From bloodjournal.hematologylibrary.org at Sanquin on August 23, 2012. For personal use only.

Prepublished online July 26, 2012;

doi:10.1182/blood-2012-06-437772

Tissue factor positive neutrophils bind to injured endothelial wall and

initiate thrombus formation
Roxane Darbousset, Grace M. Thomas, Soraya Mezouar, Corinne Frère, Rénaté Bonier, Nigel Mackman,
Thomas Renné, Françoise Dignat-George, Christophe Dubois and Laurence Panicot-Dubois

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Blood First Edition Paper, prepublished online July 26, 2012; DOI 10.1182/blood-2012-06-437772
From bloodjournal.hematologylibrary.org at Sanquin on August 23, 2012. For personal use only.

Tissue factor positive neutrophils bind to injured endothelial wall and

initiate thrombus formation

Running title: Neutrophils initiate thrombus formation in vivo.

Roxane Darbousset1,2, Grace M. Thomas3, Soraya Mezouar1,2, Corinne Frère1,2, Rénaté

Bonier1,4, Nigel Mackman5, Thomas Renné6, Françoise Dignat-George1,2, Christophe

Dubois1,2, Laurence Panicot-Dubois1,2.

1
Aix Marseille Université, Campus Santé, 27 Boulevard Jean Moulin, Marseille, France.
2
INSERM UMR-S1076, Faculty of Pharmacy, 27 Boulevard Jean Moulin, 13385 Marseille,

France. 3Immune Disease Institute and Program in Cellular and Molecular Medicine,

Children’s Hospital Boston; Department of Pediatrics, Harvard Medical School, 3 Blackfan

circle, Boston, MA 02215, USA.4INSERM UMR-911, 27 Boulevard Jean Moulin, 13385

Marseille, France.5Division of Hematology/oncology, Department of Medicine, 917 Mary

Ellen Jones Building, University of North Carolina, Chapel Hill, NC 27599, USA.
6
Department of Molecular Medicine and Surgery and Center of Molecular Medicine,

Karolinska Institute and University Hospital, 17176, Stockholm, Sweden.

Corresponding author: Christophe Dubois, INSERM UMR-S1076, Faculty of Pharmacy, 27

Boulevard Jean Moulin, 13385 Marseille, France. E-mail: christophe.dubois@univ-amu.fr;

Phone: +33491835600; Fax: +33491835602.

Copyright © 2012 American Society of Hematology

 From bloodjournal.hematologylibrary.org at Sanquin on August 23, 2012. For personal use only.

Abstract:

For a long time, blood coagulation and innate immunity have been viewed as interrelated

responses. Recently, the presence of leukocytes at the sites of vessel injury has been described.

Here we analyzed interaction of neutrophils, monocytes and platelets in thrombus formation

following a laser-induced injury in vivo. Neutrophils immediately adhered to injured vessels,

preceding platelets, by binding to the activated endothelium via LFA-1-ICAM-1 interactions.

Monocytes rolled on a thrombus 3 to 5 minutes post-injury. The kinetics of thrombus

formation and fibrin generation were drastically reduced in low tissue factor mice whereas the

absence of factor XII had no effect. In vitro, tissue factor was detected in neutrophils. In vivo,

the inhibition of neutrophil binding to the vessel wall reduced the presence of tissue factor

and diminished the generation of fibrin and platelet accumulation. Injection of wild-type

neutrophils into low tissue factor mice partially restored the activation of the blood

coagulation cascade and accumulation of platelets. Our results show that the interaction of

neutrophils with endothelial cells is a critical step preceding platelet accumulation for

initiating arterial thrombosis in injured vessels. Targeting neutrophils interacting with

endothelial cells may constitute an efficient strategy to reduce thrombosis.

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Introduction:

Following vessel injury, a rapid accumulation of platelets and activation of the blood

coagulation cascade are critical steps to prevent blood loss. Also, blood polymorphonuclear

neutrophils (PMNs) play a major role to limit intrusion of microorganisms into the

bloodstream. For a long time, different observations have linked blood coagulation and innate

immunity. In 1882, Bizzozero first observed that the formation of a thrombus involved both

platelets and leukocytes1. More recently, the presence of leukocytes accumulating at the sites

of vessel injury has been described2,3. Clinical studies have shown that traditional vascular

risk factors are associated with leukocyte activation and may predispose to thrombogenesis4,5.

PMNs may contribute to fibrin generation directly by exposing active tissue factor (TF)6

and/or indirectly by inactivating the major tissue factor inhibitor, tissue factor pathway

inhibitor (TFPI)2. PMNs may also locally assemble proteins of the factor XII-driven contact

pathway7. Recent advances in digital microscopy make it possible to observe, in real-time, the

formation of a thrombus following a vessel injury in living mice8. In this model of laser-

induced injury, the subendothelial matrix is not exposed to the blood circulation9. Thrombin is

locally generated and platelet-rich thrombi are formed following activation of the endothelial

cells10. Von Brühl et al have recently showed the importance of monocyte, PMN and platelet

cross-talk in a mouse model of deep vein thrombosis (DVT)3. In this model, neutrophils are

indispensable for propagation of the clotting cascade by binding factor XII (FXII) and by

supporting its activation through the release of neutrophil extracellular traps (NETs)11.

Following exposure of the subendothelial matrix, neutrophils may, by interacting with

platelets, inactivate TFPI thus leading to the activation of the tissue factor-dependent

coagulation cascade2. Vascular smooth muscle-derived TF is also critical for arterial

thrombosis in mice12. Following a laser-induced injury, a previous study suggested that

monocyte-derived microparticles contribute to thrombus formation in arteries by delivering

 From bloodjournal.hematologylibrary.org at Sanquin on August 23, 2012. For personal use only.

tissue factor to the site of injury and interacting with accumulating platelets13,14. However, the

relative contribution of neutrophils and monocytes in microvascular thrombosis remains

unclear. Using the laser-injury system with a high-definition, high-speed camera, we

analyzed interaction of neutrophils, monocytes and platelets in thrombus formation in the

microvasculature of mice. Our results demonstrate that neutrophils by binding to the vessel

wall are the main source of blood-borne tissue factor in the early phase of thrombus

formation.

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Material and Methods:

Mice.

Wild-type C57BL/6J mice were from Janvier Elevage (Le Genest Saint Isle, France).

CX3CR1GFP C57BL/6J mice were obtained from the Jackson Laboratory. FXII-/- mice and
15 16 17
low TF mice have been previously described . All animal care and experimental

procedures were performed as recommended by the European Community guidelines and

approved by the French Ministry of Agriculture (agreement no. 13.382, L. Panicot-Dubois).

Antibodies and reagents.

The rat anti–mouse CD41 antibody (clone MWReg30; 0,2μg/g mouse weight) and R300

antibody were used to deplete circulating platelets18 were obtained from Emfret (Eibelstadt,

Germany). Alexa Fluor 488 rat anti–mouse, Lysosomal-Associated Membrane Protein 1

(LAMP-1, CD107a) antibody (clone 1D4B; 0,25 μg/g mouse weight) and Alexa Fluor 647

anti-mouse CD54 (ICAM-1) antibody (clone YN1/1.7.4; 0,5 μg/g mouse weight) were from

Biolegend (Ozyme Biolegend, St Quentin, France). PE rat anti-mouse Ly-6G antibody (clone

1A8; 0,1 μg/g mouse weight), purified rat anti-mouse Ly-6G and Ly-6C antibody (clone

RB6-8C5), Alexa Fluor 647 rat anti-mouse CD11b (Mac-1) antibody (clone M1/70; 1 μg/g

mouse weight), blocking rat monoclonal anti–mouse P-selectin antibody (clone RB40.34,),

purified hamster anti-mouse ICAM-1 antibody (clone 3E2), rat anti-mouse CD11a (LFA-1)

antibody (clone M17/4), APC rat anti–mouse CD45 antibody (clone 30-F11), rat anti–mouse

TER-119 antibody (clone TER-119; 0,59 μg/g mouse weight) and rat IgG2a isotype control

were from BD Biosciences (Le Pont de Claix, France). Blocking anti-mouse ICAM-1 and

LFA-1 antibodies were respectively used at 4 μg/g and 2 μg/g of mouse weight. Isolectin B4

used to label the endothelial wall19, DIO (Iodide of (dodecyl-4 aminostyrile)-4 N-

methylpyridium) was obtained from Life Technology (Saint Aubin, France). The mouse

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monoclonal antibody directed against human, rat and mouse TF (ab104513) was from Abcam.

1H1 antibody directed against mouse TF was obtained from Dr. Daniel Kirchhofer20. A

mouse anti-human fibrin II γ-chain antibody (clone NYBT2G1) was from Accurate Chemical

(Westbury, NY, USA).

Fab fragments from the anti-CD41 antibody were generated using the Immuno-Pure Fab

Preparation Kit (Pierce Thermofisher, Brebieres, France). Fab fragments, antibodies and

control isotypes were conjugated to dye light Fluor 488 or 647 according to the

manufacturer’s instruction (Pierce Thermofisher). Prostaglandin I2 was obtained from

Merck. Apyrase grade VI, saponin and BSA were purchased from Sigma-Aldrich (Saint

Quentin, France). Corn trypsin inhibitor was obtained from Cryopep (Mauguio, France).

Flow cytometry.

Blood was collected from mice in a citrate solution (ACD: 85 mM trisodium citrate, 67 mM

citric acid, 111.5 mM glucose, pH 4.5) in the presence of 0.5 mM prostacyclin and 0.02 U/ml

apyrase as previously described21. Analyses of blood and platelet-rich plasma samples were

performed on a flow cytometer (Gallios; Beckman Coulter, Villepinte, France) using

appropriate antibodies to label white blood cells22.

Isolation of leukocytes.

Leukocytes were extracted from spleen of wild-type mice as previously described by Stem

Cells technologies. Isolated leukocytes were counted in Malassez’s cell and labeled with DIO

(Iodide of (dodecyl-4 aminostyrile)-4 N- methylpyridium) according to the manufacturer

instruction (Vybrant Kit, life technologies). Purified labeled cells were resuspended in saline

buffer at a concentration of 1.109 cells/ml and were infused in the blood of a recipient mouse

at a concentration of 2.106cells/ mouse.

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Isolation of neutrophils.

Neutrophils from bone marrow or peripheral blood were isolated as previously described23

using GR-1 coupled magnetic beads (anti-Ly-6G Microbead kit, Milteny Biotech, Paris,

France).

Depletion of neutrophils.

Depletion of neutrophils from wild-type mice was performed by intravenous injection of 5

μg/g mouse of rat anti-Ly-6G/C (RB6-8C5; eBioscience, Paris, France) as previously

described24. Control mice were injected with PBS. Neutropenia was evaluated 24 hours

postinjection by flow cytometry using a PE-conjugated anti-mouse neutrophils mAb.

Fluorescence microscopy.

Purified neutrophils were washed with PBS and fixed for 30 minutes at 4°C in PBS

containing 2% paraformaldehyde. Once fixed, cell suspensions were cytocentrifugated for 10

minutes at 500 rpm in a shandom cytospin 4 (Thermo Fisher Scientific). Cells were

permeabilised using PBS containing 0.3% saponin for 20 minutes. Then, cells were blocked

for 20 minutes at 4°C with BSA 1%, saponin 0.3% in PBS buffer. Cells were incubated at

4°C for 2 hours with the appropriate dilution of antibodies and 0.3% saponin, followed by

incubation with an Alexa 488- conjugated secondary antibody. Between each step, cells were

rinsed with PBS containing 0,3% saponin and were observed on a microscope (Nikon Eclipse

TE2000-U, Nikon, Champigny sur Marne, France).

Western blotting.

Western blotting was performed after SDS polyacrylamide (11%) gel electrophoresis.

Proteins were transfered onto a nitrocellulose membrane using an iBlot system (Life

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Technology) according to the manufacturer’s instruction. Transfer was verified by staining

membranes with Ponceau red. Detection of the labeled proteins was performed by

chemiluminescence using ECL from GE Healthcare (Orsay, France).

Intravital microscopy.

Intravital videomicroscopy of the cremaster muscle microcirculation was performed as

previously described8 using an Intelligent Imaging Innovations system (Intelligent Imaging

Innovations GmbH). Briefly, mice were pre-anesthetized with intraperitoneal ketamine (125

mg/kg; Panpharma, Fougères, France), xylazine (12.5 mg/kg; Bayer, Romans-sur-Isère,

France), and atropine (0.25 mg/kg; Lavoisier, Paris, France). A tracheal tube was inserted and

the mouse maintained at 37°C on a thermo-controlled rodent blanket. To maintain anesthesia,

pentobarbital (CEVA, Libourne, France) was administered through a cannula placed in the

jugular vein. After the scrotum was incised, the testicle and surrounding cremaster muscle

were exteriorized onto an intravital microscopy tray. The cremaster preparation was

superfused with a thermo-controlled (37°C) bicarbonate-buffered saline throughout the

experiment. Microvessel data were obtained using an Olympus AX microscope with a 60x or

100x 0.9 NA water immersion objective. The fluorescence microscopy system has previously

been described25. Digital images were captured with a Cooke Sensicam CCD camera in 640 x

480–pixel format or with a CoolSnapEZ HD camera in 1392x1040-pixel format.

Laser-induced injury.

Antibodies or exogenously labeled mouse leukocytes were infused through the jugular vein

into the circulation of an anesthetized mouse. Vessel wall injury was induced with a nitrogen

dye laser (Micropoint; Photonics Instruments, Bozeman, MT, USA) focused through the

microscope objective, parafocal with the focal plane and aimed at the vessel wall9. Typically

6
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1 or 2 pulses were required to induce vessel wall injury. For all the experiments performed

involving injection through the jugular vein of antibodies or cells, 3 to 5 mice were studied

for each condition and a minimum of 10 thrombi induced per mouse. In all experiments new

thrombi were formed upstream of earlier thrombi to avoid any contribution from thrombi

generated earlier. There were no characteristic trends in thrombus size or thrombus

composition in sequential thrombi generated in a single mouse during an experiment. Image

analysis was performed using Slidebook (Intelligent Imaging Innovations). Fluorescence data

were captured digitally at up to 50 frames per second and analyzed as previously described to

determine the median of fluorescent intensity signal overtime8.

Statistics.

Significance was determined by the Wilcoxon’s rank-sum test for the in vivo experiments 8.

Differences were considered significant at P < 0.05.

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Results:

Leukocytes accumulate at the site of arterial injury independent of platelets:

In the second following vessel injury, cells of 10 to 20 μm in diameter were seen to

accumulate at the injury site. These cells were also detectable 3 to 5 seconds following the

injury when platelets started to accumulate, as well as 80 to 120 seconds post-injury when the

thrombus was at its maximal size and 3 minutes later when it was stabilized (Fig. 1A).

Fluorescently labeled leukocytes infused into mice were detected at the site of injury with

similar kinetics (Fig. 1B). In addition, a fluorescent signal was observed in the second

following the injury when a Mac-1 (Macrophage 1 Antigen, CD11b) but not an irrelevant

antibody (Fig. 1C) was used. Also, when the red blood cell-specific (RBC) TER-119 antibody

was infused RBCs were detected in the circulating blood but not at the site of thrombus (Fig.

S1A). The kinetics of Mac-1 positive cells indicates that leukocytes rapidly accumulate at the

site of laser-induced injury in vivo (Fig. S1B). Following the depletion of the circulating

platelets, no signal corresponding to platelet accumulation was detected at the site of injury.

However, the kinetics of leukocyte accumulation was identical in the presence or the absence

of platelets (Fig. 1D). We concluded that leukocytes accumulated before platelets at the site of

laser-induced injury by interacting with activated endothelial cells.

LFA-1 expressing leukocytes interact with ICAM-1 expressed on injured vascular wall:

Recently, Atkinson et al. determined that endothelial cells were activated following the use of

a dye laser26. We confirmed these results and found that both LAMP-1 (Lysosomal-

Associated Membrane Protein 1, CD107a) and ICAM-1 (Inter-Cellular Adhesion Molecule 1,

CD54) were detected on endothelial cells immediately after the injury (Fig. S1C). This laser-

induced activation of the endothelium was a prerequisite for the interaction of leukocytes

because the fluorescently labeled circulating leukocytes did not interact with resting

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endothelium in vivo. Blocking ICAM-1 abolished the accumulation of leukocytes at the site of

laser-induced injury (Fig. 2A). Similar results were observed after blocking the ICAM-1

ligand, Leukocyte Function Antigen-1 (LFA-1, CD11a), expressed by leukocytes (Fig. 2B).

Neutrophils but not monocytes accumulate at the site of injury:

To determine if monocytes were rapidly binding to the injured endothelium, thrombus

formation was studied in CX3CR1GFP mice. CX3CR1GFP mice coexpress GFP (Green

Fluorescent Protein) with CX3CR1 so that their monocytes, monocytes-derived

microparticles, macrophages and dendritic cells (DCs) are all fluorescently labeled27.

Exogenous monocytes and monocyte-derived microparticles were previously shown to play a

role in thrombus formation13. The kinetics of thrombus formation was similar in CX3CR1GFP

and wild-type mice (Fig. 3A). Although macrophages and monocytes were detected in the

cremaster tissue and circulating in veins, no GFP signal was detected at the site of thrombus

formation during the first 3 minutes following a laser-induced injury (Fig. 3B and movie S1).

Three to 5 minutes following the injury, GFP-expressing cells were detected rolling on the

thrombus and bound to it 25 min later (Fig. 3B). Altogether, these results indicate that

monocytes are not required for the formation of the platelet thrombus in arteries.

Neutrophils are the main population of granulocytes and constitute about 20% of total

leukocytes circulating in the blood of mice. The 1A8 antibody is directed against Ly-6G,

which is mainly expressed in circulation by neutrophils, although about 20% of circulating

monocytes, identified as inflammatory monocytes, also expressed this marker (Fig. S2A).

Following infusion of the 1A8 antibody into wild-type mice, a fluorescent signal was detected

at the site of injury (Fig. 4A). The signal appeared before any platelets could be detected and

it increased over time (Fig. 4B). To confirm that neutrophils were the subpopulation of

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leukocytes accumulating at the site of injury, experiments in CX3CR1GFP mice were

performed. Neutrophils were detected by infusion of 1A8 antibody; monocytes, monocyte-

derived microparticles, DCs and macrophages were detected by the expression of GFP;

platelets were labeled by infusion of an anti-CD41 antibody; and inflammatory monocytes

expressed GFP and were labeled by 1A8. We observed that neutrophils, but neither

monocytes nor inflammatory monocytes, accumulated at the site of injury in the seconds

following the injury (Fig. 4C and movie S2). Three to 5 minutes following the injury,

inflammatory monocytes were detected rolling over the thrombus (Fig. 4D). We conclude that

neutrophils were the exclusive circulating cells that bound to the activated endothelium in the

second following the arterial injury, before platelets. Previous studies have shown that
2,28,29
leukocytes participate in the activation of the blood coagulation cascade . Following a

laser-induced injury, thrombin is the main platelet agonist involved in thrombus

formation10,15,25. We confirmed that thrombus formation and fibrin generation were largely

reduced in low TF mice (Fig. S2B). PMNs assemble the factor XII (FXII) driven contact

pathway that initiates the intrinsic pathway of coagulation30. We compared the kinetics of

platelet accumulation and fibrin generation in wild-type and FXII-/- mice16. Our results

indicate that both thrombus formation and fibrin generation were similar in wild-type mice

and FXII-/- mice (Fig. 4E). These data were confirmed in wild-type mice infused with corn

trypsin inhibitor (CTI, an inhibitor of activated FXII). CTI affected neither the kinetics of

thrombus formation nor fibrin generation following laser-induced injury in wild-type mice

(Fig. S3). Altogether, these results indicated that following a laser-induced injury TF was the

main trigger leading to the generation of thrombin and the formation of a thrombus.

The presence of PMNs is required for the activation of coagulation by the tissue factor

pathway:

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To determine if PMNs are required for activation of coagulation, we compared thrombus

formation and fibrin generation before and after blocking of ICAM-1 to prevent neutrophil

binding to activated endothelial cells. Thrombus formation and fibrin generation were both

significantly reduced when the binding of neutrophils to the endothelium was inhibited (Fig.

5A and 5B). These results were confirmed in wild-type mice following a pretreatment with

the GR-1 antibody. In such mice, 99% of circulating PMNs were depleted in comparison with

wild-type mice (Fig. S4A). Following a laser-induced injury thrombus formation and fibrin

generation was significantly reduced (Fig. S4B and S4C). Altogether, these results indicate

that the interaction of PMNs with the injured endothelial wall is required for the activation of

coagulation by the TF pathway.

PMNs are the main source of tissue factor present at the site of injured vessel wall:

Previous studies have shown that in vitro neutrophils contain TF and may express it at the cell

surface3,16. In the present study, using two different anti-TF antibodies, a band corresponding

to TF was observed by western-blot in PMNs purified from the bone marrow and from blood

(Fig. 6A left panel). The presence of TF in purified PMNs was confirmed in vitro by

immunofluorescence only following permeabilization the cells (Fig. 6A, right panel),

indicating that naïve PMNs do not express TF at the cell surface. In vivo, TF was detected at

the site of injury in the blood circulation immediately after a laser-induced injury using two

different antibodies directed against mouse TF (Fig. 6B and Fig S5). When PMNs binding

was prevented by the use of ICAM-1 blocking antibody, the signal corresponding to TF was

significantly reduced (Fig. 6B and Fig. S5), indicating that in vivo, following the interaction

with endothelial cells, PMNs expressed TF at the cell surface. To determine if the interaction

of PMNs with the endothelial cells is required and sufficient for thrombus formation and

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fibrin generation, purified PMNs from wild-type mice were infused into low TF mice.

Following infusion of PMNs, fibrin generation and platelet accumulation were significantly

increased after a laser-induced injury (Fig. 6C and 6D). These results indicate that PMNs

expressing TF is the main source of TF needed for platelet thrombus formation and fibrin

generation following a laser-induced injury in vivo.

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Discussion:

Our results demonstrated that the interaction of neutrophils with the injured

endothelial cells, through LFA-1/ICAM-1 interactions, is the first step leading to the

generation of fibrin and thrombus formation in the laser-induced injury model of cremaster

arterioles. Altogether, these data identify neutrophils as a key partner involved in thrombosis

by providing TF prior to platelet accumulation. In addition to this new key role in hemostasis,

blood neutrophils are crucial players in the innate immunity, participating in the antimicrobial

machinery. This crosstalk between innate immunity and blood coagulation may represent an

efficient strategy to prevent both infection and bleeding.

The traffic signals for neutrophil and monocyte localization during inflammation

function in a three steps process. This molecular model was described according to the

observations that at sites of inflammation leukocytes first roll on the vessel wall and then

become firmly adhered at a single location on the vessel wall before the last step diapedesis32.

Tethering brings leukocytes into proximity with chemoattractants that are present at sites of

injury/inflammation. Chemoattractants activate integrins. Activated integrins can then interact

with their ligands and induce the firm adhesion of leukocytes at the site of injury. The

immediate adhesion of neutrophils to the vessel wall was mainly dependent on interactions

between LFA-1 expressed by neutrophils and ICAM-1 expressed by activated endothelial

cells. This interaction between an integrin and its ligand may occur spontaneously as is the

case for the firm adhesion of platelets to immobilized fibrinogen. Indeed, different studies

have shown that chemoattractants stimulate strong, integrin-mediated adhesion to bilayers

containing ICAM-1 under static conditions but not in shear flow conditions33. Also, it is still

unknown whether chemoattractants can act in the bloodstream, where they would be rapidly

diluted and swept downstream by the blood flow34. The selective and varied responses of

PMNs and monocytes to injury can be explained by their receptivity to the distinct

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combinations of molecular signals such that LFA-1/ICAM-1 and P-selectin/PSGL-1. Our

observations indicate that neutrophils, under arteriole flow conditions, stably attach to the

vessel wall in the seconds following the injury. Different publications have shown that

neutrophils have the ability to roll at tenfold higher shear stress in vivo than in vitro 35,36. This

may occurred via the formation of slings, recently described as the essential mechanism

involved in high shear rolling of neutrophils37. Lastly, it is possible that only a subpopulation

of neutrophils that have, for instance, performed a reverse transmigration are able to adhere

rapidly at the site of injury.

The use of real-time digital intravital microscopy has helped the comprehension of

cellular mechanisms involved in thrombus formation. However, how the blood coagulation

cascade is activated following an injury has remained unclear. Falati et al. proposed that

monocyte-derived microparticles that expose TF accumulate at the site of injury via

interactions between P-selectin expressed on activated platelets and PSGL-1 expressed on

monocyte-derived microparticles13. However, these experiments were performed by infusion

of exogenously isolated monocyte-derived microparticles and failed to show that such

endogenous microparticles exist and indeed interact with a platelet thrombus in vivo. When

we studied thrombus formation in CX3CR1GFP mice, we did not observe any signal

corresponding to an accumulation of endogenous monocyte-derived microparticles at the site

of injury. We concluded that the accumulation of monocyte-derived microparticles was not

essential for thrombus formation and fibrin generation. This discrepancy may be explained by

the fact that Falati et al. used exogenous purified, labeled monocyte-derived microparticles13.

We concluded that neutrophils are the main source of blood borne tissue factor in the model

of laser-induced injury. Infusion of neutrophils only partially restores thrombus formation in

low TF mice. This may indicate that other sources of TF are involved following a laser-

induced injury, including the vascular wall15. Thus, following a laser-induced injury,

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neutrophils may represent the initial source of blood-borne TF needed to generate thrombin.

Vascular TF could be involved for optimal thrombus growth. Additional studies are needed to

discriminate the contribution of blood-borne versus vascular TF in this model of thrombosis.

A candidate identified as a possible activator of TF in vivo was the enzyme protein

disulfide isomerase (PDI). Indeed, inhibition of PDI reduced fibrin generation and thrombus

formation38,39. However, PDI plays roles in platelet activation that may explain this result. For

instance, PDI is needed to maintain the platelet integrin αIIbβ3 in the right configuration,

leading to inside-out and outside-in signaling40. PDI may also participate with neutrophils in

TF activation. Indeed, PMNs may express inactive TF that could be activated by PDI secreted

by activated endothelial cells and platelets39. We observed that active TF is expressed at the

surface of PMNs at the site of injury. According to our observations, neutrophils represent the

main source of blood-borne TF in the laser model of thrombosis. However, the cellular source

of blood-borne TF remains controversial41. It is possible, as previously suggested, that TF was

transferred from monocytes or monocyte-derived microparticles to PMNs6.

Massberg et al. have recently shown that blood activated neutrophils can inactivate

TFPI via the released of proteases at the site of injury2. In this model, the subendothelial

matrix is exposed to blood leading to the formation of a platelet thrombus. Here, we

demonstrate, using the laser model of injury, that neutrophil interaction with endothelial cells

is required for thrombus formation. This interaction may not occur when the subendothelial

matrix is exposed to the blood circulation. Neutrophils may thus not be able to support the

first steps of thrombus formation in this model. However, their presence was described to be

important for formation of an occlusive thrombus 3. In the present study, we focused on the

initial steps leading to thrombus formation following a laser-induced injury. We observed a

rapid and a constant recruitment of neutrophils. Thus, interactions of neutrophils with

platelets may occur independently of the model of injury. This interaction may be involved

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for the optimal growth of a thrombus in cardiovascular diseases, such as myocardial infarction,

as well as in infectious disease. Alternatively, in the ferric chloride model, neutrophils may

also bind to the endothelial cells surrounding the site of injury. Hypothetically, this may lead

to the formation of multiple microthrombi, similar to the one observed after a laser-induced

injury, and will ultimately induce the occlusion of the artery observed in this model of injury.

Apart from the TF pathway, activation of coagulation may also occur through the

FXII–dependent contact pathway16. Using FXII-/- mice, we observed that the extrinsic

pathway was the exclusive pathway involved in the first steps leading to the initiation phase

of coagulation after a dye laser-injury. Previous publications have reported that neutrophils

may serve as a matrix for the activation of the contact phase7. It is, therefore, possible that in

models of thrombus formation that are dependent on FXII activation, neutrophils may also

play a critical role. In our model, which is strictly dependent on TF activation, PMNs played a

major role as the unique source of TF. Additionally PMNs may also inactivate TFPI. It is also

possible that neutrophils, by producing NETs42, could directly activate TF. Further studies are

needed to identify the exact molecular mechanisms involved in TF activation and TFPI

inactivation by PMNs.

Although neutrophils seem to be involved in both arterial and venous injuries, their

role may not be the same in different vessel beds. Indeed, in a mouse model of deep vein

thrombosis3, Von Brühl et al. demonstrate by intravital two-photon microscopy that blood

monocytes and neutrophils crawling along and adhering to the venous endothelium provide

the initiating stimulus for DVT development. The authors show that thrombus-resident

neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII)

and by supporting its activation through the release of NETs. In this model of venous

thrombosis, platelets promote leukocyte recruitment and stimulating neutrophil-dependent

coagulation. In our model of arteriole thrombosis, neutrophils participate to the activation of

16
 From bloodjournal.hematologylibrary.org at Sanquin on August 23, 2012. For personal use only.

the extrinsic pathway of coagulation via tissue factor, independent of the factor XII and of the

presence of platelets and monocytes at the site of injury. Inhibiting PMN from interacting

with the endothelium at the site of injury prevents both accumulation of platelets and

activation of the blood coagulation cascade. Our results identified that the interaction of

neutrophils with the injured endothelial cells, through LFA-1/ICAM-1 interactions, is the first

step leading to the generation of fibrin and thrombus formation.

Several vascular risk factors are associated with proinflammatory alterations, including

leukocyte activation, and predispose cerebral vasculature to thrombogenesis on inflammatory

stimulation4. For example, accumulation of inflammatory cells within the vascular wall starts

early during atherogenesis. During later disease stages, their activation can lead to plaque

rupture and thrombus formation, increasing the risk of stroke and acute coronary syndromes.

Moreover, the association of an acute inflammatory reaction with unstable angina has been

previously established and a variety of experimental studies suggested that neutrophil-

induced endothelial dysfunction occurred near the site of coronary obstruction5,43. Our model

of injury is strictly dependent on endothelial activation26. We now show that participation of

neutrophils is essential for thrombus formation. This may indicate that the laser model mimics

what may occur following thrombus formation induced by – or in relation with- inflammatory

events, such as after lung inflammation44, Shiga toxin thrombotic mechanisms involved in

hemolytic uremic syndrome45 or thrombus formation in cerebral microvessels involved in

sickle cell disease (SCD)46. Our results indicate that preventing the binding of PMNs to the

vessel wall could potentially represent a therapeutic strategy to prevent arterial thrombosis

and inflammation. Targeting neutrophils adhesion interacting with endothelial cells may thus

efficiently reduce the risks of a stroke and prevent acute coronary syndromes or acute cerebral

ischemia.

17
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Acknowledgments

The project was supported by the FRM association (Fondation pour la Recherche Médicale,

grant to C. Dubois). This work was supported by grants from the institutional funding from

INSERM, the Aix-Marseille University, the ANR (Agence Nationale pour la Recherche, grant

number ANR-09-JCJC-0053 to C. Dubois) and the ARC association (Agence pour la

Recherche sur le Cancer, grant to C. Dubois).

The authors are indebted to Stephane Robert (INSERM UMR-S1076) for technical assistance.

The authors thank Dr Markus Riederer (Actelion, Switzerland) for its critical scientific review

of the work and Ben Atkinson (Intelligent Imaging Innovation) for its technical support of the

intravital system.

Authorship contributions

RD, SM, RD and CF contributed in the design and performance of the research, in the

analysis of data and contributed to the writing of the paper; GMT, NM, TR, FDG, CD and

LPD contributed to the experimental design, analysis of data and writing of the paper.

Conflicts of interest

The authors declare there are no competing financial interests.

18
 From bloodjournal.hematologylibrary.org at Sanquin on August 23, 2012. For personal use only.

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 From bloodjournal.hematologylibrary.org at Sanquin on August 23, 2012. For personal use only.

Legends to figures

Figure 1. Real-time imaging of leukocytes accumulation at the site of injury in wild-type

mice. (A) Representative images of cells (white arrows) accumulating at the site of injury

(n=32; 3 mice). (B) Exogenous labeled leukocytes (blue) were detected at the site of injury

(upper Panel). (C) Accumulation of Mac-1 (blue), but not the irrelevant antibody, was

detected at the site of thrombus formation (white arrows) (middle panel). Images are

representative of 32 thrombi performed in 3 mice for each condition. (D) Detection of

Leukocytes before (upper panel) or after (lower panel) depletion of platelets. The Graphs

depict the median of the maximal integrated fluorescence intensity of CD41(left panel) and

Mac-1 (right panel) corresponding to platelets and leukocytes accumulation, respectively (31

thrombi; 3 mice) * P<0.05.

Figure 2. Leukocytes accumulation is dependent of LFA-1/ ICAM-1 interactions.

Leukocytes (blue) accumulation at site of injury (white arrow) in absence (upper panel) or

presence (lower panel) of ICAM-1 (B) or LFA-1 (C) blocking antibody. Graph represents

medians of Mac-1 integrated fluorescence intensity before (upper curve, 33 thrombi; 3 mice)

and after (lower curve, 31 thrombi; 3 mice) infusion of ICAM-1 (B) or LFA-1 (C) blocking

antibody.

Figure 3. Kinetics of monocytes accumulation at the site of injury. (A) kinetics of platelet

accumulation in wild-type mice (32 thrombi, 3 mice) and in CX3CR1GFP mice (31 thrombi, 3

mice). (B) Representative images of a thrombus in CX3CR1GFP mice. GFP-monocytes (green)

and platelets (red) accumulation were observed at the site of injury.

24
 From bloodjournal.hematologylibrary.org at Sanquin on August 23, 2012. For personal use only.

Figure 4. In vivo imaging of platelets, neutrophils and monocytes during thrombus

formation. (A, B) Representative images of Ly-6G (A) or platelets and Ly-6G neutrophils

(B) at site of injury in wild-type mice (26 thrombi, 3 mice). (C) Thrombus formation in mice

expressing CX3CR1GFP after infusion of CD41 and Ly-6G antibody. Monocytes (green),

platelets (red) and neutrophils (blue) are visualized over time after laser injury. (D)

Representative images showing an inflammatory monocyte rolling on a thrombus. The

monocyte is depicted in white (composite of blue + green + red) or in light blue (composite of

dark blue and green). Platelets are in red, neutrophils are in dark blue, monocytes,

macrophages and DC are depicted in green. (E) Platelets and fibrin generation in wild-type

mice and FXII-/- mice. Graphs represent medians of fibrin integrated fluorescence intensity in

wild-type mice (29 thrombi, 3 mice) and FXII-/- mice (30 thrombi, 3 mice) over time.

Figure 5. Neutrophils activate the coagulation TF pathway leading to thrombus and

fibrin formation. (A) Platelet accumulation at the site of injury in wild-type mice in absence

or presence of ICAM-1 blocking antibody (32 thrombi, 3 mice for each condition). Graph

represents medians of platelets integrated fluorescence intensity in presence or absence of

ICAM-1 blocking antibody over time. (B) Fibrin generation in wild-type mice in presence or

absence of ICAM-1 blocking antibody. Graph represents medians of fibrin integrated

fluorescence intensity in WT mice in presence (28 thrombi in 3 mice) or absence (40 thrombi

in 3 mice) of ICAM-1 blocking antibody over time.

Figure 6. Neutrophils contain tissue factor in vitro and expressed it in vivo at the site of

laser-induced injury. (A) In vitro detection of TF in neutrophils; Western blot analysis of

protein extracts from purified Panc02 mouse pancreatic cancer cells, bone marrow

neutrophils, circulating neutrophils and washed platelets with two anti-mouse TF antibodies

25
 From bloodjournal.hematologylibrary.org at Sanquin on August 23, 2012. For personal use only.

(left panel). Immunofluorescence microscopy of neutrophils (PMNs) isolated from mice with

an antibody directed against mouse tissue factor with an Alexa 488-conjugated secondary

antibody. Negative control was performed with an irrelevant antibody and the secondary

antibody. Original magnification, ×200; n = 3 (right panel). (B) Representatives pictures

showing the visualization of TF in the bloodstream of wild-type mice at the site of laser-

induced injury in presence or absence of ICAM-1 blocking antibody using the mouse anti-

tissue factor antibody 1H1. TF is shown in yellow. Graph represents the sum of the medians

of TF integrated fluorescence intensity in wild-type mice in presence (32 thrombi in 3 mice)

or absence (31 thrombi in 3 mice) of ICAM-1 blocking antibody. (C) Fibrin generation and

thrombus formation at the site of laser-dye injury in low TF mice before and after infusion of

isolated neutrophils (PMNs). Fibrin is depicted in green, platelets are in red. (D) Graph

depicts medians of fibrin and platelets integrated fluorescence intensity in low TF mice (32

thrombi, 3 mice) and low TF mice following infusion of PMNs (31 thrombi, 3 mice).

26
Figure 1 From1bloodjournal.hematologylibrary.org
s 5 sat Sanquin on August 23, 2012.
80 sFor personal use only. 180 s
A.

1s 60 s 120 s
B.

C. 1s 60 s 120 s 180 s 240 s

Mac-1
antibody

Irrelevant
antibody

D. 1s 60 s 120 s 180 s 240 s

Before platelet
depletion

After platelet
depletion

CD41 (Platelets) Mac-1 (Leukocytes)

450 600
integrated fluorescence

integrated fluorescence

400
Median of maximum

Median of maximum

500
(arbitrary units)

(arbitrary units)

350
300 400
intensity

intensity

250
200 300
150 200
100
100
50
0 0
Before platelet After platelet Before platelet After platelet
depletion depletion depletion depletion
Figure 2 From bloodjournal.hematologylibrary.org
A. 1s 60 s at Sanquin on 120
August
s 23, 2012. For personal
180 s use only. 240 s

Before blocking
ICAM-1

After blocking
ICAM-1

350
Integrated fluorescence

300
(arbitrary units)

250
intensity

Before blocking ICAM-1

200
150
100
50 After blocking ICAM-1
0
0 50 100 150 200 250 300 350
Elapsed time (seconds)

B. 1s 60 s 120 s 180 s 240 s

Before
blocking
LFA-1

After
blocking
LFA-1

350
Integrated fluorescence intensity

300
Before blocking LFA-1
250
(arbitrary units)

200

150

100
After blocking LFA-1
50

0
0 50 100 150 200 250 300 350
Elapsed time (seconds)
Figure 3 From bloodjournal.hematologylibrary.org at Sanquin on August 23, 2012. For personal use only.

A.
300
Integrated fluorescence intensity

250
(arbitrary units)

200

150

100 Wild-type mice

50
CX3CR1GFP mice
0
0 50 100 150 200 250 300 350
Elapsed time (seconds)

B.
1s 60 s 120 s 180 s 240 s 30 min
Figure 4 From bloodjournal.hematologylibrary.org at Sanquin on August 23, 2012. For personal use only.
A. 1s 20 s 60 s 120 s 180 s 240 s

1A8
antibody

B. 1s 20 s 60 s 120 s 180 s 240 s

1A8 +
CD41
antibodies

C.
1s 20 s 60 s 120 s 180 s 240 s

D. 180 s 181 s 185 s 190 s 195 s

E. 1s 60 s 120 s 180 s 240 s

Wild-type
mice

F XII -/-
mice

500
Integrated fluorescence

400
FXII -/- mice
(arbitrary units)

300
intensity

Wild-type mice
200
100
0
0 50 100 150 200 250 300 350
Elapsed time (seconds)
Figure 5 From bloodjournal.hematologylibrary.org at Sanquin on August 23, 2012. For personal use only.

A. 1s 60 s 120 s 180 s 240 s

Before
blocking
ICAM-1

After
blocking
ICAM-1

350
Integrated fluorescence

300
(arbitrary units)

250
intensity

200
150
Before blocking ICAM-1
100
50
After blocking ICAM-1
0
0 100 200 300 400 500
Elapsed time (seconds)
B.
1s 60 s 120 s 180 s 240 s

Before
blocking
ICAM-1

After
blocking
ICAM-1

200
Integrated fluorescence

Before blocking ICAM-1

(arbitrary units)

150
intensity

100

50
After blocking ICAM-1
0
0 50 100 150 200 250 300
Elapsed time (seconds)
 Tissue Factor antibody Irrelevant antibody
Figure 6 From bloodjournal.hematologylibrary.org at Sanquin on August 23, 2012. For personal use only.

A.
(1H1)

(ab104513)

500

Median of maximum integrated

Tissue Factor (1H1)
B. 1s 60 s 120 s 240 s

fluorescence intensity
400

TF 300
*
200
TF + 100
ICAM-1
0
Wild-type mice Wild-type mice
treated with
C. ICAM-1
1s 60 s 120 s 180 s 240 s

Fibrin in low TF mice

Fibrin in low TF mice +

PMNs

Platelets in low TF mice

Platelets in low TF mice +

PMNs

D.
integrated fluorescence

Fibrin Platelets
Median of maximum
integrated fluorescence

200 250
Median of maximum

160 200
intensity
intensity

120 150
80 100
*
40
* 50
0
0
Low TF mice Low TF mice with
Low TF mice Low TF mice with
exogenous
exogenous
neutrophils
neutrophils