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\begin{document}

\title{Molecular Imaging of Arterial and Venous Thrombosis}



\author[1]{Xiaowei Wang}%
\author[2]{Melanie Ziegler}%
\author[2]{James McFadyen}%
\author[1]{Karlheinz Peter}%
\affil[1]{Baker IDI Heart and Diabetes Institute}%
\affil[2]{Baker Heart Research Institute - BHRI}%


\vspace{-1em}



  
  \date{May 04, 2024}


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\selectlanguage{english}
\begin{abstract}
Thrombosis contributes to one in four deaths worldwide and is the cause
of a large proportion of mortality and morbidity. A reliable and rapid
diagnosis of thrombosis will allow for immediate therapy, thereby
providing significant benefits to patients. Molecular imaging is a
fast-growing and captivating area of research, in both preclinical and
clinical applications. Major advances have been achieved by improvements
in three central areas of molecular imaging: 1) Better markers for
diseases, with increased sensitivity and selectivity; 2) Optimised
contrast agents with improved signal to noise ratio; 3) Progress in
scanner technologies with higher sensitivity and resolution. Clinically
available imaging modalities used for molecular imaging include,
magnetic resonance imaging (MRI), X-ray computed tomography (CT),
ultrasound, as well as nuclear imaging, such as positron emission
tomography (PET) and single photon emission computed tomography (SPECT).
In the preclinical imaging field, optical (fluorescence and
bioluminescent) molecular imaging has provided new mechanistic insights
in the pathology of thrombembolic diseases. Overall, the advances in
molecular imaging, driven by the collaboration of various scientific
disciplines, have substantially contributed to an improved understanding
of thrombotic disease, and raises the exciting prospect of earlier
diagnosis and individualised therapy for cardiovascular diseases. As
such, these advances hold significant promise to be translated to
clinical practice and ultimately to reduce mortality and morbidity in
patients with thromboembolic diseases.%
\end{abstract}%



\sloppy


\textbf{Molecular imaging for thrombosis}

Thrombosis is a complex event, usually a result of undesired and
uncontrolled platelet adhesion and aggregation, as well as increased
thrombin formation and fibrin generation, within a vessel (Ziegler et
al., 2019; Montague et al., 2020). Therefore, biomarkers of platelet
activation and fibrin generation have attracted major attention as
molecular targets for molecular imaging of arterial and venous
thrombosis. As microthrombosis plays an important role in
ischemic-reperfusion (I/R) injury, these targets have also been used to
image, for example cardiac I/R injury (Ziegler et al., 2019).

\textbf{Platelet imaging}

Platelets express several platelet-typical receptors on their surface.
For molecular imaging, those receptors are of particular interest, which
can be used to differentiate between resting and activated platelets.
Thereby, selective targeting of activated platelets present in thrombi
as compared to non-activated platelets in the circulation can be
achieved. The two most attractive biomarkers for platelet activation
used in molecular imaging are P-selectin and Glycoprotein (GP) IIb/IIIa.

\emph{P-selectin}

Upon platelet activation P-selectin, which is also known as CD62P, is
rapidly mobilised from alpha granules to the platelet surface membrane,
thereby allowing for the discrimination between non-activated and
activated platelets. The expression of P-selectin also leads to
leukocyte recruitment into the vessel via P-selectin glycoprotein
ligand-1 (PSGL-1) (Furie and Furie, 2004). By conjugating microbubbles
with either a P-selectin monoclonal antibody or a PSGL-1 recombinant
targeting ligand, Davidson et al. visualised enhanced ultrasound imaging
in the post-ischemic regions using a hind-limb ischemia murine model, as
well as myocardial I/R injury in both a murine and a non-human primate
model (Davidson et al., 2012, 2014). These microbubbles produced an
increase in signal enhancement at the 90 min and 3 h time-points
post-cardiac I/R injury (Figure 1) (Davidson et al., 2012). The area of
contrast enhancement on ultrasound correlated with the area at risk of
ischemia on histology.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Several research groups have used fucoidan, a polysaccharide that has a
high affinity towards P-selectin, functionalised to contrast agents for
visualisation of thrombi via SPECT, PET, MRI and ultrasound imaging
(Rouzet et al., 2011; Saboural et al., 2014; Suzuki et al., 2015; Li et
al., 2019). Using rat models, Rouzet et al. demonstrated the binding of
fucoidan radiolabeled with \textsuperscript{99m}Tc to the intraluminal
thrombus in an elastase rat model of abdominal aortic aneurysm (AAA).
Histologic analysis using Masson trichrome and immunostaining using
P-selectin showed co-registration with the autoradiography
of\textsuperscript{99m}Tc-fucoidan uptake within the mural thrombi
(Figure 2) (Rouzet et al., 2011). Using the same AAA rodent model,
Suzuki et al. investigated the binding of ultrasmall superparamagnetic
iron oxide (USPIO) particles coated in fucoidan to intraluminal thrombi
via MRI. The successful targeting of USPIO-FUCO resulted in hyposignals
at the location of the thrombi, whereas no change of signal was observed
using the non-binding USPIOs control (Figure 3) (Suzuki et al., 2015).
Via SPECT imaging, the group also observed an uptake
of\textsuperscript{99m}Tc-fucoidan in the intramyocardial vasculature in
a cardiac I/R injury rat model (Figure 4) (Rouzet et al., 2011; Saboural
et al., 2014). The uptake of radioactivity in the mid ventricular
section of the heart (area at risk) also matched their results from
immunohistological staining of P-selectin (Rouzet et al., 2011).
However, P-selectin can be shed from the surface of activated platelets
and is also expressed on other cells such as endothelial cells.
Therefore, it is not specific for platelets, thereby limiting its
suitability as a target epitope of activated platelets.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\emph{Glycoprotein (GP) IIb/IIIa}

GPIIb/IIIa receptor, also known as integrin
\selectlanguage{greek}α\selectlanguage{english}\textsubscript{IIb}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3} and CD41/CD61, is the most
abundant receptor on the membrane surface of platelets with 60,000 to
80,000 receptors per platelet. The expression of GPIIb/IIIa is
platelet-specific and upon platelet activation, the GPIIb/IIIa receptor
undergoes a conformational change from a low affinity to a high affinity
receptor for its natural ligand fibrinogen/fibrin. The
arginine-glycine-aspartic (RGD) peptide mimics the binding epitopes of
GPIIb/IIIa's natural ligand fibrinogen, and has therefore been used for
the detection of vascular thrombosis via a range of imaging technologies
(Schumann et al., 2002; Klink et al., 2010; Zhou et al., 2011; Hu et
al., 2012; Unger et al., 2014; Guo et al., 2015; Kang et al., 2015; Rix
et al., 2016). Hu et al, demonstrated that microbubbles conjugated with
cyclic RGD peptides, facilitated the visualisation of thrombosis via
molecular ultrasound imaging (Figure 5) (Hu et al., 2012). In this
study, the authors demonstrated that brightness-mode ultrasound imaging
could be used to visualise the echogenic thrombi in the abdominal artery
of a rodent model. Colour Doppler imaging was also used to confirm the
vascular stenosis created by the thrombi (Hu et al., 2012).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Using a cyclic RGD peptide, P975 conjugated to a Gd-DOTA chelate, Klink
et al. demonstrated enhanced MRI signal of the carotid artery \emph{in
vivo} using a murine model of arachidonic acid induced thrombosis.
T1-weighted MRI was used to image the black blood of the healthy left
carotid and an occluded right carotid artery. After injection of P975,
increased in Gd signal intensity was observed at 30 min and 120 min,
where no signal enhancement was noted for the untargeted Gd or in the
healthy carotid artery (Klink et al., 2010). Two other radiolabelled
RGD-based constructs have proceeded to clinical trials for the diagnosis
of deep vein thrombosis (DVT) via SPECT imaging (Muto et al., 1995;
Lister-James et al., 1996; Taillefer et al., 1999, 2000; Klem et al.,
2000). Unfortunately, both \textsuperscript{99m}Tc-P280
and\textsuperscript{99m}Tc-DMP444 have been discontinued because of
their low sensitivity and specificity for the diagnosis of PE,
contributed by prolonged radioactivity readout in both the chest region
and the blood pool, as well as their low uptake by the thrombus
(Carretta et al., 1999; Taillefer et al., 2000; Oliveira and Caravan,
2017). However, RGD-based contrast agents have two major limitations. 1)
As RGD peptides do not provide specificity for activated GPIIb/IIIa ,
RGD-based contrast agents will bind to all circulating platelets.
Thrombus imaging can be only achieved by the comparison of the static
thrombus signal to the moving blood signal. 2) Several other integrins
act as receptors for RGD containing ligands (Wang and Peter, 2017).
Indeed, this has been used for molecular imaging approaches since RGD
peptides have been used to target the vitronectin receptor (integrin
\selectlanguage{greek}α\selectlanguage{english}\textsubscript{v}\selectlanguage{greek}β\selectlanguage{english}\textsubscript{3}, CD51/CD61) (Golestani et al.,
2015; Sun Yoo et al., 2015) and are widely used in cancer imaging
(Alonso et al., 2009; Melemenidis et al., 2015; Withofs et al., 2015).

Using the antibody fragment abciximab (Reopro\selectlanguage{ngerman}®), Alonso et al.
demonstrated that preincubation of abciximab-microbubbles with human
thrombi generated \emph{ex vivo} resulted in enhanced ultrasound signals
after i.v. injection. Visualisation of the human clot in the artery was
achieved using ultrasound imaging, and as a negative control, upon
ultrasonic bubble destruction, the clot lost its echogenicity/brightness
(Alonso et al., 2007). However, Reopro has been developed as an
anti-platelet drug and is known to bind to GPIIb/IIIa on all human
platelets, independent of their activation state (Coller, 1999; Schwarz
et al., 2002). The use of Reopro as an imaging agent faces the same
limitations as RGD-based contrast agents with binding to all circulating
platelets and therefore the excistence of a circulating blood background
signal. Also abciximab blocks fibrinogen binding and therefore inhibits
the function of circulating platelets, potentially resulting in an
increased bleeding risk (Peter et al., 2000; Lele et al., 2001).

The development of a single-chain antibody (scFv) that specifically
targets the activated form of the GPIIb/IIIa receptor
(scFv\textsubscript{anti-GPIIb/IIIa}) has provided the unique
opportunity to do molecular imaging of activated platelets. This
approach has been successfully used to image diseases with central
involvement of activated platelets such as thrombosis, inflammation and
malignant diseases (Wang and Peter, 2017; Yap et al., 2017). This
scFv\textsubscript{anti-GPIIb/IIIa} does not bind to the resting
platelets (Figure 6) in the circulation and does not cause bleeding
complications (Schwarz et al., 2006; Wang et al., 2012). The
scFv\textsubscript{anti-GPIIb/IIIa} has been successfully used with a
wide range of contrast agents, such as ultrasound microbubbles (Wang et
al., 2012, 2014, 2016) and MPIOs (von Elverfeldt et al., 2014), and
demonstrated highly favourable binding capabilities to microthrombi
under flow conditions (Figure 7 and 8).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}

Using an acute mouse thrombosis model, where the carotid artery is
exposed to ferric-chloride to induce thrombosis, thrombi were
successfully visualised via MRI (von zur Muhlen et al., 2008; Ta et al.,
2011, 2017), nuclear imaging (Heidt et al., 2011; Ardipradja et al.,
2014; Hagemeyer et al., 2014), ultrasound (Wang et al., 2012, 2014,
2016), and optical imaging (Lim et al., 2017). Upon the injection of
microparticles, iron oxide (MPIOs) coupled with
scFv\textsubscript{anti-GPIIb/IIIa}, these targeted MPIOs were shown to
bind at the site of blood clots, resulting in a decrease of signal
intensity in T2-weighted MRI (Figure 9) (von zur Muhlen et al., 2008).
The authors further demonstrated that this diminution in signal could be
reversed after the injection of urokinase as a fibrinolytic drug,
indicating successful thrombolysis (Figure 10). Similarly,
scFv\textsubscript{anti-GPIIb/IIIa} radiolabelled with Indium-111
(\textsuperscript{111}In) and \textsuperscript{18}F has been used for
the imaging of activate platelets in thrombi using SPECT (Figure 11 and
Video 1) (Heidt et al., 2011) and PET (Ardipradja et al., 2014),
respectively. In another murine study, Ziegler et al. radiolabelled
scFv\textsubscript{anti-GPIIb/IIIa} with \textsuperscript{64}Cu, and
visualised the accumulation of activated platelets in ischemic and then
reperfused myocardium (Ziegler et al., 2016). This PET imaging approach
showed an uptake of radioactivity in the myocardium of mice, after just
a- 10 min period of ischemia (Figure 12).(Ziegler et al., 2016).
Noteably, after 10 min there was no increase of classical markers of
cardiac ischemia, indicating that the imaging of platelets provides a
means of detection of cardiac ischaemia with an unprecedented
sensitivity. Using ultrasound, Wang \emph{et al.} demonstrated direct
and immediate visualisation of thrombi using microbubbles coupled with
scFv\textsubscript{anti-GPIIb/IIIa} (Figure 13) (Wang et al., 2012).
Realtime molecular ultrasound imaging can be used to image the success
or failure of thrombolysis (Figure 14), and importantly, the dislodgment
of a thrombus can be directly visualised (Video 2) (Wang et al., 2012).
Lim et al. conjugated near-infrared dyes onto
scFv\textsubscript{anti-GPIIb/IIIa}, which allowed early and sensitive
detection of thrombi using \emph{in vivo} three-dimensional optical
imaging (Lim et al., 2017).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image12/image12}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image13/image13}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image14/image14}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image15/image15}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image16/image16}

\end{center}
\end{figure}

An increased fluorescence signal was also detected in the lung area,
indicating successful imaging of PE in a murine model (Figure 15) (Lim
et al., 2017). In another \emph{in vivo} study of PE, these activated
GPIIb/IIIa targeted MPIOs demonstrated focal accumulation within the
edematous area in the lung via T2 MRI (Figure 16) (Heidt et al., 2016).
Using the same particles, von Elverfeldt et al. successfully imaged
platelet-driven inflammation in the myocardium of an I/R injury mouse
model (von Elverfeldt et al., 2014). This activated-platelet targeted
MPIOS compared favourably to a standard gadolinium imaging of myocardial
necrosis (Figure 17) (von Elverfeldt et al., 2014).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image17/image17}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image18/image18}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image19/image19}

\end{center}
\end{figure}

\textbf{Fibrin imaging}

Fibrin is another crucial target that is frequently used for molecular
imaging of thrombosis. Botnar\emph{et al.} developed a novel
fibrin-binding gadolinium-labelled peptide (EP-1873) for MRI of
thrombosis and tested this in an experimental rabbit model of plaque
rupture (Botnar et al., 2004). This probe was further optimised
(EP-2104R) and successfully validated for MRI-based diagnosis of
coronary thrombosis and PE (Spuentrup et al., 2005a, 2005b; Overoye-Chan
et al., 2008). In another study using a murine model of venous
thrombosis in the inferior vena cava, Andia et al. demonstrated the
maximal increased of signal at day 7 of thrombosis and gradual reduction
from day 10 onwards (Andia et al., 2014). After radiolabelling
\textsuperscript{64}Cu with EP-2104R, the construct was injected into a
rat model of crush injury-induced thrombosis. A clear increase in uptake
of radioactivity was observed in the location of the thrombi via PET
imaging (Ay et al., 2014). Furthermore, the administration of
fibrinolytic therapy resulted in a loss of radioactivity signal,
indicating that the thrombi have been thrombolysed, indicating the
suitability of this imaging approach for the monitoring of success or
failure of thombolytic therapy (Ay et al., 2014). Bimodal imaging
performed with \textsuperscript{64}Cu radiolabeled EP-2104R provided
direct visualisation of thrombi via PET and MRI (Uppal et al., 2011).
This method also allowed the detection of multisite thrombi located in
the carotid artery and the femoral vein in a rat model via a whole-body
PET scan (Blasi et al., 2015). In another study, EP-2104R was
radiolabelled with \textsuperscript{68}Ga or\textsuperscript{111}In,
whereas a non-binding control was radiolabeled with
\textsuperscript{64}Cu, for multimodal SPECT/PET/CT imaging (Oliveira et
al., 2015). \textsuperscript{125}I-fibrinogen was injected prior to
ferric chloride induced thrombosis of the common carotid artery.
Multimodal imaging showed a hot spot corresponding to
the\textsuperscript{125}I-fibrinogen labelled thrombi with
fibrin-targeted\textsuperscript{68}Ga or \textsuperscript{111}In
isotopes, but not the non-binding radioisotopes (Oliveira et al., 2015).

Using EP-2104R, Hara et al. synthesised a fibrin-binding peptide (FTP11)
conjugated to a near-infrared dye and demonstrated successful \emph{in
vivo} optical imaging of thrombi in a jugular DVT murine model (Figure
18) (Hara et al., 2012). Using the same construct, the group further
demonstrated successful imaging of fibrin deposition on stents that were
implanted in rabbits using invasive optical coherence tomography (Figure
19) (Hara et al., 2015). EP-2104R has also been evaluated in Phase II
trials, where molecular MRI was performed on patients who were
previously diagnosed with thrombosis in the arteries, veins and/or the
heart (Spuentrup et al., 2008; Vymazal et al., 2009). Nevertheless, none
of these imaging products has reached approval for clinical use (Lanza
et al., 2019).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image20/image20}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image21/image21}

\end{center}
\end{figure}

Using a fibrin-binding peptide (FibPep) radiolabelled with 111In,
Starmans et al. demonstrated the successful location of thrombi in the
carotid artery of an acute murine thrombosis model using SPECT (Starmans
et al., 2013). In the same animal model, the group also demonstrated
significantly increased thrombus uptake when they injected iron oxide
nanoparticle-micelles, which were conjugated with Fibpep, via magnetic
particle imaging (Starmans et al., 2015).

\textbf{Conclusion}

Molecular imaging holds strong promise for early and sensitive diagnosis
of thromboembolic diseases. The choice of epitopes and biomarkers for
molecular targeting, as well as the selection of imaging modalities and
their respective contrast agents, are especially important for clinical
translation. A better understanding of thrombotic diseases and their
progression will provide the clinicians with information to determine
the pharmacological treatment most suited for an individual, taking us
closer towards personalised medicine. Furthermore, the ability to
determine the success or failure of antithrombotic or fibrinolytic
treatment will also enable better decisions for invasive interventions
to be performed. Therefore, molecular imaging of thrombi has an enormous
potential to provide benefits for patients suffering from thromboembolic
diseases.

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