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64 Cu-FBP8-PET/MR whole-body imaging. With a single method 64 Cu-FBP8 is able to detect thrombus in the right carotid artery (top) and right femoral vein (bottom). Activity in urine (U) and feces (F) denoted by blue arrowheads; L = liver; W = surgical wound. Reprinted from ref. 40 with permission. Copyright © 2014 by American Heart Association.
Source publication
The development of new methods to image the onset and progression of thrombosis is an unmet need. Non-invasive molecular imaging techniques targeting specific key structures involved in the formation of thrombosis have demonstrated the ability to detect thrombus in different disease state models and in patients. Due to its high concentration in the...
Citations
... To examine our hypothesis that the "staged" targeted delivery approach can further improve site-specificity and minimize untoward effects by first actively guiding nanocarriers and then stabilizing their binding to the site of arterial injury, we first demonstrated that MNPs can be endowed with fibrin binding capacity by particle surface modification with a short proline-rich peptide exhibiting a high affinity for the C-terminal portion of the γ-chain of fibrin (GPRPP). This peptide was previously applied in the context of thrombus detection and for the monitoring of thrombogenesis and fibrinolysis [11][12][13][14][15][16] and was shown to bind fibrin with high avidity due to >10 15 binding sites per mg of fibrin protein [11]. The high avidity and the multivalent binding mechanism of peptide-functionalized MNPs can both be instrumental for extending the retention of the magnetic carriers at the site of stent placement. ...
The injury-triggered reocclusion (restenosis) of arteries treated with angioplasty to relieve atherosclerotic obstruction remains a challenge due to limitations of existing therapies. A combination of magnetic guidance and affinity-mediated arterial binding can pave the way to a new approach for treating restenosis by enabling efficient site-specific localization of therapeutic agents formulated in magnetizable nanoparticles (MNPs) and by maintaining their presence at the site of arterial injury throughout the vulnerability period of the disease. In these studies, we investigated a dual-targeted antirestenotic strategy using drug-loaded biodegradable MNPs, surface-modified with a fibrin-avid peptide to provide affinity for the injured arterial wall. The MNPs were characterized with regard to their magnetic properties, efficiency of surface functionalization, disassembly kinetics, and interaction with fibrin-coated substrates. The antiproliferative effects of MNPs formulated with paclitaxel were studied in vitro using a fetal cell line (A10) exhibiting the defining characteristics of neointimal smooth muscle cells. Animal studies examined the efficiency of combined (physical/affinity) MNP targeting to stented arteries in Sprague Dawley rats using fluorimetric analysis and fluorescent in vivo imaging. The antirestenotic effect of the dual-targeted therapy was determined in a rat model of in-stent restenosis 28 days post-treatment. The results showed that MNPs can be efficiently functionalized to exhibit a strong binding affinity using a simple two-step chemical process, without adversely affecting their size distribution, magnetic properties, or antiproliferative potency. Dual-targeted delivery strongly enhanced the localization and retention of MNPs in stented carotid arteries up to 7 days post-treatment, while minimizing redistribution of the carrier particles to peripheral tissues. Of the two targeting elements, the effect of magnetic guidance was shown to dominate arterial localization (p = 0.004 vs. 0.084 for magnetic targeting and peptide modification, respectively), consistent with the magnetically driven MNP accumulation step defining the extent of the ultimate affinity-mediated arterial binding and subsequent retention of the carrier particles. The enhanced arterial uptake and sustained presence of paclitaxel-loaded MNPs at the site of stent deployment were associated with a strong inhibition of restenosis in the rat carotid stenting model, with both the neointima-to-media ratio (N/M) and % stenosis markedly reduced in the dual-targeted treatment group (1.62 ± 0.2 and 21 ± 3 vs. 2.17 ± 0.40 and 29 ± 6 in the control animals; p < 0.05). We conclude that the dual-targeted delivery of antirestenotic agents formulated in fibrin-avid MNPs can provide a new platform for the safe and effective treatment of in-stent restenosis.
... Previously, selective fibrin targeting within the microthrombi was limited due to 98% structural similarities between fibrin and fibrinogen and a high concentration (2.5-3 mg/mL) of fibrinogen in the blood [43]. However, in recent times specificity for fibrin over fibrinogen has been achieved by several techniques such as selective antibodies, small peptides, and tissue plasminogen activator (tPA) [44]. Since fibrin is predominant in all vascular thrombotic obstructions, including arterial, venous, acute, and chronic, by microembolization, targeting fibrin can more effectively resolve MVO. ...
Rationale: Microvascular obstruction (MVO) following percutaneous coronary intervention (PCI) is a common problem associated with adverse clinical outcomes. We are developing a novel treatment, termed sonoreperfusion (SRP), to restore microvascular patency. This entails using ultrasound-targeted microbubble cavitation (UTMC) of intravenously administered gas-filled lipid microbubbles (MBs) to dissolve obstructive microthrombi in the microvasculature. In our prior work, we used standard-sized lipid MBs. In the present study, to improve upon the efficiency and efficacy of SRP, we sought to determine the therapeutic efficacy of fibrin-targeted phase shift microbubbles (FTPSMBs) in achieving successful reperfusion of MVO. We hypothesized that owing to their much smaller size and affinity for thrombus, FTPSMBs would provide more effective dissolution of microthrombi when compared to that of the corresponding standard-sized lipid MBs.
Methods: MVO in the rat hindlimb was created by direct injection of microthrombi into the left femoral artery. Definity MBs (Lantheus Medical Imaging) were infused through the jugular vein for contrast-enhanced ultrasound imaging (CEUS). A transducer was positioned vertically above the hindlimb for therapeutic US delivery during the concomitant administration of various therapeutic formulations, including (1) un-targeted MBs; (2) un-targeted phase shift microbubbles (PSMBs); (3) fibrin-targeted MB (FTMBs); and (4) fibrin-targeted PSMBs (FTPSMBs). CEUS cine loops with burst replenishment were obtained at baseline (BL), 10 min post-MVO, and after each of two successive 10-minute SRP treatment sessions (TX1, TX2) and analyzed (MATLAB).
Results: In-vitro binding affinity assay showed increased fibrin binding peptide (FBP) affinity for the fibrin clots compared with the untargeted peptide (DK12). Similarly, in our in-vitro model of MVO, we observed a higher binding affinity of fluorescently labeled FTPSMBs with the porcine microthrombi compared to FTMBs, PSMBs, and MBs. Finally, in our hindlimb model, we found that UTMC with FTPSMBs yielded the greatest recovery of blood volume (dB) and flow rate (dB/sec) following MVO, compared to all other treatment groups.
Conclusions: SRP with FTPSMBs achieves more rapid and complete reperfusion of MVO compared to FTMBs, PSMBs, and MBs. Studies to explore the underlying physical and molecular mechanisms are underway.
... Other PET tracers have also been investigated for their potential to visualize different aspects of thrombosis including 68 Ga-DOTA-fibrin-binding-probe ( 68 Ga-FBP) which specifically targets fibrin, a major component of thrombus formation [50,51] and 18 F-GP1 which binds with the GPIIb/IIIa receptor found in activated platelets and involved in platelet aggregation during thrombus formation [52]. The use of thrombus-targeted radiotracers can help identify patients with unstable plaques and elevated thrombotic risk, guiding adjustments in their medical treatment approaches or early preventive intervention. ...
Purpose of Review
Current non-invasive tests for evaluating patients with peripheral artery disease (PAD) have significant limitations for early detection and management of patients with PAD and are generally focused on the evaluation of large vessel disease. PAD often involves disease of microcirculation and altered metabolism. Therefore, there is a critical need for reliable quantitative non-invasive tools that can assess limb microvascular perfusion and function in the setting of PAD.
Recent Findings
Recent developments in positron emission tomography (PET) imaging have enabled the quantification of blood flow to the lower extremities, the assessment of the viability of skeletal muscles, and the evaluation of vascular inflammation and microcalcification and angiogenesis in the lower extremities. These unique capabilities differentiate PET imaging from current routine screening and imaging methods.
Summary
The purpose of this review is to highlight the promising role of PET in the early detection and management of PAD providing a summary of the current preclinical and clinical research related to PET imaging in patients with PAD and related advancement of PET scanner technology.
... In addition, fibrin itself, is the fibrinolytic substrate of recombinant tissue plasminogen activator (rt-PA), the most important choice for thrombosis-related disease treatments [16]. Afterwards, fibrin will constantly degrade to non-fibrinolytic collagen and elastic fibers with the evolution of thrombotic compositions, predicting a decrease of thrombolysis efficiency [17,18]. Therefore, fibrin is a well-deserved biomarker for identification of the thrombosis initiation as well as distinguishing thrombus those can be efficiently fibrinolytic. ...
Abstact
Anastomotic thrombosis prevalently causes anastomosis failure, accompanied with ischemia and necrosis, the early diagnosis of which is restricted by inherent shortcomings of traditional imaging techniques in clinic and lack of appropriate prodromal biomarkers for thrombosis initiation. Herein, a fresh thrombus-specific molecular event, protein disulfide isomerase (PDI) is innovatively chosen as the activating factor, and a thrombosis targeting and PDI-responsive turn-on near infrared II (NIR-II) fluorescence nanoprobe is firstly developed. The supramolecular complex-based nanoprobe IR806-PDA@BSA-CREKA is fabricated by assembling NIR-II emitting cyanine derivative IR806-PDA with bovine serum albumin (BSA), which could ameliorate the stability and pharmacokinetics of the nanoprobe, addressing the contradiction in the balance of brightness and biocompatibility. The NIR–II–off nanoprobe exhibits robust turn-on NIR-II fluorescence upon PDI-specific activation, in vitro and in vivo. Of note, the constructed nanoprobe demonstrates superior photophysical stability, efficient fibrin targeting peptide-derived thrombosis binding and a maximum signal-to-background ratio (SBR) of 9.30 for anastomotic thrombosis in NIR-II fluorescent imaging. In conclusion, the exploited strategy enables positive visualized diagnosis for anastomotic thrombosis and dynamic monitoring for thrombolysis of fresh fibrinolytic thrombus, potentially contributes a novel strategy for guiding the therapeutic selection between thrombolysis and thrombectomy for thrombosis treatment in clinic.
... Fibrin is the end-product of the coagulation cascade and is one of the most widely pursued targets of radiotracers for thrombus imaging (46). As fibrin is present in developing and mature thrombi, and absent from circulating blood, specific detection relies on target-selective binding to distinguish it from fibrinogen, its similar, widely circulating precursor (46). ...
... Fibrin is the end-product of the coagulation cascade and is one of the most widely pursued targets of radiotracers for thrombus imaging (46). As fibrin is present in developing and mature thrombi, and absent from circulating blood, specific detection relies on target-selective binding to distinguish it from fibrinogen, its similar, widely circulating precursor (46). Early fibrin-targeted molecular diagnostic procedures were performed with radiolabeled fibrinogen and later with radiolabeled antifibrin antibodies and antibody fragments. ...
... However, the field has since moved in the direction of fibrin-binding peptides, given their generally more favorable production, binding, and pharmacokinetic characteristics. Numerous fibrin-binding peptides have been developed and labeled for MR, PET, PET/MR, and SPECT imaging in preclinical thrombosis models (46)(47)(48). One such fibrin-targeted PET radiotracer, 64 Cu-labeled fibrin-binding probe 8 ( 64 Cu-FBP8), demonstrated highly accurate carotid artery and femoral vein thrombus detection in rats (97.6%; 95% CI, 92-100) (49). ...
Cardiovascular imaging is evolving in response to systemwide trends toward molecular characterization and personalized therapies. The development of new radiotracers for PET and SPECT imaging is central to addressing the numerous unmet diagnostic needs that relate to these changes. In this 2-part review, we discuss select radiotracers that may help address key unmet clinical diagnostic needs in cardiovascular medicine. Part 1 examined key technical considerations pertaining to cardiovascular radiotracer development and reviewed emerging radiotracers for perfusion and neuronal imaging. Part 2 covers radiotracers for imaging cardiovascular inflammation, thrombosis, fibrosis, calcification, and amyloidosis. These radiotracers have the potential to address several unmet needs related to the risk stratification of atheroma, detection of thrombi, and the diagnosis, characterization, and risk stratification of cardiomyopathies. In the first section, we discuss radiotracers targeting various aspects of inflammatory responses in pathologies such as myocardial infarction, myocarditis, sarcoidosis, atherosclerosis, and vasculitis. In a subsequent section, we discuss radiotracers for the detection of systemic and device-related thrombi, such as those targeting fibrin (e.g., 64Cu-labeled fibrin-binding probe 8). We also cover emerging radiotracers for the imaging of cardiovascular fibrosis, such as those targeting fibroblast activation protein (e.g., 68Ga-fibroblast activation protein inhibitor). Lastly, we briefly review radiotracers for imaging of cardiovascular calcification (18F-NaF) and amyloidosis (e.g., 99mTc-pyrophosphate and 18F-florbetapir).
... Other diagnostic tools used are ultrasound, X-ray, CT, or MRI, which are all based on structural changes or the cessation of blood flow rather than the molecular composition of thrombi. The development and improvement of molecular imaging techniques are essential to visualise thrombi at an early stage, enable whole-body or multisite imaging, improve diagnostic specificity and sensitivity, and monitor clinical outcomes [7][8][9][10]. ...
The efficacy of thrombolysis is inversely correlated with thrombus age. During early thrombogenesis, activated factor XIII (FXIIIa) cross-links α2-AP to fibrin to protect it from early lysis. This was exploited to develop an α2-AP-based imaging agent to detect early clot formation likely susceptible to thrombolysis treatment. In this study, this imaging probe was improved and validated using 111In SPECT/CT in a mouse thrombosis model. In vitro fluorescent- and 111In-labelled imaging probe-to-fibrin cross-linking assays were performed. Thrombus formation was induced in C57Bl/6 mice by endothelial damage (FeCl3) or by ligation (stenosis) of the infrarenal vena cava (IVC). Two or six hours post-surgery, mice were injected with 111In-DTPA-A16 and ExiTron Nano 12000, and binding of the imaging tracer to thrombi was assessed by SPECT/CT. Subsequently, ex vivo IVCs were subjected to autoradiography and histochemical analysis for platelets and fibrin. Efficient in vitro cross-linking of A16 imaging probe to fibrin was obtained. In vivo IVC thrombosis models yielded stable platelet-rich thrombi with FeCl3 and fibrin and red cell-rich thrombi with stenosis. In the stenosis model, clot formation in the vena cava corresponded with a SPECT hotspot using an A16 imaging probe as a molecular tracer. The fibrin-targeting A16 probe showed specific binding to mouse thrombi in in vitro assays and the in vivo DVT model. The use of specific and covalent fibrin-binding probes might enable the clinical non-invasive imaging of early and active thrombosis.
... Panning counterselection campaigns by incubating phages with fibrinogen eliminated non-specific binders while positive selection panning rounds against fibrin and immobilized DD(E) fragments were used to isolate small cyclic peptide families, referred to as Tn6, Tn7, and Tn10, that differ in the size of the central disulfide-linked macrocyclic ring and the sequences (Kolodziej et al. 2012). In all three of these peptides classes (Tn6, Tn7, and Tn10), the disulfide bridge is critical for fibrin binding (Kolodziej et al. 2012;Oliveira and Caravan 2017). Tn6 and Tn7 peptides were used as conjugates together with MRI contrast agents (Stefanelli and Barker 2015). ...
... Tn6 and Tn7 peptides were used as conjugates together with MRI contrast agents (Stefanelli and Barker 2015). Later, it was discovered that EP-2104R belongs to the Tn6 family (Oliveira and Caravan 2017). The Caravan group further developed an elegant strategy for fibrin targeting using a modified EP-2104R. ...
... Since PDI catalyzes disulfide exchange in the integrin α 2B β 3 that allows the platelet to bind fibrinogen, they hypothesized that in the presence of activated platelets and PDI, the peptide would undergo disulfide formation and cyclization, producing a fibrin targeting probe in situ. A linear EP-2104R analogue prodrug had no affinity for the fibrin DD(E) fragment, but in the presence of PDI, disulfide rearrangement re-established fibrin binding properties (Oliveira and Caravan 2017). ...
The coagulation cascade represents a sophisticated and highly choreographed series of molecular events taking place in the blood with important clinical implications. One key player in coagulation is fibrinogen, a highly abundant soluble blood protein that is processed by thrombin proteases at wound sites, triggering self-assembly of an insoluble protein hydrogel known as a fibrin clot. By forming the key protein component of blood clots, fibrin acts as a structural biomaterial with biophysical properties well suited to its role inhibiting fluid flow and maintaining hemostasis. Based on its clinical importance, fibrin is being investigated as a potentially valuable molecular target in the development of coagulation therapies. In this topical review, we summarize our current understanding of the coagulation cascade from a molecular, structural and biophysical perspective. We highlight single-molecule studies on proteins involved in blood coagulation and report on the current state of the art in directed evolution and molecular engineering of fibrin-targeted proteins and polymers for modulating coagulation. This biophysical overview will help acclimatize newcomers to the field and catalyze interdisciplinary work in biomolecular engineering toward the development of new therapies targeting fibrin and the coagulation system.
... Thus fibrin could predict tumor growth and is prognostic factor for survival in clinical therapy. Although there are several approach for the detection of fibrin in the Table 1 [11][12][13][14][15][16][17]. The most of these methods, which employ molecular imaging technology, can detect micron insoluble fibrin, instead of molecular level. ...
A highly sensitive and specific ELISA-like chemiluminescence method for detection of fibrin has been developed. In the sensing platform, the homing peptide (CREKA), as recognition molecule, which can specially recognize the fibrin on microtiter plate, combined with G-quadruplex-based DNAzyme to form the probe of G-quadruplex-hemin DNAzyme-CREKA. After the sample solution was coated on the plates, the probe was crosslinked with fibrin through the interaction of CREKA and fibrin. Finally, luminol–H2O2 chemiluminesecence (CL) reaction was exploited for quantitative analysis of fibrin. The liner range for fibrin detection was from 0.112 pmol L⁻¹ to 5.6 pmol L⁻¹ with the detection limit of fibrin as low as 0.04 pmol L⁻¹, based on a signal-to-noise ratio (S/N) of 3. Furthermore, on the basis of the high amplification efficiency of the rolling circle amplification.
(RCA) reaction, the method enabled to analyze fibrin with a detection limit corresponding to 0.06 fmol L⁻¹, whose sensitivity increased 3 orders of magnitude than that of above method in the absence of RCA reaction. In particular, combined with the separation and washing steps of ELISA, the proposed method possessed higher selectivity, high-throughput and low cost, which shows promise for applications in clinical diagnosis.
... In the past, molecular targeting of fibrin was limited by the challenge of discerning the fibrin from fibrinogen, a component that shares 98% of its structure with fibrin and presents in high concentrations (2.5-3 mg/ml) in the blood (Standeven et al., 2005). Recently, specificity for fibrin over fibrinogen has been achieved by several molecular entities such as selective antibodies, small peptides, and tissue plasminogen activator (tPA) (Oliveira and Caravan, 2017). ...
Microbubbles with enhanced ultrasound represent a potentially potent evolution to the administration of a free drug in the treatment of thrombotic diseases. Conformational and expressional changes of several thrombotic biological components during active coagulation provide epitopes that allow site-specific delivery of microbubble-based agents to the thrombus for theranostic purpose. Through the interaction with these epitopes, emerging high-affinity small molecular ligands are able to selectively target the thrombi with tremendous advantages over traditional antibody-based strategy. In this mini-review, we summarize recent novel strategies for microbubble-based targeting of thrombus through epitopes located at activated platelets and fibrin. We also discuss the challenges of current targeting modalities and supramolecular carrier systems for their translational use in thrombotic pathologies.
... Noninvasive, sensitive, and specific imaging techniques, including magnetic resonance imaging (MRI) and positronemitted tomography, have also been used to study thrombus composition in patients and animal models. 65,66 Imaging of AIS thrombi revealed information about clot length, clot density, and clot burden score. 67 Other studies used proteomic approaches to identify thrombi constituents. ...
Thrombosis is a major complication of cardiovascular disease, leading to myocardial infarction, acute ischemic stroke (AIS), or venous thromboembolism. Thrombosis occurs when a thrombus forms inside blood vessels disrupting blood flow. Developments in thrombectomy to remove thrombi from vessels have provided new opportunities to study thrombus composition which may help to understand mechanisms of disease and underpin improvements in treatments. We aimed to review thrombus compositions, roles of components in thrombus formation and stability, and methods to investigate thrombi. Also, we summarize studies on thrombus structure obtained from cardiovascular patients and animal models. Thrombi are composed of fibrin, red blood cells, platelets, leukocytes, and neutrophil extracellular traps. These components have been analyzed by several techniques, including scanning electron microscopy, laser scanning confocal microscopy, histochemistry, and immunohistochemistry; however, each technique has advantages and limitations. Thrombi are heterogenous in composition, but overall, thrombi obtained from myocardial infarction are composed of mainly fibrin and other components, including platelets, red blood cells, leukocytes, and cholesterol crystals. Thrombi from patients with acute ischemic stroke are characterized by red blood cell- and platelet-rich regions. Thrombi from patients with venous thromboembolism contain mainly red blood cells and fibrin with some platelets and leukocytes. Thrombus composition from patients with myocardial infarction is influenced by ischemic time. Animal thrombosis models are crucial to gain further mechanistic information about thrombosis and thrombus structure, with thrombi being similar in composition compared with those from patients. Further studies on thrombus composition and function are key to improve treatment and clinical outcome of thrombosis.
