly eliminated by the kidneys and this results in rapid systemic clearance of the activity. The affinity for fibrin is sufficient such that activity in the thrombus persists over ≥5 hours resulting in high target:background ratios that increase with time. The fast systemic clearance is a major pharmacokinetic advantage for the small FBP7 probe, especially in comparison with other thrombus-targeted imaging approaches (such as antibodies that exhibit long half-lives in blood causing low target-to-background ratios).33,34 Compared with other commonly used PET radionuclides (eg, fluorine-18), copper-64 has a longer half-life (110 minutes versus 12.7 hours, respectively). This means that the probe can be synthesized in advance and be ready to use when needed or even produced remotely and then shipped on demand. The latter feature is useful for institutions that are geographically isolated from a cyclotron. Although a longer half-life can raise radiation exposure concerns, FBP7 showed rapid whole body elimination with little activity retained in the kidneys and liver at the 5-hour time point. However, because of its chemical properties, fast clearance, and the rapid target-specific accumulation, our fibrin-binding peptide could be readily labeled with radionuclides with shorter half-lives (eg, fluorine-18, gallium-68), although we expect blood clearance to be slower in humans compared with rats because of lower cardiac output. The peptide used in FBP7 is a derivative of that used in the MR probe EP-2104R. EP-2104R was evaluated in small phase II trial for the detection of thrombus in the deep veins, the lungs, cardiac chambers, ascending aorta, and carotid arteries.35 Although EP-2104R showed promise in thrombus detection, clots were more conspicuous at 2 hours or later postprobe injection. For MR imaging, where a baseline image is required, this resulted in 2 imaging sessions and is not ideal for patient workflow or for comparing the preprobe and postprobe images. The PET technique does not suffer this limitation as a baseline scan is not required. There is also a much lower safety risk of the PET probe because of the much lower mass dose compared with MR imaging, although this is offset by the radiation exposure with PET. In our study, we found about 2- to 3-fold lower thrombus:background ratios for the intracranial clots compared with the extracranial thrombi when measured by PET imaging. However, ex vivo autoradiography showed comparable ipsi:contra ratios between the intra- and extracranial thrombi. Histology also revealed that the intracranial clots were fibrin rich. This discrepancy between the PET imaging and autoradiography is due to a partial volume effect related to the vessel size (internal diameter in adult rats are >600 μm for extracranial ICA, ≈200–360 μm for intracranial ICA and MCA),36 where the thrombus is smaller than the resolution of the micro-PET camera used for the present study (≈1.2 mm). For smaller thrombi, this partial volume loss is more pronounced and will ultimately limit the size of thrombus that can be detected. However, the high target:background achieved with FBP7 already demonstrates that we can still detect pathology well below the resolution limit of the PET camera (eg,
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704 Circ Cardiovasc Imaging July 2014 the MCA thrombus occupies at most 1/10th of a PET voxel). Combining PET with CT also provided valuable anatomic context that enables confident diagnosis of small thrombi.
Study Limitations There are some limitations in the present study, including the small sample size and the animal models, which do not entirely mimic the clinical setting. We did not perform any specific experiment to assess whether FBP7 is able to visualize clots older than 24 hours, but previous works have shown that fibrin-targeted probes can detect aged and organized thrombi.37 Thrombus aging is a dynamic process and time-dependent changes in thrombus composition (eg, fibrin content) affect clot stability and thus thrombolysis.38 Future studies are needed to assess the time window of efficacy for FBP7, as well as its potential to discriminate between different stages of thrombus formation based on fibrin content.
Conclusions In this study, we demonstrated the feasibility of PET imaging of thrombosis and thrombolysis using a new fibrin-binding probe. FBP7 imaging detected both nonocclusive (mural) and occlusive thrombi; furthermore, FBP7 enabled both visualization and quantification of in vivo thrombolysis after rtPA treatment. Indeed, FBP7 represents a promising candidate for translation in clinical testing.
Acknowledgments Prof. Carolyn J. Anderson of the University of Pittsburgh Medical Center is gratefully acknowledged for a gift of the cross-bridged chelator CB-TE2A used in this study.
Sources of Funding This work was supported by HL109448 (to P. Caravan) from the National Heart, Lung, and Blood Institute, and the micro-PET/ SPECT/CT system was funded by RR029495 (to A.L. Brownell) from the National Center for Research Resources. This research was carried out in whole at the Athinoula A. Martinos Center for Biomedical Imaging at the Massachusetts General Hospital, using resources provided by the Center for Functional Neuroimaging Technologies, P41RR14075, a P41 Regional Resource supported by the Biomedical Technology Program of the National Center for Research Resources, National Institutes of Health.
Disclosures P. Caravan has equity in Factor 1A, LLC, the company holding the patent rights to the peptide used in this work. The other authors report no conflicts.
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patterns, risk factors, and outcomes of stroke recurrence in the South London Stroke Register. Stroke. 2003;34:1457–1463. 3. Sipola P, Hedman M, Onatsu J, Turpeinen A, Halinen M, Jäkälä P, Vanninen R. Computed tomography and echocardiography together reveal more high-risk findings than echocardiography alone in the diagnostics of stroke etiology. Cerebrovasc Dis. 2013;35:521–530. 4. Markel A, Weich Y, Gaitini D. Doppler ultrasound in the diagnosis of venous thrombosis. Angiology. 1995;46:65–73. 5. Kronzon I, Tunick PA. Aortic atherosclerotic disease and stroke. Circulation. 2006;114:63–75. 6. Srichai MB, Junor C, Rodriguez LL, Stillman AE, Grimm RA, Lieber ML, Weaver JA, Smedira NG, White RD. Clinical, imaging, and pathological characteristics of left ventricular thrombus: a comparison of contrast-enhanced magnetic resonance imaging, transthoracic echocardiography, and transesophageal echocardiography with surgical or pathological validation. Am Heart J. 2006;152:75–84. 7. Litmanovich D, Bankier AA, Cantin L, Raptopoulos V, Boiselle PM. CT and MRI in diseases of the aorta. AJR Am J Roentgenol. 2009; 193:928–940. 8. Beyer T, Townsend DW, Czernin J, Freudenberg LS. The future of hybrid imaging-part 2: PET/CT. Insights Imaging. 2011;2:225–234. 9. Beyer T, Freudenberg LS, Czernin J, Townsend DW. The future of hybrid imaging-part 3: PET/MR, small-animal imaging and beyond. Insights Imaging. 2011;2:235–246. 10. Lindner JR. Molecular imaging of thrombus: technology in evolution. Circulation. 2012;125:3057–3059. 11. Ciesienski KL, Caravan P. Molecular MRI of Thrombosis. Curr Cardiovasc Imaging Rep. 2010;4:77–84. 12. Kolodziej AF, Nair SA, Graham P, McMurry TJ, Ladner RC, Wescott C, Sexton DJ, Caravan P. Fibrin specific peptides derived by phage display: characterization of peptides and conjugates for imaging. Bioconjug Chem. 2012;23:548–556. 13. Overoye-Chan K, Koerner S, Looby RJ, Kolodziej AF, Zech SG, Deng Q, Chasse JM, McMurry TJ, Caravan P. EP-2104R: a fibrin-specific gadolinium-Based MRI contrast agent for detection of thrombus. J Am Chem Soc. 2008;130:6025–6039. 14. Botnar RM, Perez AS, Witte S, Wiethoff AJ, Laredo J, Hamilton J, Quist W, Parsons EC Jr, Vaidya A, Kolodziej A, Barrett JA, Graham PB, Weisskoff RM, Manning WJ, Johnstone MT. In vivo molecular imaging of acute and subacute thrombosis using a fibrin-binding magnetic resonance imaging contrast agent. Circulation. 2004;109:2023–2029. 15. Uppal R, Catana C, Ay I, Benner T, Sorensen AG, Caravan P. Bimodal thrombus imaging: simultaneous PET/MR imaging with a fibrin-targeted dual PET/MR probe–feasibility study in rat model. Radiology. 2011;258:812–820. 16. Ciesienski KL, Yang Y, Ay I, Chonde DB, Loving GS, Rietz TA, Catana C, Caravan P. Fibrin-targeted PET probes for the detection of thrombi. Mol Pharm. 2013;10:1100–1110. 17. Boros E, Rybak-Akimova E, Holland JP, Rietz T, Rotile N, Blasi F, Day H, Latifi R, Caravan P. Pycup-A Bifunctional, Cage-like Ligand for (64) Cu Radiolabeling. Mol Pharm. 2014;11:617–629. 18. Zhang RL, Chopp M, Zhang ZG, Jiang Q, Ewing JR. A rat model of focal embolic cerebral ischemia. Brain Res. 1997;766:83–92. 19. Zhang ZG, Zhang L, Ding G, Jiang Q, Zhang RL, Zhang X, Gan WB, Chopp M. A model of mini-embolic stroke offers measurements of the neurovascular unit response in the living mouse. Stroke. 2005;36:2701–2704. 20. Loening AM, Gambhir SS. AMIDE: a free software tool for multimodality medical image analysis. Mol Imaging. 2003;2:131–137. 21. Uppal R, Ay I, Dai G, Kim YR, Sorensen AG, Caravan P. Molecular MRI of intracranial thrombus in a rat ischemic stroke model. Stroke. 2010;41:1271–1277. 22. Buxton DB, Antman M, Danthi N, Dilsizian V, Fayad ZA, Garcia MJ, Jaff MR, Klimas M, Libby P, Nahrendorf M, Sinusas AJ, Wickline SA, Wu JC, Bonow RO, Weissleder R. Report of the National Heart, Lung, and Blood Institute working group on the translation of cardiovascular molecular imaging. Circulation. 2011;123:2157–2163. 23. Dobrucki LW, Sinusas AJ. PET and SPECT in cardiovascular molecular imaging. Nat Rev Cardiol. 2010;7:38–47. 24. Fayad ZA, Mani V, Woodward M, Kallend D, Abt M, Burgess T, Fuster V, Ballantyne CM, Stein EA, Tardif JC, Rudd JH, Farkouh ME, Tawakol A; dal-PLAQUE Investigators. Safety and efficacy of dalcetrapib on atherosclerotic disease using novel non-invasive multimodality imaging (dalPLAQUE): a randomised clinical trial. Lancet. 2011;378:1547–1559. 25. Sirol M, Aguinaldo JG, Graham PB, Weisskoff R, Lauffer R, Mizsei G, Chereshnev I, Fallon JT, Reis E, Fuster V, Toussaint JF, Fayad ZA.
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Ay et al Molecular Imaging of Thrombosis and Thrombolysis 705 Fibrin-targeted contrast agent for improvement of in vivo acute thrombus detection with magnetic resonance imaging. Atherosclerosis. 2005;182:79–85. 26. Spuentrup E, Fausten B, Kinzel S, Wiethoff AJ, Botnar RM, Graham PB, Haller S, Katoh M, Parsons EC Jr, Manning WJ, Busch T, Günther RW, Buecker A. Molecular magnetic resonance imaging of atrial clots in a swine model. Circulation. 2005;112:396–399. 27. Spuentrup E, Buecker A, Katoh M, Wiethoff AJ, Parsons EC Jr, Botnar RM, Weisskoff RM, Graham PB, Manning WJ, Günther RW. Molecular magnetic resonance imaging of coronary thrombosis and pulmonary emboli with a novel fibrin-targeted contrast agent. Circulation. 2005;111:1377–1382. 28. Halperin JL, Hart RG, Kronmal RA, McBride R, Pearce LA, Sherman DG. Warfarin versus aspirin for prevention of thromboembolism in atrial fibrillation: Stroke Prevention in Atrial Fibrillation II Study. Lancet. 1994;343:687–91. 29. Mohr JP, Thompson JL, Lazar RM, Levin B, Sacco RL, Furie KL, Kistler JP, Albers GW, Pettigrew LC, Adams HP, Jr., Jackson CM, Pullicino P. A comparison of warfarin and aspirin for the prevention of recurrent ischemic stroke. N Engl J Med. 2001;345:1444–51. 30. Guercini F, Acciarresi M, Agnelli G, Paciaroni M. Cryptogenic stroke: time to determine aetiology. J Thromb Haemost. 2008;6:549–554. 31. Garrison JC, Rold TL, Sieckman GL, Figueroa SD, Volkert WA, Jurisson SS, Hoffman TJ. In vivo evaluation and small-animal PET/CT of a prostate cancer mouse model using 64Cu bombesin analogs: side-by-side comparison of the CB-TE2A and DOTA chelation systems. J Nucl Med. 2007;48:1327–1337.
32. Boswell CA, Sun X, Niu W, Weisman GR, Wong EH, Rheingold AL, Anderson CJ. Comparative in vivo stability of copper-64-labeled crossbridged and conventional tetraazamacrocyclic complexes. J Med Chem. 2004;47:1465–1474. 33. Cerqueira MD, Stratton JR, Vracko R, Schaible TF, Ritchie JL. Noninvasive arterial thrombus imaging with 99mTc monoclonal antifibrin antibody. Circulation. 1992;85:298–304. 34. Olafsen T, Wu A. Antibody vectors for imaging. Semin Nucl Med. 2010;40:167–181. 35. Vymazal J, Spuentrup E, Cardenas-Molina G, Wiethoff AJ, Hartmann MG, Caravan P, Parsons EC Jr. Thrombus imaging with fibrin-specific gadolinium-based MR contrast agent EP-2104R: results of a phase II clinical study of feasibility. Invest Radiol. 2009;44:697–704. 36. Nordborg C, Fredriksson K, Johansson BB. The morphometry of consecutive segments in cerebral arteries of normotensive and spontaneously hypertensive rats. Stroke. 1985;16:313–320. 37. Sirol M, Fuster V, Badimon JJ, Fallon JT, Moreno PR, Toussaint JF, Fayad ZA. Chronic thrombus detection with in vivo magnetic resonance imaging and a fibrin-targeted contrast agent. Circulation. 2005;112:1594–1600. 38. Brommer EJ, van Bockel JH. Composition and susceptibility to thrombolysis of human arterial thrombi and the influence of their age. Blood Coagul Fibrinolysis. 1992;3:717–725.
Clinical Perspective FBP7 can be used to detect source thrombus in patients admitted to the hospital with cardiovascular or neurological symptoms typical of thrombosis. In these patients, etiologic work-up includes the parallel use of several imaging modalities that may be expensive and time consuming, causing delays in secondary prevention strategies. Instead of performing sequential imaging studies using ultrasound, transesophageal echocardiogram, computed tomography/magnetic resonance imaging, a bimodal positron emission tomographic scan using FBP7 as a molecular contrast agent can help visualizing both the thrombus causing the symptoms (eg, an embolus lodged in the lungs or in the brain), the original thrombus (eg, a thrombus in the deep vein of the leg, carotid artery, or the aortic arch), and tissue ischemia in 1 session. Another possible application of FBP7 is in combination with angiography (eg, positron emission tomographic–magnetic resonance angiography) for patients undergoing thrombolytic therapy. In these cases, in addition to image post–recombinant tissue-type plasminogen activator recanalization with angiography, it would be also possible to detect the presence of nonocclusive residual thrombi in the vascular bed. This can provide additional information to predict the chance of possible recurrent thrombotic episodes.
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SUPPLEMENTAL MATERIAL
In vivo molecular imaging of thrombosis and thrombolysis using a fibrin-binding PET probe
Ilknur Ay*, MD PhD; Francesco Blasi*, PharmD PhD; Tyson A. Rietz, BS; Nicholas J. Rotile, BA; Sreekanth Kura, MS, Anna Liisa Brownell, PhD, Helen Day, MS; Bruno L. Oliveira, PhD; Richard J. Looby, BS; Peter Caravan, PhD
Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital East, 149 13th Street, Charlestown, Massachusetts 02129, USA
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SUPPLEMENTAL METHODS
Reagents CB-TE2A [C16H30N4O4 (2,2'-(1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diyl)diacetic acid)] was a gift of Prof. Carolyn Anderson, University of Pittsburgh Medical Center. 64CuCl2 was obtained from the University of Wisconsin, Medical Physics Department. All other chemicals were purchased commercially and used without further purification.
High Performance Liquid Chromatography (HPLC) Preparative HPLC purification was performed on a Varian Prostar system with two Prostar 210 pumps and a Prostar 325 UV/Vis detector, using a Phenomenex Luna C18 column (250 x 21.2 mm, 10 μm). Liquid chromatography-electrospray mass spectrometry (LC-MS) was performed using an Agilent 1100 Series apparatus with a Phenomenex Luna C18 column (100 mm × 2 mm, 5 μm). This system is equipped with an LC/MSD trap and a Daly conversion dynode detector. Radio-HPLC analyses were obtained on an analytical Agilent 1100 Series system using a Phenomenex Luna C18 column (150 x 4.6 mm, 5 μm; to check radiochemical yields and purity) or a MetaChem Polaris C18 (150 x 4.6 mm, 5 μm; for analysis of urine and blood samples). Four different HPLC methods were used depending on whether HPLC was being used for preparative purification (Method 1), to assess purity (Method 2), to check radiochemical yields and purity (Method 3) or to analyze urine and blood metabolites (Method 4). For all the methods the mobile phase A was H2O with 0.1% trifluoroacetic acid (TFA) and mobile phase B was CH3CN with 0.1% TFA. Method 1 (flow rate of 15 mL/min): 0 – 3 min, 5% B; 3 – 15 min, 5 to
2
95% B; 15 – 18 min, 95% B; 18 – 19 min, 95 to 5% B; 19 – 22 min, 5% B. Method 2 (flow rate of 0.8 mL/min): 0 – 1 min, 5% B; 1 – 10 min, 5 to 95% B; 10 – 12 min, 95% B; 12 – 12.5 min, 95 to 5% B; 12.5 – 15 min, 5% B. Method 3 (flow rate of 1.0 mL/min): 0 – 12 min, 5 to 80% B; 12 - 12.1 min, 80 to 5% B; 12.1 – 13 min, 5% B. Method 4 (flow rate of 1 mL/min): 0 – 17 min, 0 to 100% B; 17 – 17.1 min, 100 to 0% B; 17.1 – 18 min, 0% B.
Probe Synthesis The cyclic disulfide peptide precursor F.H.C.Hyp.Y(3-CI).D.L.C.H.I.L.PXD (Hyp=L-4hydroxyproline, Y(3-Cl)=L-3-chlorotyrosine, PXD=paraxylenediamine) was prepared with the use of a rink amide MBHA resin, Fmoc coupling strategy, and aqueous DMSO cyclization. This peptide (24 mg), CB-TE2A (17.2 mg, 3eq), and Pybop (3eq) were added to 5 ml DMF while the pH of the reaction (monitored by spotting the reaction on a wet pH stick) was adjusted to ca. 6 with diisopropylethylamine. The reaction was stirred at RT for 16 h. The reaction was concentrated to an oil and purified using Method 1. Lyophilization of fractions containing the desired product provided 10 mg of white powder (theoretical MW for [M + 2H]2+ = 1089.3, observed 1089.0).1 The peptide-(CB2)2 ligand (25 μL, 1.0 mM in 10mM in NH4OAc) was diluted in 400 μL of 40 mM NH4OAc, pH 4.5.
CuCl2 was added (0.5 - 5 mCi) and heated at 95 C for 60 minutes.
64
The reaction was monitored by radio-HPLC, which showed 90 - 95% conversion to the desired product. EDTA (1 mL of 10 mM solution) was added and the mixture stirred an additional 20 min to scavenge any unchelated copper ion. The compound was purified using Method 3, concentrated via rotary evaporation, and reconstituted with 10 mM NH4OAc and saline (1:1). This purified material was again checked by HPLC (Method 3) for final radiochemical purity.
3
In vitro stability assay FBP7 was incubated with citrated rat plasma (Innovative Research Inc) at 37°C. Aliquots (100 μL) were collected after 0, 2 and 16 hours of incubation and mixed with 100 μL ice-cold methanol to precipitate plasma protein. Proteins were then sedimented by centrifugation (10,000 g x 5 min) and the supernatant was injected into HPLC (Method 4).1
DD(E) Binding Assay The affinity of FBP7 to the soluble fibrin fragment DD(E) was assessed in the presence and absence of heparin using a fluorescence polarization assay as previously described.1, 2 Here we used FBP7 labeled with natural abundance 63,65Cu. This assay measures the displacement of a tetramethylrhodamine-labeled peptide (TRITC-Tn6) from DD(E) upon addition of FBP7 by observing the corresponding change in fluorescence anisotropy. The Kd of the TRITC-Tn6 probe binding to DD(E) is 0.92 µM2, and based on this Kd and the observed displacement as a function of FBP7 concentration, an inhibition constant Ki for FBP7 can be determined.2 The experiment was performed at room temperature using a probe concentration of 0.1 μM in the following assay buffer: Tris base (50 mM), NaCl (100 mM), CaCl2 (2 mM), Triton X-100 (0.01%), pH=7.8. For the heparin competition, the assay buffer was mixed in 33 IU sodium heparin blood collection tubes to allow for a 5U/ml final concentration. The anisotropy measurements were made using a TECAN Infinity F200 Pro plate reader equipped with the appropriate filter set for tetramethylrhodamine (excitation 535 nm, emission 590 nm). A series of solutions of FBP7 were prepared through serial dilutions from a stock solution. These solutions were then added to a mixture of the DD(E) protein and TRITC-Tn6 peptide in assay buffer with or without heparin. The final concentrations of DD(E) and fluorescent probe used in these experiments were 2 μM
4
and 0.1 μM, respectively. All measurements were performed in a 384-well plate. The inhibition constant, Ki of FBP7, were then calculated using least-squares regression and the known Kd of the fluorescent probe.
Biodistribution analysis Serial blood samples were collected from the femoral artery at 0, 2, 5, 10, 15, 30, 60, 120, 240, and 300 min post-injection into EDTA blood tubes. Blood was weighed and radioactivity in the blood was measured on a gamma counter (Packard, CobraII Auto gamma) to assess clearance of total activity. To quantify organ and body fluid distribution of copper-64, blood, chest and abdominal organs, brain, left rectus femoris muscle, and left femur were collected from all animals after euthanasia. Additionally, the right common carotid artery containing the intramural thrombus and the contralateral common carotid artery were collected from animals that had carotid crush injury. The tissues were weighed and radioactivity in each tissue was measured using a gamma counter, along with an aliquot of the injected FBP7 solution.1 The radioactivity was reported as percent injected dose per gram (%ID/g) which was calculated by dividing the counts of copper-64 per gram of tissue by the total counts of the injected dose, all decay corrected to the time of injection.
5
Autoradiography Autoradiography of the common carotid arteries and the brain sections was performed using a multipurpose film and a Perkin-Elmer Cyclone Plus Phosphor Imager and PerkinElmer OptiQuant 5.0 software. Exposure time was between 1-10 min.1
Metabolic Stability Assay Blood samples collected at 2, 15, 120 and 240 min were immediately centrifuged for 10 min at 2000 rpm and the supernatant passed through a 0.22-μm Millipore filter and injected onto an analytical HPLC column at a flow rate of 1 mL/min using Method 4. The eluent was collected every 0.5 min and the activity of each fraction was measured by the gamma counter. The HPLC analysis was performed in duplicate or triplicate. Data obtained from the gamma counter were plotted to reconstruct the HPLC chromatograms.1
Functional Fibrin Binding Assay Human fibrinogen (American Diagnostica) was dialyzed against Tris (50 mM, pH 7.4), sodium chloride (150 mM), and sodium citrate (TBS·citrate; 5 mM), and the fibrinogen concentration was adjusted to 5 mg/mL (based on the absorbance at 280 nm and ε280 = 1.512 L/g/cm). After adding CaCl2 (7 mM), 50 μL of this fibrinogen solution was dispensed into a 96-well polystyrene microplate (Immulon-II). Human thrombin solution (50 μL; 2 U/mL in TBS) was added into each well to clot the fibrinogen yielding final fibrin concentration close to 2.5 mg/mL (7.3 μM based on fibrinogen MW = 340 kDa). The plates were incubated at 37°C overnight and evaporated to dryness. Blood plasma that was obtained after centrifugation (2000 rpm for 20 min at 4°C) diluted in TBS (1:1) was incubated in fibrin immobilized wells as well as in empty
6
wells for 2h on a shaker at 300 rpm and room temperature. The counts in the supernatant in both the fibrin-containing and empty wells were measured on a gamma counter and divided by the weight of plasma to determine the concentration of unbound probe and total probe, respectively. The amount of copper-64 containing species bound to fibrin was calculated from [bound] = [total] − [unbound]. The amount of functional probe in the blood at time T was determined by taking the ratio of the % bound to fibrin at time T compared to the % bound at T = 0, and multiplying this ratio by the measured total copper-64 %ID/g in the blood.1
Histopathology Ipsilateral (right) and contralateral (left) common carotid arteries were collected two hours after crush injury (n=2); brains were harvested one hour after embolic stroke induction (n=2). Samples were carefully rinsed in Phosphate Buffer, embedded in OCT mounting media (Tissue-Tek), and snap-frozen in -45°C isopentane. The brains and the arteries were cryosectioned (20 μm thickness) and processed for Hematoxylin and Eosin staining according to the standard protocol. For fibrin immunostaining, adjacent slices were incubated with a mouse anti-fibrin alpha-chain antibody (Abcam, U45). Griffonia Simplicifolia Lectin 1 (Vector Laboratories) was used to stain the vascular endothelium; Hoechst 33342 (Sigma) was used as nuclear marker. Negative control experiments were performed by omitting the primary antibody anti-fibrin U45, resulting in the absence of specific fibrin immunostaining. Images were acquired using a Nikon TE-2000 microscope equipped with epifluorescence illumination.
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Supplemental Figures and Figure Legends
Figure I. Synthesis and chemical structure of FBP7.
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Figure II. Displacement of TRITC-Tn6 peptide (0.2 μM) from DD(E) by FBP7 in absence (clear symbols) and presence of heparin (5U/mL, solid symbols). The reduction in fluorescence anisotropy is used to calculate the inhibition constant (Ki). The binding of FBP7 to DD(E) in presence (Ki = 620 nM) or absence (Ki = 660 nM) of heparin was comparable.
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Figure III. (A) Data showing blood clearance (clear symbols) of FBP7 and result of fibrinbinding assay (solid symbols). (B) Radio-HPLC analysis from serial blood draws showing the percentage of intact probe at 2, 15, 120 and 240 min post-injection. (C) Representative radioHPLC tracing of plasma and urine showing high levels of probe and just few metabolites up to 4 hours post-injection.
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Figure IV. Ex vivo autoradiography (upper panels) of crushed (RC) and contralateral (LC) common carotid arteries at 2 hours and 5 hours post-injection confirmed high thrombus uptake of FBP7. Lower panels show the photographs of arteries taken before autoradiography.
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B
A
C
D
Figure V. Macroscopical inspection of the carotid arteries suggests thrombosis at the level of the ipsilateral common carotid artery, but not contralaterally (center panel). Hematoxylin and Eosin staining reveals the presence of arterial thrombosis after carotid crush injury (A, top). High magnification microphotograph shows that mural thrombi are mainly localized at the level of the endothelial cells layer (A, arrows, bottom); this finding is consistent with previously reported models of mural thrombosis3. Fibrin immunostaining performed on adjacent histological sections reveals an immunopositive signal (green) in proximity of the endothelial cells layer (B, arrows), consistent with fibrin deposition. Histopathology did not reveal the presence of mural thrombi (C) or fibrin (D) in the contralateral common carotid artery. Green, fibrin; red, endothelial cells layer; blue, nuclei. Arrows in A, mural thrombi; arrows in B, fibrin deposits. Scale bars: 0.2 (A and C, top), 0.05 (A and C, bottom), and 0.025 mm (B and D).
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Figure VI. Hematoxylin and Eosin staining reveals an occlusive thrombus in the right middle cerebral artery of rats subjected to embolic stroke (top panel). Insets (middle panels) show high magnification (40X) images of the contralateral (left) and ipsilateral (right) cerebral arteries. The entire lumen of the right middle cerebral artery is occluded by the embolus. Adjacent sections were immunostained for fibrin (bottom panels). An immunopositive signal (green) was present in the lumen of the ipsilateral middle cerebral artery (right), but not contralaterally (left). Green, fibrin; red, endothelial cells layer; blue, nuclei. Scale bars: 0.2 (top) and 0.05 mm (middle and bottom).
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SUPPLEMENTAL VIDEO LEGENDS
Video I. 3D reconstruction of a representative rat following carotid crush injury and FBP7 injection. Carotid thrombus is clearly visible and distinguishable from background organs and tissues. A superficial hyperintense area is also present at the level of the surgical incision.
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SUPPLEMENTAL REFERENCES
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Ciesienski KL, Yang Y, Ay I, Chonde DB, Loving GS, Rietz TA, Catana C and Caravan
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Kolodziej AF, Nair SA, Graham P, McMurry TJ, Ladner RC, Wescott C, Sexton DJ and
Caravan P. Fibrin specific peptides derived by phage display: characterization of peptides and conjugates for imaging. Bioconjug Chem. 2012;23:548-56. 3.
Yamashita A, Furukoji E, Marutsuka K, Hatakeyama K, Yamamoto H, Tamura S, Ikeda
Y, Sumiyoshi A and Asada Y. Increased vascular wall thrombogenicity combined with reduced blood flow promotes occlusive thrombus formation in rabbit femoral artery. Arterioscler Thromb Vasc Biol. 2004;24:2420-2424.
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