Histological comparison of arterial thrombi in mice and men and the influence of Cl-amidine on thrombus formation
Histological comparison of arterial thrombi in mice and men and the influence of Cl-amidine on thrombus formation
Julia Novotny 0 1
Sue Chandraratne 0 1
Tobias Weinberger 0 1
Vanessa Philippi 0 1
Konstantin Stark 0 1
Andreas Ehrlich 0 1
Joachim Pircher 0 1
Ildiko Konrad 0 1
Paul Oberdieck 0 1
Anna Titova 0 1
Qendresa Hoti 0 1
Irene Schubert 0 1
Kyle R. Legate 0 1
Nicole Urtz 0 1
Michael Lorenz 0 1
Jaroslav Pelisek 1 2
Steffen Massberg 0 1
Marie- Luise von BruÈ hl 0 1
Christian Schulz 0 1
0 Medizinische Klinik und Poliklinik I, Ludwig-Maximilians-Universit aÈt , Munich, Germany , 2 Walter-Brendel- Centre of Experimental Medicine, Ludwig-Maximilians-Universit aÈt , Munich, Germany, 3 DZHK ( German Centre for Cardiovascular Research), partner site Munich Heart Alliance , Munich, Germany , 4 Department of Applied Physics, Center for NanoSciences, Ludwig-Maximilians-Universit aÈt , Munich , Germany
1 Editor: Nades Palaniyar, Hospital for Sick Children , CANADA
2 Department of Vascular and Endovascular Surgery, Klinikum rechts der Isar der Technischen UniversitaÈ t , Munich , Germany
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This study was supported by the SFB 914
(SM, KS, CS), the DFG-Forschergruppe 923 (SM)
as well as the FP7 programme (PRESTIGE; SM),
the SFB 1123 (SM, KS), LMU excellent (KS), the
Deutsche Herzstiftung e.V. (TW, PO), the DZHK
(German Centre for Cardiovascular Research) and
by the BMBF (German Ministry of Education and
Research). PO is recipient of the
Medical treatment of arterial thrombosis is mainly directed against platelets and coagulation
factors, and can lead to bleeding complications. Novel antithrombotic therapies targeting
immune cells and neutrophil extracellular traps (NETs) are currently being investigated in
animals. We addressed whether immune cell composition of arterial thrombi induced in
mouse models of thrombosis resemble those of human patients with acute myocardial
Methods and results
In a prospective cohort study of patients suffering from AMI, 81 human arterial thrombi were
harvested during percutaneous coronary intervention and subjected to detailed histological
analysis. In mice, arterial thrombi were induced using two distinct experimental models,
ferric chloride (FeCl3) and wire injury of the carotid artery. We found that murine arterial
thrombi induced by FeCl3 were highly concordant with human coronary thrombi regarding
their immune cell composition, with neutrophils being the most abundant cell type, as well
as the presence of NETs and coagulation factors. Pharmacological treatment of mice with
the protein arginine deiminase (PAD)-inhibitor Cl-amidine abrogated NET formation,
reduced arterial thrombosis and limited injury in a model of myocardial infarction.
Neutrophils are a hallmark of arterial thrombi in patients suffering from acute myocardial infarction and in mouse models of arterial thrombosis. Inhibition of PAD could represent an
Doktorandenstipendium der Deutschen
Herzstiftung. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
interesting strategy for the treatment of arterial thrombosis to reduce neutrophil-associated
tissue damage and improve functional outcome.
Ischemic heart diseases account for 7.0 million deaths per year worldwide [
]. In AMI patients,
arterial thrombosis is typically triggered by rupture of an atherosclerotic plaque within
coronary arteries [
]. Plaque rupture is associated with disintegration of the endothelial layer and
subsequent exposure of the subendothelial extracellular matrix (ECM) [
]. ECM provides a
strong activation signal to circulating blood cells and the coagulation system, and triggers a
complex series of events that ultimately culminate in the formation of an occluding arterial
thrombus. Although the number of deaths due to AMI has decreased [
], efficient strategies to
prevent thrombotic complications of coronary atherosclerosis are still lacking.
Platelets are of major importance in the pathophysiology of thrombus formation. They
initiate the thrombotic cascade by forming platelet aggregates in a process that depends on
platelet integrins, particularly glycoprotein IIb-IIIa [
]. In addition, platelets support the local
recruitment of immune cells to nascent thrombi at the site of plaque rupture. The subsequent
crosstalk between platelets and recruited immune effectors then results in activation of blood
], which propagates thrombus formation and growth in a process termed
]. The latter represents a physiological process supported by immune cells and
specific thrombosis-related molecules generating an intravascular scaffold that facilitates the
recognition, containment and destruction of pathogens, thereby protecting host integrity.
However, if uncontrolled, immunothrombosis constitutes the pathophysiologic basis of vessel
thrombosis in the absence of pathogens [
]. Monocytes and neutrophils are of particular
importance for immunothrombosis, as they deliver tissue factor (TF) and promote TF
activation to initiate local coagulation. Further, neutrophils provide extracellular traps (NETs), DNA
matrices that act as strong procoagulant surfaces propagating local coagulation [6±8].
Our current knowledge on the cellular and molecular mechanisms underlying arterial
thrombosis largely derives from mouse models [9±11]. In mice arterial injury can be induced
by several techniques [
], including FeCl3 exposure or wire injury. However, it is unknown to
date whether these mouse models reflect the phenotype of human coronary thrombi and thus
are suitable to obtain data relevant for human pathophysiology. Therefore, we directly
compared human coronary artery thrombi of AMI patients to thrombi generated in two distinct
mouse models of arterial thrombosis. We found arterial thrombi induced by FeCl3 to be highly
concordant with human coronary thrombi regarding their cellular composition. We also
identified key features of immunothrombosis, specifically neutrophils and NETs, in both human
and murine arterial thrombi. Application of Cl-amidine, a pharmacological inhibitor of
peptidylarginine deiminase (PAD), into mice reduced arterial thrombosis and myocardial injury.
Our findings support recent data on immunothrombosis and suggest that the cross-talk
between immune cells and coagulation could represent a target for the treatment of arterial
Materials and methods
Retrieval of human thrombi
In this study, we included patients with acute myocardial infarction (AMI) undergoing
percutaneous coronary intervention (PCI) that displayed thrombotic occlusion of a coronary artery
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upon cardiac catheterization. Acute myocardial infarction was defined as ST-segment
elevation (STEMI) in the ECG or positive troponin test and/or unstable angina pectoris without
ST-segment changes (Non-ST-segment elevation myocardial infarction (NSTEMI)). STEMI
was defined as ST-elevation in 2 concordant leads according to current ESC guidelines [
]. In all cases there was an indication for removing the thrombotic material from the
coronary artery (cases of stent-thrombosis were not included) according to the guidelines valid
during the time of patient enrolment. For this procedure a ProntoTM thrombectomy catheter
device (Vascular Solutions, Minneapolis, USA) was used. Approval for this study was obtained
from the Ethics Committee of the University of Munich (Project number 4007/11a), and
written informed consent was obtained in accordance with the Declaration of Helsinki.
Definition of thrombus age
We defined thrombus age as period of time between the onset of symptoms according to the
patients' statement and the time point of interventional thrombus removal. In 50 out of 81
patients, the onset of AMI symptoms could be determined precisely, thus allowing the
calculation of thrombus age.
Time points of thrombus analysis in mice were driven by the thrombus age calculated in
human patients according to their presentation. The mouse model was adapted to match this
timing. In 24 out of 50 human patients (approximately 50%) the coronary thrombus was
removed within 6 hours after symptom onset. Therefore, we decided to determine the kinetics
of leukocyte recruitment after 3 and 6 hours and carried out in-depth histological analysis at
those time points.
All mice were on C57BL6/J background. Specific pathogen-free mice were obtained from
Charles River. All mice used for experiments were between 8 and 14 weeks old and weighed
between 21 and 25 g. Each experimental group was weight- and sex-matched so that
experiments were carried out on male and female mice with similar weight and in equal distribution
between groups. All procedures were performed on anaesthetized animals. Animals were
sacrificed by cervical dislocation under deep anaesthesia induced with fentanyl, midazolam and
medetomidine. Animal experiments were carried out according to the guidelines from
Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific
purposes. All experimental procedures on animals met the requirements of the German
legislation on protection of animals and were approved by the Government of Bavaria (Regierung
von Oberbayern), Germany (reference numbers 55.2-1-54-2532-176-09,
55.2-1-54-2532-18211 and 55.2-1-54-2532-76-13).
Assessment of arterial thrombosis after ferric chloride exposure
For thrombus induction in the carotid artery in vivo, we used wire injury (see below) or local
application of FeCl3 [
]. In brief, mice were anesthetized using 2% isoflurane and
intraperitoneal injection of fentanyl (0.05 mg/kg), midazolam (5.0 mg/kg) and medetomidine (0.5 mg/
kg). Thereafter, the common carotid artery was exposed. Platelets were either labeled in vivo
via intravenous infusion (tail vein catheter) of a fluorescently labeled antibody (X488,
DyLight488-labeled, Emfret analytics, WuÈrzburg Germany, 7 μL per mouse in 100 μL sterile PBS), or
by infusion of platelets labeled ex vivo with 2',7'-dichlorofluorescein (DCF). For ex vivo
labelling, murine platelets were collected by cardiac puncture and isolated from citrated whole
blood as reported previously [
]. Subsequently, platelets were labelled with DCF. After
adjustment to a final concentration of 150x103 platelets / 200μl the fluorescently labelled platelet
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suspension was injected via a tail vein catheter. To induce arterial thrombosis, a filter paper
(0.5±1.0 mm) saturated with 10% FeCl3 was applied for 3 minutes at the lateral side of the
carotid artery adventitial surface (close to the carotid bifurcation) [
]. To investigate
leukocyte accumulation over time, thrombus growth was allowed for up to six hours before the
vessels were excised.
Determination of arterial thrombosis after wire-induced arterial denudation
Wire-induced endothelial disruption was performed as described previously [
]. In brief,
animals were anesthetized and platelets were isolated from donor mice and labelled with DCF
]. In recipient mice, the right carotid artery was exposed via a midline neck incision. The
common, external and internal carotid arteries were identified and the right internal carotid
artery was subsequently ligated with 8±0 silk suture (Ethicon). Additional 8±0 silk ties were
looped around the common and external carotid arteries to prevent potential blood loss
during the procedure. After transverse arteriotomy of the right internal carotid artery a 0.014-inch
flexible angioplasty guide wire was introduced and advanced 10 mm towards the aortic arch.
Endothelial denudation injury of the right common carotid artery was performed by passing
through the wire three times in a rotating motion to cause endothelial denudation. After
removal of the wire, the right internal carotid artery was untied and thrombus growth was
allowed for three hours. Subsequently animals were sacrificed and arteries were excised for
Ischemia-reperfusion injury in mice
Myocardial ischemia reperfusion injury was performed as previously described [
brief, mice were anaesthetized using 2% isoflurane and intraperitoneal injection of fentanyl
(0.05 mg/kg), midazolam (5.0 mg/kg) and medetomidine (0.5 mg/kg). Mice were then placed
on a heated operating table in a supine position and ventilated using a small animal ventilator
(MiniVent, HUGO SACHS, March, Germany) after endotracheal intubation. Access to the
heart was gained using a lateral thoracotomy in the second intercostal space. After
visualization of the left anterior descending artery (LAD), a suture (8±0 polyamid) was passed
underneath the LAD approximately 1 mm distal of the left auricle and tied to a loose double knot. A
PE-10 tube was placed between the heart and the knot. The knot was tightened and secured
with a second slipknot. Ischemia was confirmed by appearance of a pale color in the
myocardium distal to the ligation. After ischemia for 60 minutes, the PE tube and ligation was
removed and reperfusion was confirmed by return to a reddish color of the myocardium.
After chest closure anesthesia was antagonized using atipamezol (3.75 mg/kg) and flumazenil
(0.72 mg/kg), and mice were extubated when spontaneous breathing was sufficient.
Hemodynamic parameters were measured in vivo on day 7 after ischemia. Mice were
anaesthetized as described above, fixated on a temperature controlled operating table, intubated
and ventilated (MiniVent, Hugo Sachs, March, Germany). A 1.4 French impedance
micromanometer catheter (Millar Instruments, Houston, TX, USA) was introduced into the left
ventricle via the right carotid artery and pressure volume loops were recorded. Raw
conductance volumes were corrected for parallel conductance. Hemodynamic measurements as
well as data analyses were performed using PVAN analysis software (Hugo Sachs, March,
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To evaluate infarct size, mice were euthanized after cardiac catheterization and perfused with
10 ml of 4% formaldehyde solution. Infarct size was determined according to the previous
]. In brief, hearts were excised and fixated in 4% formaldehyde solution for 24
hours. Thereafter, they were cut into three 2 mm thick transverse slices (from the base to the
apex) and embedded in paraffin. 5 μm thick sections of each slice were cut and mounted on
positively charged glass slides and stained using Masson's trichrome staining. Mean fibrosis
area was quantified on transverse slices by a researcher blinded to the treatment groups. Infarct
size was determined as area of fibrosis correlated to the area of the left ventricle (including
Inhibition of NETosis
We used the peptidylarginine deiminase (PAD) inhibitor
N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-l-ornithine amide (Cl-amidine, Calbiochem, #506282) to disrupt NET formation
]. Cl-amidine was dissolved in PBS and a dosage of 10mg/kg was administered. For the
study of carotid artery injury Cl-amidine was injected into the tail vein of mice 30 min before
ferric chloride exposure [
]. Injection of its vehicle (PBS) served as control. Further, we
compared the effect of Cl-amidine on thrombus formation to heparin (100U/kg body weight)
injected into the tail vein 10 minutes before ferric chloride exposure . In the myocardial
ischemia-reperfusion model Cl-amidine was administered intraperitoneally at onset of
ischemia and at time of reperfusion. A third dose of Cl-amidine was administered 12 hours after
reperfusion. Vehicle (PBS) injection served as control.
Intravital epifluorescence microscopy
To maintain a physiological temperature, anesthetized animals were placed on a custom
heating mat. The skin of the animals' neck was opened and the carotid artery was carefully
prepared. Stained platelets were injected into the tail vein. Thereafter, thrombus growth in the
carotid artery of Cl-amidine and vehicle treated mice was induced using the FeCl3 method as
described above. In parallel, imaging was started to record the initiation of thrombus
formation as well as the duration of arterial occlusion (time from thrombotic vessel occlusion until
restoration of blood flow). The time to thrombotic occlusion of the carotid artery downstream
of the site of injury was defined as the time required for complete arrest of blood flow in the
center of the vessel after removal of the filter paper. While imaging, a prewarmed (37ÊC)
solution of isotonic (0.9%) saline was used to continuously cover exposed regions of the vessel.
Measurements were carried out using a Leica DM 6 FS microscope equipped with an Andor
Zyla sCMOS camera, or a high-speed widefield Olympus BX51WI fluorescence microscope
equipped with a long-distance condenser, a 10x objective with an Olympus MT20
monochromator and an ORCA-ER CCD Camera (Hamamatsu). Leica or Olympus (Cell^R) software
were used for image recording and analysis.
Human thrombi were harvested during percutaneous coronary intervention as described
above. For haematoxylin and eosin (H&E) histology and immunofluorescence stainings,
human thrombi were immediately submerged in liquid nitrogen after retrieval and stored at
-80ÊC. Murine carotid arteries were harvested and rinsed with PBS, embedded in O.C.T.
compound and frozen at -80ÊC. Both human thrombi and murine vessels were cut into 5 μm thick
sections using a cryotome. Specimens were fixed in 4% formaldehyde solution for 4 min,
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washed in PBS, and blocked with serum or 5 μg/ml anti-mouse CD16/32 (eBioscience) and 1%
BSA (PAA Laboratories) in PBS for 30 min. Sections were incubated with primary antibodies
(Table 1) for one hour at room temperature, and then washed in PBS + 0.1% Tween. Detection
was performed with fluorescent secondary antibodies (Table 1).
Leukocytes were identified by using an anti-CD45 antibody in both species. Neutrophils
were identified by expression of neutrophil elastase (NE) in humans and mice [
], or using the
mouse Ly6G antigen. In mice, monocytes/mononuclear phagocytes were labelled using
], whereas in humans we used the marker CD14 [
]. The fibrinogen
antibody detected both fibrinogen and fibrin. Murine lymphocytes were identified using
CD45R for B-cells and CD3 for T-cells. 1 μg/ml Hoechst 33342 (Invitrogen) was used to stain
DNA and Histone H3 antibody was used to visualize histones and NET formation. For control
stainings, we used a matching isotype control in combination with the fluorescent secondary
antibody. All immunofluorescence images, including controls, were stained with a nuclear dye
(DAPI or Hoechst, as indicated).
Human lymphocytes were analyzed by immunohistochemistry. Tissues were stained with
antibodies against CD20 to identify B-cells and CD3 to identify T-cells, respectively. Primary
antibodies were visualized by Peroxidase/DAB ChemMate Detection Kit (DAKO) according
to the manufacturer's instructions. Samples were mounted using an anti-fade mounting
medium (DAKO) and sealed with a coverslip.
Microscopy and immunofluorescence analysis
Images were acquired using either a Zeiss Imager M2 Axio epifluorescence microscope, or
a Leica DMRB epifluorescence microscope with a Zeiss AxioCam and processed with an
AxioVision software (Zeiss). Neutrophils and NETs were counted in four fields of view using a
40x objective (176x131 μm). Monocytes, T- and B- lymphocytes were counted in the whole
thrombus area. The results were extrapolated to cells/mm2 or NETs/100 leukocytes. In our
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experiments the following prerequisites had to be fulfilled for quantification of NET
formation: 1) presence of filamentary structured extracellular DNA, 2) this DNA had to originate
from cells stained positively for a neutrophil marker, and 3) respective filamentary structures
had to be decorated with a marker for neutrophil granule proteins (like NE) or citrullinated
histone H3. To investigate thrombus coverage of coagulation factors (fibrin/fibrinogen, FXII,
TF) and platelet area, overview images of the thrombi were acquired and analysed using Zeiss
imaging software and ImageJ.
All data are shown as mean ± standard deviation (SD). Baseline characteristics were performed
with SPSS. Statistical analyses were carried out using SigmaPlot1 12.0 and GraphPad (Prism
6). To determine differences between groups, data were analysed using a two-tailed unpaired
StudentÂs t-test or one-way ANOVA for multiple comparisons. A value of P<0.05 was
Phenotype of human coronary artery thrombi
To evaluate the phenotype of human thrombi in arterial thrombosis we collected specimens of
AMI patients undergoing emergency PCI and thrombus aspiration (Table 2). In total, we
analysed 81 thrombi of 81 patients (one thrombus per patient). Mean age of the patients enrolled
was 62.9 ± 11.2 years. The majority of patients were male (80.2%), presented with STEMI
(58%) and one- or three-vessel coronary artery disease (38.3% and 39.5%, respectively). Only
few patients had an ejection fraction <30% (5.1%). The most common cardiovascular risk
factors were hypercholesterolemia (present in 70% of patients) and hypertension (71.3%). Only
9% of the patients were on dual antiplatelet therapy when admitted to hospital (Table 2). In 50
cases we were able to determine the exact time point of symptom onset, allowing close
approximation of thrombus age (for details see materials and methods section). Out of these 50
patients, 62% arrived at the hospital within 12 hours of symptom onset, 20% arrived within
12±24 hours, and 18% arrived later than 24 hours (Fig 1A).
In histological and immunohistochemical analyses, human coronary thrombi displayed a
heterogeneous morphology with compact, cell-rich regions and areas with fewer cells and less
density (Fig 1B). Apart from platelets, leukocytes were present in large numbers in thrombi,
supporting a role for inflammatory cells in arterial thrombosis in humans.
Thrombus-associated leukocytes were mostly found in clusters or layers (Fig 1C). Further analysis revealed that
neutrophils represented the major fraction with 73.4 ± 26%, whereas monocytes constituted
17.9 ± 6% (Fig 1D). Within the first 12 hours after symptom onset, the number of leukocytes
increased and correlated moderately with thrombus age (R2 = 0.3364) (Fig 1E). Together this
suggests that in addition to platelets, neutrophils and monocytes were the most abundant cell
subsets that accumulated in thrombi of patients suffering from AMI. This is in line with
previous work on thrombi isolated from AMI patients [
FeCl3-induced arterial thrombi closely resemble the phenotype of human
As outlined above, various techniques exist to induce arterial thrombosis in mice. In this study
we analysed the thrombus phenotype in the two most frequently used models (wire-injury and
FeCl3) and compared it to arterial thrombi in humans. Similar to humans, CD41+ platelets
were a major cellular constituent of mouse thrombi, consistent with their important role in
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Fig 1. Characteristics of human arterial thrombi. (A) Pie chart shows the distribution of thrombus age. 50
out of 81 patients described the precise onset of AMI symptoms, which allowed the calculation of thrombus
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age following its removal during PCI. The majority of human thrombi (with precise onset of symptoms) was
younger than 24h. (B) Leukocyte accumulation in human thrombi. Representative images of HE staining
(n = 3). Bars, 200μm (top image) and 50μm (bottom image). (C) Immunohistochemical visualization of
leukocytes (CD45, green, n = 3), neutrophils (NE, red, n = 81) and monocytes (CD14, green, n = 11). Nuclei
are counterstained with Hoechst (including controls). Control (isotype) or secondary antibody alone. Bars,
10μm. (D) The graph shows the quantification of monocytes (n = 11) and neutrophils (n = 81) in human
thrombi. Results are shown as mean ± SD. (E) Correlation between human thrombi younger than 12h and the
number of leukocytes (n = 33).
arterial thrombogenesis (Fig 2A, S1 Fig). FeCl3 exposure resulted in large, compact thrombi
that closely resembled the phenotype of thrombi from AMI patients. In contrast, experimental
thrombi obtained with wire denudation were considerably smaller, less dense and did not
occlude the entire lumen of the artery. Thus, mechanical injury by wire denudation was a less
efficient trigger of thrombosis compared to FeCl3, and these thrombi were markedly different
to those found in human coronary artery thrombosis (Fig 2A).
In addition to platelets, we found large numbers of leukocytes accumulating in murine
arterial thrombi. Similar to human thrombi, CD45+ leukocytes in FeCl3-induced arterial
thrombosis were distributed in clusters or layers (Fig 2B), and showed a substantial increase over
time. Because of the close similarities in morphology and cellular composition of thrombus
specimen obtained from human coronary arteries and FeCl3-treated mouse carotid arteries,
we compared these thrombi in more detail. Leukocyte content at different time points of
thrombosis and the increase in cell numbers was well comparable between mice and men
(Figs 1E and 2C). In thrombi retrieved after 3 hours, we quantified 902 leukocytes/mm2 in
humans (3h from symptom onset to thrombus retrieval) and 857 leukocytes/mm2 in mice (3h
after FeCl3 application), (Figs 1E and 2C). Further, neutrophils were the predominant
leukocyte subset also within mouse arterial thrombi, constituting 81.1 ± 19%, while monocytes
accounted for 15.4 ± 8% of leukocytes (Fig 2D). B and T lymphocytes represented only minor
leukocyte fractions in both human and murine arterial thrombi (S2 Fig). We next addressed
fibrinogen/fibrin (FGN) deposition and found comparable immunofluorescence stainings in
mouse and human thrombi (Fig 3A and 3B). Due to the fragmented morphology and smaller
size of the thrombi generated in the wire injury model, we were not able to determine the
composition of these thrombi in more detail. Taken together, murine thrombi generated by
FeCl3exposure closely resembled the phenotype of thrombi harvested from AMI patients containing
the major effectors of immunothrombosis, including platelets, fibrinogen/fibrin, and immune
Presence of NETs in arterial thrombi of mice and men
Neutrophils can exhibit strong pro-coagulatory properties by releasing DNA matrices (NETs).
In line with previous reports [
], we found extracellular DNA in 23% (19 out of 81) of
human coronary thrombi (Fig 4A). Interestingly, the amount of NETs relative to the thrombus
leukocyte count was similar between human AMI specimens and FeCl3-induced murine
arterial thrombi (Fig 4B). Of these 19 patients with NETs, thrombus age could be determined in
10 patients (Fig 4C). The amount of netting neutrophils increased with thrombus age and
displayed similar kinetics between mice and men (Fig 4C).
Cl-amidine inhibits arterial thrombosis
A functional role of NETs in thrombosis has previously been demonstrated in
atherosclerosisprone mice [
]. To address the role of NETs in FeCl3-induced arterial thrombosis, we
visualized thrombus formation using intravital microscopy in the presence and absence of
Cl10 / 24
Fig 2. Characteristics of mouse arterial thrombi induced by FeCl3 injury or wire denudation in mice. (A) Immunohistological images
of platelet aggregate area (red) in arterial thrombi (n = 3/group) and control stainings. Bars, 100μm. Control (isotype) or secondary antibody
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alone. (B) Comparison of leukocyte recruitment to the mouse carotid artery 3h after FeCl3 exposure or wire denudation (n = 3/group).
Representative images show immunohistochemical staining for leukocytes (CD45, green) and their subsets, as distinguished by expression
of neutrophil elastase (NE, red) for neutrophils and CD68 (red) for blood monocytes. Nuclei were counterstained with Hoechst (including
controls). Bars, 10μm. Control (isotype) or secondary antibody alone. (C) Association between number of leukocytes and thrombus age
(n = 3/group). Mean ± SD. (D) Quantification of monocyte and neutrophil subsets within mouse thrombi 3h after FeCl3 exposure (n = 3/
group). Mean ± SD.
amidine, which impairs NETosis through inhibition of peptidylarginine deiminase (PAD) [
]. Cl-amidine reduced FeCl3-induced arterial thrombosis (Fig 5A and 5B). More specifically,
the time until thrombotic occlusion of the carotid artery was prolonged (Cl-amidine 19.9
min ± 7.6 vs. vehicle 13.3 min ± 4.0, n = 8) and re-establishment of blood flow was accelerated
(Cl-amidine 1.7 min ± 1.4 vs. vehicle 7.8 min ± 6.3, n = 8) (Fig 5B). Similar results for
ClFig 3. Accumulation of fibrinogen/fibrin in human and mouse arterial thrombi. (A) Representative
immunohistochemical staining of mouse and human thrombi for fibrinogen/fibrin (red) and control stainings.
Nuclei were counterstained with Hoechst (including controls). Bars: 50μm (top left and right), 200μm (bottom
left and right), 300μm (top and bottom middle). (B) Fibrinogen/fibrin-covered area in the thrombus (human
thrombi n = 6, mouse thrombi n = 3). Data are shown as mean ± SD.
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Fig 4. NETs in arterial thrombi of mice and humans. (A) Representative illustration of NETs stained for NE and DNA
(DAPI) in the early phase of arterial thrombosis. Human and mouse thrombi showed comparable morphology after 3, 6 or
12h. Extracellular DNA originates from NE+ neutrophils. Bars, 10μm. Arrows, nuclei; arrowheads, NET fibers. (B)
Quantification of NETs per 100 neutrophils in human thrombi (<12h) (n = 10) and experimental thrombosis (FeCl3) (3±6h)
(n = 5). Dots represent individual experiments; lines indicate mean values for each group. (C) Association between thrombus
age and number of NETs in mice and humans.
amidine were observed by direct labelling of platelets in vivo (Fig 5A) as compared to ex vivo
labelling and re-infusion of donor platelets (S3 Fig). Inhibition of thrombus formation by
Clamidine was robust but not as pronounced as intravenous application of 100U/kg heparin
To further address the role of Cl-amidine in thrombus formation, we carried out
immunofluorescence stainings of immune cells and coagulation proteins. Unexpectedly, we observed a
strong decrease in the number of leukocytes, including neutrophils, within arterial thrombi of
Cl-amidine treated mice. Thus, in addition to disrupting NETosis, Cl-amidine depletes
leukocytes in arterial thrombi whereas leukocyte numbers in peripheral blood remained stable (S4B
Fig). The underlying mechanism remains elusive and will need to be determined in future
work. However, absence of distinct leukocyte populations is known to affect thrombus stability
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Fig 5. Cl-amidine inhibits arterial thrombosis in mice. (A) Representative intravital microscopy images 5,
10 and 20min after FeCl3 injury in mice treated with Cl-amidine or vehicle. Platelets were labeled in vivo
(green). Bars, 200μm. (B) Time until occlusion (left) and duration of vessel occlusion (right) after FeCl3
exposure in mice treated with vehicle (n = 8) or Cl-amidine (n = 8). (C) Left: Representative histological
images (Ly6G in red, cit H3 in green, DAPI in blue) of NETs in mice treated with vehicle or Cl-amidine (n = 5/
group). Bars, 5μm. Arrowhead, NET fiber. Middle: Quantification of NETs per 100 neutrophils (n = 5/group).
Right: Quantification of leukocytes (left axis) and neutrophils (right axis) in murine arterial thrombi.
PLOS ONE | https://doi.org/10.1371/journal.pone.0190728
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Fig 6. Cl-amidine modulates thrombus composition. (A) Immunofluorescence analysis of fibrinogen (1st
row), tissue factor (2nd row) and factor XII (3rd row) in arterial thrombi of mice treated with vehicle or
Clamidine. Nuclei were counterstained with Hoechst. Controls were stained with isotype and secondary
antibody antibody together, and Hoechst, Bars: 50μm. (B) Immunofluorescence staining of coagulation
factors in % of whole thrombus area. Left: Fibrinogen-covered thrombus area (vehicle n = 5, Cl-amidine n = 5).
Middle: Tissue factor (vehicle n = 5, Cl-amidine n = 4). Right: Factor XII (vehicle n = 6, Cl-amidine n = 5).
Results are mean ± SD.
]. To define the role of Cl-amidine in more detail, we carried out immunofluorescence
stainings of key proteins of the coagulation system. Fibrinogen and FXII coverage of the thrombus
area was reduced in the presence of Cl-amidine (Fig 6), suggesting that this compound affects
not only thrombus composition in respect to NETs and leukocyte content, but also modulates
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coagulatory properties of the thrombus. Together, these findings support the concept that
Clamidine could provide an interesting strategy to inhibit arterial thrombosis.
Cl-amidine reduces myocardial injury
Coronary NET burden has been associated with infarct size in humans [
] and abrogation of
NET formation in Pad4-/- mice reduced infarct size in the early phase of myocardial ischemia
]. To address whether Cl-amidine reduces myocardial injury and whether these effects
persist in the late phase of AMI, we applied Cl-amidine in a model of myocardial
ischemia-reperfusion (I/R) injury. In detail, we performed a transient ligation of the LAD for 60 minutes and
treated these mice with either Cl-amidine (10 mg/kg) or vehicle. Cl-amidine significantly
reduced infarct size after 7 days in comparison to vehicle treated mice (Fig 7A and 7B). This
Fig 7. Cl-amidine reduces myocardial ischemia-reperfusion injury. (A) Representative masson-trichrome
stainings of myocardial sections from mice 7 days after myocardial ischemia-reperfusion injury treated with vehicle
(left) or Cl-amidine (right). Mice treated with Cl-amidine show a decrease in fibrotic tissue compared to vehicle. Bars
2mm. (B) Infarct size 7 days after myocardial ischemia-reperfusion injury in mice treated with vehicle (n = 10) and
Clamidine (n = 7). (C) Myocardial function was evaluated by measuring ejection fraction (in %) and (D) cardiac output
(in μl/min) 7 days after myocardial injury in mice treated with vehicle (n = 5) and Cl-amidine (n = 6). Dots represent
individual experiments, lines indicate mean values for each group.
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resulted in an improved cardiac function of Cl-amidine treated animals, reflected by an
increased ejection fraction (Fig 7C) and higher cardiac output (Fig 7D). Thus, Cl-amidine
improves functional outcome after myocardial injury in mice beyond the acute phase of ischemia.
Acute myocardial infarction is a leading cause of death worldwide. Platelets and coagulation
pathways are known to critically contribute to arterial thrombosis. However, the role of
immune cells and immunomodulatory molecules is less well understood. Therefore, animal
models are warranted that produce arterial thrombi closely resembling those in human
pathologies such as myocardial infarction. In our present study, we show that the mouse carotid
artery injury model based on FeCl3 exposure displays many relevant similarities to human
coronary thrombi retrieved from patients with AMI. Specifically, the percentage distribution of
immune cell populations, the time course of leukocyte accumulation as well as the kinetics of
NET formation in this mouse model resembled those of human coronary artery thrombi.
Inhibition of NETosis with Cl-amidine is associated with reduced thrombus stability in arterial
thrombosis and improved outcome in myocardial infarction. Interestingly, Cl-amidine not
only disrupted NETs but also abrogated leukocyte accumulation in arterial thrombi, indicating
that the mechanistic effects of this compound extend beyond inhibition of NETosis.
Rupture of an atherosclerotic plaque provides the primary trigger of atherothrombosis in
humans resulting in platelet adhesion, activation and aggregation [
]. Apart from platelets,
activation of coagulation and subsequent fibrin formation plays a crucial role during thrombus
growth and stabilization [2, 30±33]. Immune cells actively contribute to this process [
]. A large body of experimental work in mice has already addressed the mechanisms
underlying arterial thrombosis. However, it remained unclear whether these models adequately
reflected the situation in humans [
]. We therefore characterized common mouse models
of arterial thrombosis applying either chemical (FeCl3) or mechanical injury (wire denudation)
. Thrombi obtained through wire injury were incompact and did not match the
morphology human coronary artery thrombi. Immune cells such as neutrophils were detectable,
however, the smaller size and fragmentation of these thrombi precluded an in-depth analysis. We
then compared murine arterial thrombi generated in the FeCl3 model with human coronary
artery thrombi obtained from patients with acute myocardial infarction. We determined
whether both pathologies shared key features, such as immune cell composition, NET
formation and platelet content. Despite differences in thrombus age and localization of arterial
thrombi in mice (3±6 hours, carotid artery) and humans (< 24 hours, coronary artery), we
found major analogy between specimens of both species. During the first 12 hours the
appearance of leukocytes in both human and mouse thrombi was time dependent, with more
immune cells present in older thrombi. In general, we found numerous neutrophils and
monocytes in thrombi of mice and humans, a finding quantitatively and qualitatively
consistent among both species. Neutrophils thereby represented the most prominent leukocyte
subset, which is in line with other recent studies [
], even though human blood consists of a
larger neutrophil fraction compared to mouse blood . Mice have approximately three
times more platelets in whole blood as compared to humans [
], however, mouse platelets are
significantly smaller [
]. It has been speculated that the higher platelet count combined with a
smaller volume would result in a similar overall platelet mass in human and mouse blood .
In line with this, we detected platelets and fibrinogen/fibrin in comparable amounts in mouse
and human thrombi indicating similar contribution to thrombus development.
How do immune cells trigger arterial thrombosis? Recent evidence suggests that the
mechanisms used by immune cells to trigger coagulation in nascent arterial thrombi partially mimic
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those involved in immunothrombosis, a conserved process in which immune cells activate
procoagulant pathways to compartmentalize, retain and kill invading pathogens [
]. On the
one hand neutrophils can release neutrophil elastase (NE), which stabilizes intraluminal
thrombus development by counteracting endogenous anticoagulants (e.g. TF pathway
inhibitor) that impede intraluminal coagulation under physiological conditions [
]. In this regard,
absence of NE in mice resulted in prolonged bleeding time and reduced thrombus stability
]. In our comparison of mouse and human specimen, we determined the presence of NE in
thrombi of both species suggesting the contribution of neutrophil-derived prothrombotic
molecules to arterial thrombosis. A second prominent function of neutrophils is the formation of
]. These procoagulant DNA matrices are among the key effector molecules of
]. NETs contribute to deep vein thrombosis in mice and men [
]. They have also been identified in arterial thrombi of AMI patients, in which NET burden
was associated with infarct size [
]. Further, NET formation is enhanced by activated platelets
presenting high mobility group box 1 (HMGB1) protein to neutrophils [
]. We show here,
that in the mouse carotid artery injury model induced by FeCl3, the kinetic of NETs
accumulation (i.e. amount of NETs accumulating over time, number of NETs per thrombus area), was
similar to that of human patients with AMI. This is an important finding since it will allow
future studies in mice studying the consequences of NETosis and targeting NET formation
in arterial thrombosis. Further, inhibition of NETosis with the PAD-inhibitor Cl-amidine
reduced thrombus stability in the FeCl3 model. This resulted in earlier reperfusion of the
occluded vessel as observed by intravital microscopy. In line with this, we did not find NETs
in thrombi of these mice. Surprisingly, Cl-amidine treatment not only abrogated NET
formation but also diminished the number of leukocytes within arterial thrombi. This observation
seems to be a local effect on the forming thrombus since leukocyte counts in peripheral blood
remained stable, which is in line with the literature [
]. It could be possible that
Cl-amidine induces apoptosis in thrombus cells. In fact, it has recently been suggested that PAD
inhibition activates the tumor suppressor gene OKL38 thereby inducing apoptosis . The
precise mechanism will need to be addressed in more detail in future work. Together, our
findings suggest that Cl-amidine not only functions as a potent inhibitor of NET formation but
also exerts additional effects on components of the immune system.
NETs can bind effectors of blood coagulation, such as TF and FXII, thereby providing a
platform to support their activation [
7, 50, 51
]. TF initiates the extrinsic pathway of
coagulation and leads to fibrin formation after vascular injury [
]. Its binding to NET structures
promotes TF exposure at the site of plaque rupture [
]. In this study, Cl-amidine did not affect
TF immunofluorescence in arterial thrombi, suggesting that NETs were not essential for
exposure of TF in this setting. In fact, various immune cells participate in the storage and exposure
of TF, and may contribute to thrombosis [54±56]. However, we observed reduced staining for
FXII and fibrinogen in arterial thrombosis of mice treated with Cl-amidine. The initiator
protease of the intrinsic pathway of coagulation FXII is activated by binding to negatively charged
surfaces such as NETs [
7, 40, 57
]. Thus, the interaction of NETs could lead to the initiation of
the FXII-coagulation pathway thereby triggering fibrin formation, as previously shown for
venous thrombosis [
]. Factor XIIa regulates the structure of the fibrin clot independently of
thrombin generation through direct interaction with fibrin [
]. Consequently, formation and
stabilization of arterial thrombi is reduced in FXII-deficient mice [
]. The data presented
here could suggest that the contact pathway-mediated fibrin formation is more important in
NET-induced thrombosis. However, our findings are based solely on immunofluorescence
analysis. Quantitative data on the protein amount of TF and FXII in the presence and absence
of netting neutrophils, as well as measurements of their enzymatic activity, are warranted
before such conclusions can be drawn.
18 / 24
As expected from patient cohorts with cardiovascular disease, individuals presenting with
AMI were mostly male, old age and presented with several cardiovascular risk factors. We
could not identify typical characteristics of patients or thrombi which predisposed for the
presence of NETs, however, the study was not designed to specifically assess this question. Another
limitation of our study is related to the detection of NETs in human thrombi. NETs were
found in only 23% (n = 19) of patient specimen. This could be due to the early, mostly
pre-hospital, application of heparin in AMI patients. In fact, the anticoagulant is known for its direct
interaction with NETs, causing their degradation both in vitro [
] and in vivo [
the numbers and morphology of NETs in the specimen staining positive for extracellular DNA
were similar to that quantified in the mouse FeCl3 model.
To test whether treatment with Cl-amidine also conferred a beneficial effect in myocardial
infarction beyond the acute phase, we performed an I/R model with transient ligation of the
proximal LAD. After 1 week, we found the infarct area to be significantly reduced in the
Clamidine group, which translated into improved cardiac function. Our data add to previous
studies in PAD4-deficient mice showing a reduction of myocardial injury in the early phase
(< 24 hours) of infarction [
]. Thus, inhibition of NETosis by Cl-amidine, or potentially
other inhibitors [
], may provide a novel treatment strategy in arterial thrombosis. Because
this strategy does not seem to affect hemostasis [
], it could provide an improved risk
(bleeding)±to±benefit (prevention of ischemic endpoints) profile as compared to conventional
In conclusion, we have demonstrated that in regard to their cellular composition (i.e.
platelets, leukocyte subsets) and prothrombotic molecules (i.e. NETs, fibrin, FXII), FeCl3-induced
arterial thrombi in mice most closely resemble those of human AMI patients, suggesting that
this experimental animal model is more suitable than the one induced by mechanical injury to
study the role of immune cells in arterial thrombus development. Further, our study points to
yet untargeted molecular pathways and cellular players other than platelets. Current strategies
to treat or prevent arterial thrombosisÐwhich consist of a combination of antiplatelet and
anticoagulant agentsÐinhibit major haemostatic pathways and thus share the inherent
weakness of affecting haemostasis. Targeting of key molecules in immunothrombosis could help to
maximize the efficacy of therapies that prevent arterial thrombosis without increasing the
incidence of bleeding complications.
S1 Fig. Platelet accumulation in mouse and human thrombi. (A-B) Immunhistological
images of platelet aggregate area in arterial thrombi received from humans (n = 6) and mice
(n = 3) after FeCl3 injury. Analyses of platelet distribution and thrombus composition showed
a comparable morphology. Bars, 100μm. (B) Corresponding quantification of platelet
aggregate area. Data are shown as mean ± SD.
S2 Fig. Lymphocytes in mouse and human thrombi. (A) Immunohistological images of
lymphocytes in arterial thrombi received from humans (n = 6 per group) and mice (n = 3 per
group) after FeCl3 injury and control stainings. Bars, 50μm. Arrowheads, positive cells. (B)
Analysis of lymphocytes shows a comparable small population between patients and
FeCl3treated mice. Data are shown as mean ± SD.
S3 Fig. Murine arterial thrombosis following infusion of platelets labeled ex vivo. (A-B)
Imaging of FeCl3-induced arterial thrombosis in mice receiving ex vivo labeled platelets.
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Isolated platelets were stained with DCF (green) and infused into recipient mice, in which
carotid injury was induced. Mice were treated with Cl-amidine or vehicle. (A) Representative
intravital microscopy images 5, 10 and 20min after FeCl3 injury. Bars, 200μm. (B) Time until
occlusion (left) and duration of vessel occlusion (right) after FeCl3 exposure. Mice were either
untreated (n = 7), or treated with vehicle (n = 9) or Cl-amidine (n = 9). Data are shown as
mean ± SD. Results are comparable to experiments in which platelets were directly labeled in
vivo (Fig 4).
S4 Fig. Comparison of heparin to Cl-amidine in mouse arterial thrombosis. (A) Time until
occlusion and duration of vessel occlusion after FeCl3 exposure in mice treated with vehicle
(n = 8) or Cl-amidine (n = 8) or 100U/kg body weight heparin (n = 3). (B) Quantification of
leukocytes in peripheral blood of mice before (n = 5) and 2h after (n = 5) application of
Clamidine. Data are shown as mean ± SD.
S5 Fig. FXII immunofluorescence in human thrombi. Immunohistochemical staining for
FXII (green) in human specimen. Human coronary thrombi (top) and liver (bottom row),
which serves as positive control. Nuclei were counterstained with Hoechst (including isotype
control). Bars, 50μm.
We thank Renate Hegenloh, Anne-Maria Suhr, Kristin Steigerwald and Elisabeth
Kennerknecht for excellent technical assistance. Furthermore, we thank Dr. Daniel Hartmann for
providing human liver for histological analysis.
Conceptualization: Steffen Massberg, Marie-Luise von BruÈhl, Christian Schulz.
Data curation: Julia Novotny, Sue Chandraratne, Anna Titova, Marie-Luise von BruÈhl.
Formal analysis: Julia Novotny, Sue Chandraratne, Tobias Weinberger, Marie-Luise von
Funding acquisition: Christian Schulz.
Investigation: Julia Novotny, Sue Chandraratne, Tobias Weinberger, Vanessa Philippi,
Konstantin Stark, Andreas Ehrlich, Joachim Pircher, Ildiko Konrad, Paul Oberdieck, Qendresa
Hoti, Irene Schubert, Kyle R. Legate, Nicole Urtz, Michael Lorenz, Jaroslav Pelisek,
MarieLuise von BruÈhl, Christian Schulz.
Methodology: Tobias Weinberger, Andreas Ehrlich, Joachim Pircher, Anna Titova, Michael
Lorenz, Jaroslav Pelisek.
Project administration: Steffen Massberg, Christian Schulz.
Resources: Steffen Massberg.
Supervision: Julia Novotny, Sue Chandraratne, Tobias Weinberger.
Validation: Julia Novotny, Sue Chandraratne.
Visualization: Julia Novotny, Sue Chandraratne, Marie-Luise von BruÈhl, Christian Schulz.
20 / 24
Writing ± original draft: Christian Schulz.
Writing ± review & editing: Konstantin Stark, Joachim Pircher, Steffen Massberg,
Luise von BruÈhl, Christian Schulz.
21 / 24
J. 2006; 20(7):956±8. Epub 2006/03/31. doi: fj.05-4763fje [pii] https://doi.org/10.1096/fj.05-4763fje
22 / 24
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