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JACC Cardiovasc Imaging. Author manuscript; available in PMC 2013 Jun 1.
Published in final edited form as:
PMCID: PMC3376390
NIHMSID: NIHMS377251

Molecular imaging of fibrin deposition in deep vein thrombosis using a new fibrin-targeted near-infrared fluorescence (NIRF) imaging strategy

Tetsuya Hara, MD, PhD,1 Brijesh Bhayana, PhD,2 Brian Thompson, PhD,1,2,3 Chase W. Kessinger, PhD,1 Ashok Khatri, PhD,4 Jason R. McCarthy, PhD,3 Ralph Weissleder, MD PhD,3 Charles P. Lin, PhD,2,3 Guillermo J. Tearney, MD, PhD,2,* and Farouc A. Jaffer, MD, PhD1,2,*

Abstract

Objectives

To develop and validate a new fibrin targeted imaging agent that enables high-resolution near-infrared fluorescence (NIRF) imaging of deep venous thrombosis (DVT).

Background

Near-infrared fluorescence (NIRF) imaging of fibrin could enable highly sensitive and noninvasive molecular imaging of thrombosis syndromes in vivo.

Methods

A fibrin-targeted peptide was conjugated to an NIR fluorophore Cy7, termed FTP11-Cy7. The NIRF peptide is based on a fibrin-specific imaging agent that has completed phase II clinical magnetic resonance imaging (MRI) trials. In vitro binding of FTP11-Cy7 to human plasma clots was assessed by fluorescence reflectance imaging (FRI). Next, FTP11-Cy7 was intravenously injected in mice with femoral DVT induced by topical 7.5% ferric chloride treatment. Intravital fluorescence microscopy (IVFM), and noninvasive fluorescence molecular tomography(FMT)-computed tomography (CT) were performed in mice (n = 32 total) with DVT, followed by histological analyses.

Results

In vitro human clot-binding analyses showed a 6-fold higher NIRF clot target-to-background ratio (TBR) of FTP11-Cy7 than free Cy7 (6.3 ± 0.34 vs. 1.2 ± 0.03, p < 0.0001). The thrombus TBR of acute and sub-acute femoral DVT with FTP11-Cy7 obtained by IVFM was >400% higher than control free Cy7. Binding of FTP11-Cy7 to thrombi was blocked by a 100-fold excess of unlabeled competitor peptide both in vitro and in vivo (p < 0.001 for each). Histological analyses confirmed that FTP11-Cy7 specifically accumulated in thrombi. Noninvasive FMT-CT imaging of fibrin in jugular DVT via FMT-CT demonstrated strong NIRF signal in thrombi compared to sham-operated jugular veins (mean TBR = 3.5 ± 0.7 vs. 1.5 ± 0.3, p < 0.05).

Conclusions

The fibrin-targeted NIRF agent FTP11-Cy7 avidly and specifically binds human and murine thrombi, and enables sensitive, multimodal intravital and noninvasive NIRF molecular imaging detection of acute and sub-acute murine DVT in vivo.

Keywords: fibrin, DVT, molecular imaging, fluorescence, optical imaging

Introduction

Thrombosis and thrombo-embolic diseases such as myocardial infarction, stroke, pulmonary embolism, and deep venous thrombosis (DVT) are major causes of morbidity and mortality worldwide (1). Clinical detection of thrombosis syndromes often utilizes ultrasound, X-ray computed tomography (CT), or magnetic resonance imaging (MRI). However such imaging approaches require the presence of a relatively large thrombus to render a diagnosis of a thrombotic disorder. In many cases, for example plaque rupture, stent thrombosis, or early or resolving DVT, a much more sensitive measure of early thrombosis is required. Furthermore, thrombus detection for most current imaging modalities is structurally related to the cessation of blood flow. It could therefore prove beneficial to harness a molecular marker of fibrin to characterize thrombus or microthrombus type/composition, and to guide fibrinolytic therapy of fibrin-rich thrombi.

Fibrin, a trimeric molecule consisting of α-, β-, and γ-chain, is present in both arterial and venous thrombi at micromolar concentrations, and is minimally present in the circulating blood, rendering it a favorable imaging target for in vivo detection (2). Previous studies have investigated fibrin-targeted agents for nuclear imaging (3,4), and more recently, a fibrin-specific gadolinium-based agent (EP-2104R) for MRI (58) that has been tested in clinical trials (9,10).

However only a few robust fibrin-targeted agents exist for in vivo optical, and specifically, near-infrared fluorescence (NIRF), imaging. Attractive features of NIRF imaging includes high-resolution imaging capabilities (11), as well as noninvasive (12) and intravascular imaging platforms (13,14). In addition, fluorescence-based detection is inherently more sensitive than MRI-based detection, and thus requires lower dosages and shorter washout times for injected agents (15).

In this study, we have synthesized and investigated the avidity and specificity of a new fibrin-targeted NIRF thrombus imaging agent, termed FTP11-Cy7. We report here the targeting capabilities and specificity of the NIRF agent in acute and sub-acute murine DVT, employing high-resolution intra-vital fluorescence microscopy (IVFM) and noninvasive integrated fluorescence molecular tomography–computed tomography (FMT-CT) in vivo.

Methods

Synthesis of FTP11-Cy7, a NIRF fibrin-targeted imaging agent

The fibrin-targeted fluorescent peptide (FTP11-Cy7) was synthesized in the MGH Peptide/Protein Core facility using solid-phase chemistry based on EP-2104R (8), a peptide imaging agent comprised of 11 amino acids, Tyr-D-Glu-Cys-Hyp-Tyr (3-Cl)- Gly-Leu-Cys-Tyr-Ile-Gln-NH2, and cyclized via the formation of a disulphide bond (8). Full synthetic details are provided in the Appendix. Linearity of the NIRF signal intensity generated from FTP11-Cy7 was assessed by fluorescence reflectance imaging (FRI) at various concentrations (50 μL sample volume).

In vitro clot binding

Human plasma clots were created from fresh frozen plasma (FFP) in 96 well plates (16). FFP was obtained using an IRB-approved human subject protocol. Each well received 90 μL of FFP, 5 μL of 0.4 M CaCl2, and 5 μL of thrombin (0.1 U/μL PBS). The plate was incubated at 37°C for 90 minutes to clot the plasma. Thereafter, 0.02 nmol of fluorophore was added to respective wells and then incubated at 37 °C for an additional 30 minutes. In competitive binding assays, unlabeled FTP11 at 10-fold or 100-fold excess concentration was added 30 minutes prior to the incubation of FTP11-Cy7 with clots. The clots were then washed twice with PBS and centrifuged at 500 rpm for 10 minutes. They were then washed twice more with PBS, and then imaged by FRI (excitation/emission 740/790 nm). Experiments were performed in quadruplicate.

Animal studies

Animal studies were approved by the Hospital Subcommittee of Research Animal Care. For in vivo studies, C57BL/6 mice were anesthetized using an intraperitoneal (i.p.) ketamine (50 mg/ml, 330 μL), xylazine (100 mg/ml, 50 μL), and sterile saline (380 μL) mixture. Each mouse received a 50 μL i.p. induction dose of the resulting mixture, followed by 10–20 μL i.p. hourly for continued anesthesia.

Blood half-life study of FTP11-Cy7

To determine the blood half-life of FTP11-Cy7, serial blood sampling was performed through a jugular vein. C57BL/6 mice (n = 5) received a 150 nmol/kg iv bolus of the FTP11-Cy7 dissolved in PBS. At the desired timepoints, a 50 μL volume of blood was obtained and mixed with 50 μL of heparin (1000 USP units/ml), and kept on ice until analysis. The Cy7 fluorescence of the blood at each time point was quantified by FRI. The blood NIRF signal at each timepoint was measured, and then normalized by the NIRF signal at one minute post-injection. Exponential fitting yielded the blood half-life of FTP11-Cy7 (GraphPad Prism 5.0, San Diego, CA).

Creation of deep vein thrombosis in mice

In vivo DVT were induced by topical ferric chloride injury to the femoral vein (for IVFM studies) or jugular vein (for FMT studies) (17). Under anesthesia, FeCl3 was locally applied to the vein using a 1 mm strip of Whatman No.1 filter paper soaked in 7.5% FeCl3. The filter paper was applied to the anterior surface of the vein for 5 minutes. Thereafter, the filter paper was removed and the surgical field was irrigated with PBS. For acute imaging studies, imaging was performed in 2 hour old DVT. For subacute imaging studies, imaging was performed in 3 day (72 hour) old DVT. A total of 32 mice were studied.

Intravital fluorescence microscopy (IVFM)

To image fibrin deposition in thrombi at high-resolution, IVFM of femoral DVT was performed (n = 23 mice). Mice were anesthetized as above. At 60 minutes before imaging, a fibrin-targeted agent (FTP11-Cy7, n = 5 acute DVT, n = 5 subacute DVT) or control agent (free Cy7, n = 5) was administered via retro-orbital injection. Agents were injected at a dose of 150 nmol/kg in 100 μL total volume. In in vivo blocking experiments (n = 8), unlabeled FTP11 (1500 nmol/kg) or PBS was injected 30 minutes prior to injection of FTP11-Cy7 (15 nmol/kg). Injection of 100 μL of fluorescein-labeled dextran (FITC–dextran, Sigma, St Louis, MO, 5 mg/ml, excitation/emission; 490/520 nm) provided an angiogram. IVFM studies employed a multichannel laser scanning fluorescence microscope optimized for intravital imaging (18) .The utilized net 17x objective (NA 0.9) provided an in-plane resolution of 1.4μ m/pixel. Z-stacks (20–40 slices) were obtained at 5 μm steps through the vessel. All image settings were kept constant for all time points and samples.

Fluorescence molecular tomography–computed tomography (FMT-CT)

Noninvasive integrated FMT-CT was performed as previously described (19) in n = 9 mice with sub-acute jugular vein DVT. FTP11-Cy7 was i.v. injected at 150 nmol/kg mouse one hour prior to imaging. The surgical incisions remained closed during imaging. The NIRF signal was detected with a FMT 2500 system (PerkinElmer, excitation/emission; 745 nm/785 nm). To identify thrombosed and sham-operated jugular veins, CT venograms were obtained via continuous infusion of iodinated contrast (Isovue-370, rate 55 μL/min via tail vein). To enable accurate co-registration between FMT and CT datasets, an imaging cartridge containing the anesthetized mouse was placed into a custom-machined Plexiglas holder that supplied isoflurane, warm air and optimal positioning in the CT scanner (Inveon PET-CT, Siemens). FMT-CT co-registration was performed with OsiriX shareware. Fiducials on the imaging cartridge were visualized and tagged in FMT and CT images, allowing point-based co-registration in OsiriX (19).

Fluorescence reflectance imaging (FRI)

Plasma clots, blood samples, and resected vessels underwent FRI with an NIR filter set (excitation/emission; 740/790 nm; Kodak ImageStation 4000, Carestream Health, Inc., Rochester, NY). Multiple exposure times (0.1 to 60 s) generated images that were exported to 16-bit unscaled TIFF files for further analysis with ImageJ (version 1.44o, Bethesda, MD).

Image analyses

Image analyses of IVFM data sets were performed by summation of z-stacks into a 2-dimensional summation image with ImageJ. Thrombi presented as filling defects on FITC-dextran generated angiograms. Regions-of-interest (ROIs) of the thrombus and the adjacent normal vessel were manually traced (11). Reconstruction and image analysis of FMT-CT data sets were performed with OsiriX software (64 bit, version 3.5.1) (19). Target-to-background ratios (TBRs) were calculated by dividing the mean ROI signal of target by the mean ROI signal of the background (PBS treated clot, adjacent vessel, or other organs).

Histopathology

After sacrifice, mice were perfused with 0.9% saline (20 mL) via the left ventricle. For histopathological analysis, femoral vessels were excised and embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA). Serial 6-μm cryostat sections were obtained for fluorescence microscopy. Adjacent sections were stained with hematoxylin and eosin for general morphology. NIR fluorescence microscopy was performed on fresh-frozen femoral vein sections using an upright epifluorescence microscope (Nikon Eclipse 90i, Tokyo, Japan). Fluorescence images were obtained in the NIR channel for FTP11-Cy7 (excitation/emission; 710/810 nm, exposure time 50 msec), and FITC channel for autofluorescence (excitation/emission; 480/535 nm, exposure time 50 msec).

Statistics

Results are expressed as mean ± SEM. Statistical comparisons between two groups were evaluated by the Mann-Whitney U test, and by the Kruskal-Wallis test for multiple groups followed by the Dunn’s post-test. Statistical comparisons and half-life calculations were performed with GraphPad Prism. A value of P < 0.05 was considered statistically significant.

Results

Synthesis of FTP1-Cy7, a NIRF fibrin imaging agent

A schematic representation of the FTP11-Cy7 is shown in Figure 1A. The HPLC trace demonstrated >98% purity (Figure 1B). The final product showed a molecular mass of 2062 g/mol using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) (Figure 1C), in agreement with the calculated mass. The NIRF signal of the FTP11-Cy7 peptide, as measured by FRI, related linearly to its Cy7 concentration, at concentrations relevant for imaging (Figure 1D, R2 = 0.99).

Figure 1
Synthesis and structure of FTP11-Cy7

In vitro binding assessment of FTP11-Cy7 to human plasma clots

The binding ability of FTP11-Cy7 was first evaluated in vitro in human FFP clots. After 30 minutes of incubation with individual agents, NIRF signals were measured by FRI (Figure 2). In vitro clot-binding analyses showed >500% higher clot TBRs for FTP11-Cy7 compared to control free Cy7 (6.3 ± 0.34 vs. 1.2 ± 0.03, p < 0.0001). An excess of unlabeled competitor peptide significantly blocked binding of FTP11-Cy7 in a dose-dependent manner (TBR 5.0 ± 0.45 for 10-fold excess, 2.5 ± 0.34 for 100-fold excess), indicating that the engineered NIRF peptide was clot specific.

Figure 2
NIRF imaging of fibrin in human plasma clots

Blood half-life of FTP11-Cy7

Blood clearance was assessed by serial blood sampling in mice (n = 5), followed by measurement of blood NIRF signal levels with FRI. The NIRF signal decreased rapidly from the blood, with a blood half-life of 2.82 minutes (Figure 3, 95% CI, 1.85–5.90).

Figure 3
Clearance of FTP11-Cy7in blood circulation

High-resolution IVFM of fibrin in femoral DVT

To determine whether FTP11-Cy7 could enable high-resolution imaging of fibrin deposition, murine femoral DVT were imaged using IVFM. Both acute DVT (2 hours old) and sub-acute DVT (3 days old) were studied. Thrombi were identified as filling defects on FITC-dextran angiograms. IVFM demonstrated that NIRF signal increased in both acute and sub-acute thrombi (Figure 4A and 4B). In contrast, control free Cy7 did not enhance thrombi (Figure 4C). The thrombus TBR was significantly higher in the FTP11-Cy7 groups (3.5 ± 0.3 for acute DVT, 2.7 ± 0.5 for sub-acute DVT; 0.46 ± 0.08 for free Cy7, figure 4D). We also performed in vivo blocking experiments to evaluate the in vivo binding specificity of FTP11-Cy7. Pre-injection of 100-fold excess of unlabeled FTP11 blocked the FTP11-Cy7 mediated NIRF signal enhancement of acute DVT (thrombus TBR 1.6 ± 0.11, vs. 0.99 ± 0.04 blocked group, p < 0.001, Figure 4E). Fluorescence microscopy and hematoxylin and eosin staining of the histological sections demonstrated the accumulation of NIRF signal in DVT in the FTP11-Cy7 group (Figure 5A, 5B). In contrast, control free Cy7 did not enhance thrombi (Figure 5C).

Figure 4
High-resolution IVFM detection of fibrin deposition in femoral DVT
Figure 5
Thrombus-specific binding of FTP11-Cy7 in murine venous thrombi

Noninvasive FMT-CT of FTP11-Cy7 accumulation in jugular DVT

To image fibrin deposition in DVT noninvasively, we next performed FMT-CT of thrombosed jugular veins of mice. The same mice also possessed a sham-operated contralateral right jugular vein. To assess FMT signals accurately, we co-registered fiducially-matched jugular veins from FMT and anatomical CT images (Figure 6A through 6C). The TBR of the left jugular DVT was 230% higher than the sham operated right jugular vein (mean TBR = 3.5 ± 0.7 vs. 1.5 ± 0.3, p < 0.05; Figure 6D).

Figure 6
Noninvasive molecular imaging of fibrin in jugular DVT by integrated FMT-CT

After FMT-CT, jugular veins were resected and ex vivo FRI was performed. High NIRF signal was noted in DVT of mice (Figure 7A and 7B). The TBR of the thrombosed vein was higher than that of sham-operated right jugular vein (3.9 ± 0.4 vs. 2.4 ± 0.4, p < 0.05; Figure 7C).

Figure 7
Ex vivo targeting of FTP11-Cy7 to thrombosed jugular veins

Discussions

In this investigation we demonstrate i) the synthesis and specificity validation of a new human and murine fibrin-targeted imaging agent for in vivo NIRF imaging; ii) high-resolution, efficient IVFM detection of fibrin deposition, in both acute and sub-acute murine DVT; and .iii) and noninvasive detection of fibrin-rich jugular DVT using integrated FMT-CT.

The fibrin-targeted NIRF agent is based on the peptide backbone of EP-2104R (8), an MR imaging agent that has successfully undergone phase II clinical trials (9,10), and therefore appears attractive for clinical translation. Another favorable capability of the fibrin-targeted NIRF agent is the ability to detect thrombi with over 20-fold lower injected dose (150 nmol/kg) compared to other MRI-based thrombosis imaging agents (4–10 μmol/kg (6,10,20)).

Intravital fluorescence microscopy enabled high-resolution in vivo detection of fibrin in both acute and sub-acute murine DVT, with significantly greater thrombus-to-background ratios compared to controls. In vitro and in vivo blocking studies further demonstrated that FTP11-Cy7 was thrombus specific. Capitalizing on the multispectral capabilities of IVFM, fibrin-rich thrombi were actively detected by FTP11-Cy7 enhancement, and passively corroborated by FITC-dextran angiography. In vivo, the NIRF fibrin-targeted agent bound to the luminal surface of thrombus and did not penetrate the deep interior of the clot, despite its relatively small molecular weight, consistent with greater thrombus organization in the deeper (older) regions of thrombi.

Given this data, the validated NIRF fibrin imaging agent FTP11-Cy7 may prove to be a valuable asset in the investigations that aim to track and quantify of fibrinogenesis and fibrinolysis in vivo, especially in murine model systems. Compared to an antibody-based imaging agent that required pre-injection (21), the NIRF fibrin-targeted is readily synthesizable, provides good thrombus TBRs in rapid fashion due its favorable pharmacokinetics, and demonstrates utility for acute and subacute DVT. In contrast to other fibrin imaging agents for in vivo NIRF molecular imaging of thrombosis (16), the current peptide-based fibrin agent provides improved thrombus TBRs in sub-acute thrombi in addition to acute thrombi, consistent with its high binding affinity to fibrin (8).

The NIRF fibrin-targeted agent also enabled noninvasive FMT imaging of fibrin in murine jugular DVT. FMT is a three-dimensional, quantitative tomographic imaging technique that allows reconstruction of NIR deep within the body by using an array of photon detectors and inversion algorithms (12). The addition of CT, similar to integrated SPECT-CT or PET-CT systems, allows further co-registration using high-resolution anatomical information. In vivo investigations demonstrated the ability to image fibrin in sub-acute jugular DVT, with NIRF signal colocalizing with occluded veins on CT venography (Figure 6). Sham-surgery treated veins demonstrated significantly less NIR fluorescence. Modest fibrin signal in the healing incision was also noted and expected as fibrin formation occurs during normal wound healing (22). The ability to track and quantify fibrin deposition noninvasively in DVT offers the potential to serially investigate therapies that modulate fibrin within thrombi, and could provide insights into DVT resolution and the risk of developing the post-thrombotic syndrome (23,24). From a translational perspective, larger FMT or optoacoustic systems scalable to size of the human leg are feasible (25), and thus may eventually enable clinical NIRF imaging of fibrin within DVT. In addition, such systems could be harnessed to image fibrin content in carotid plaques to inform stroke risk at baseline and during carotid arterial intervention (e.g. stenting or endarterectomy).

A more immediate clinically relevant application of the fibrin-targeted NIRF agent is intravascular NIRF imaging of fibrin deposition on implanted coronary stents. Intravascular NIRF imaging is a new high-resolution strategy to image molecular and cellular aspects of vascular disease (13,14,26). Via optimized optical fiber catheters, intravascular NIRF imaging can provide high-resolution, sensitive readouts of molecular targets in coronary-sized vessels through blood, without the need for flushing, due to relatively low blood attenuation of NIR light. In the future, the ability to image fibrin on healing coronary stents may offer new insights into pathogenesis of stent thrombosis, particularly late stent thrombosis in current and next generation drug eluting stents, especially when coupled with high-resolution structural imaging via intravascular ultrasound (14), or via single catheters integrating NIRF with optical frequency domain imaging (27), a leading modality for imaging coronary stent architecture.

Limitations

The free cyanine dye (free Cy7) and unlabeled competing FTP11 peptide was used as controls in all experiments. Although a peptide-based control agent (e.g. scrambled peptide-Cy7) could have additionally been utilized, previous publications have validated the specificity of the fibrin-targeting peptide backbone (FTP11) used in this study (58,10). Higher blocking peptide concentrations could provide greater blocking effects. Additionally, dose- and time- optimization experiments of FTP11-Cy7 could yield higher thrombus TBRs than currently achieved.

Conclusions

A new synthesized NIRF fibrin-targeted peptide avidly and specifically binds murine and human thrombi, and enables sensitive, fast multimodal intravital and noninvasive optical imaging detection of acute and subacute murine DVT in vivo.

Supplementary Material

Acknowledgments

We gratefully acknowledge Brett Marinelli, BS and Peter Waterman, BS for FMT-CT image acquisition.

Funding Sources

National Institutes of Health R01HL108229 (FAJ), R01HL076398 and R01HL093717 (GJT), American Heart Association Scientist Development Grant #0830352N (FAJ), Howard Hughes Medical Institute Career Development Award (FAJ), Broadview Ventures (FAJ), and the Society of Nuclear Medicine Wagner-Torizuka Fellowship (TH).

Abbreviations

DVTdeep vein thrombosis
FFPfresh frozen plasma
FITCfluorescein isothiocyanate
FMT-CTfluorescence molecular tomography – computed tomography
FRIfluorescence reflectance imaging
IVFMintravital fluorescence microscopy
MRImagnetic resonance imaging
NIRFnear-infrared fluorescence
ROIregion-of-interest
TBRtarget-to-background ratio

Footnotes

Disclosures: Honorarium, GE Healthcare (FAJ)

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