Tissue factor-positive neutrophils bind to injured endothelial wall and initiate thrombus formation

doi:10.1182/blood-2012-06-437772Prepublished online July 26, 2012;2012 120: 2133-2143

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

positive neutrophils bind to injured endothelial wall and−Tissue factor

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THROMBOSIS AND HEMOSTASIS

Tissue factor–positive neutrophils bind to injured endothelial wall and initiatethrombus formationRoxane Darbousset,1,2 Grace M. Thomas,3 Soraya Mezouar,1,2 Corinne Frere,1,2 Renate Bonier,1,4 Nigel Mackman,5

Thomas Renne,6 Francoise Dignat-George,1,2 Christophe Dubois,1,2 and Laurence Panicot-Dubois1,2

1Campus Sante, Aix Marseille Universite, Marseille, France; 2Faculty of Pharmacy, Inserm UMR-S1076, Marseille, France; 3Immune Disease Institute andProgram in Cellular and Molecular Medicine, Children’s Hospital Boston, and Department of Pediatrics, Harvard Medical School, Boston, MA; 4Inserm UMR-911,Marseille, France; 5Department of Medicine, Division of Hematology/Oncology, University of North Carolina, Chapel Hill, NC; and 6Department of MolecularMedicine and Surgery and Center of Molecular Medicine, Karolinska Institute and University Hospital, Stockholm, Sweden

For a long time, blood coagulation andinnate immunity have been viewed asinterrelated responses. Recently, the pres-ence of leukocytes at the sites of vesselinjury has been described. Here we ana-lyzed interaction of neutrophils, mono-cytes, and platelets in thrombus forma-tion after a laser-induced injury in vivo.Neutrophils immediately adhered to in-jured vessels, preceding platelets, bybinding to the activated endothelium vialeukocyte function antigen-1–ICAM-1 in-

teractions. Monocytes rolled on a throm-bus 3 to 5 minutes postinjury. The kinet-ics of thrombus formation and fibringeneration were drastically reduced inlow tissue factor (TF) mice whereas theabsence of factor XII had no effect. In vitro,TF was detected in neutrophils. In vivo,the inhibition of neutrophil binding to thevessel wall reduced the presence of TFand diminished the generation of fibrinand platelet accumulation. Injection ofwild-type neutrophils into low TF mice

partially restored the activation of theblood coagulation cascade and accumu-lation of platelets. Our results show thatthe interaction of neutrophils with endo-thelial cells is a critical step precedingplatelet accumulation for initiating arte-rial thrombosis in injured vessels. Target-ing neutrophils interacting with endothe-lial cells may constitute an efficientstrategy to reduce thrombosis. (Blood.2012;120(10):2133-2143)

Introduction

After vessel injury, a rapid accumulation of platelets and activationof the blood coagulation cascade are critical steps to prevent bloodloss. In addition, blood polymorphonuclear neutrophils (PMNs)play a major role in limiting intrusion of microorganisms into thebloodstream. For a long time, different observations have linkedblood coagulation and innate immunity. In 1882, Bizzozero firstobserved that the formation of a thrombus involved both plateletsand leukocytes.1 More recently, the presence of leukocytes accumu-lating at the sites of vessel injury has been described.2,3 Clinicalstudies have shown that traditional vascular risk factors areassociated with leukocyte activation and may predispose to throm-bogenesis.4,5 PMNs may contribute to fibrin generation directly byexposing active tissue factor (TF)6 and/or indirectly by inactivatingthe major TF inhibitor, TF pathway inhibitor (TFPI).2 PMNs mayalso locally assemble proteins of the factor XII–driven contactpathway.7 Recent advances in digital microscopy make it possibleto observe, in real time, the formation of a thrombus after a vesselinjury in living mice.8 In this model of laser-induced injury, thesubendothelial matrix is not exposed to the blood circulation.9

Thrombin is locally generated, and platelet-rich thrombi are formedafter activation of the endothelial cells.10 von Bruhl et al haverecently showed the importance of monocyte, PMN, and plateletcross-talk in a mouse model of deep vein thrombosis (DVT).3 Inthis model, neutrophils are indispensable for propagation of theclotting cascade by binding factor XII (FXII) and by supporting itsactivation through the release of neutrophil extracellular traps

(NETs).11 After exposure of the subendothelial matrix, neutrophilsmay, by interacting with platelets, inactivate TFPI thus leading tothe activation of the TF-dependent coagulation cascade.2 Vascularsmooth muscle–derived TF is also critical for arterial thrombosis inmice.12 After a laser-induced injury, a previous study suggested thatmonocyte-derived microparticles contribute to thrombus formationin arteries by delivering TF to the site of injury and interacting withaccumulating platelets.13,14 However, the relative contribution ofneutrophils and monocytes in microvascular thrombosis remainsunclear. Using the laser-injury system with a high-definition,high-speed camera, we analyzed interaction of neutrophils, mono-cytes, and platelets in thrombus formation in the microvasculatureof mice. Our results demonstrate that neutrophils, by binding to thevessel wall, are the main source of blood-borne TF in the earlyphase of thrombus formation.

Methods

Mice

Wild-type C57BL/6J mice were from Janvier Elevage. CX3CR1GFP

C57BL/6J mice were obtained from The Jackson Laboratory. FXII�/� miceand low TF mice have been previously described.15-17 All animal care andexperimental procedures were performed as recommended by the EuropeanCommunity guidelines and approved by the French Ministry of Agriculture(agreement no. 13.382; L.P.-D.).

Submitted June 18, 2012; accepted July 18, 2012. Prepublished online asBlood First Edition paper, July 26, 2012; DOI 10.1182/blood-2012-06-437772.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2012 by The American Society of Hematology

2133BLOOD, 6 SEPTEMBER 2012 � VOLUME 120, NUMBER 10




Antibodies and reagents

The rat anti–mouse CD41 antibody (clone MWReg30; 0.2 �g/g mouseweight) and R300 antibody used to deplete circulating platelets18 wereobtained from Emfret. Alexa Fluor 488 rat anti–mouse, lysosomal-associated membrane protein 1 (LAMP-1; CD107a) antibody (clone 1D4B;0.25 �g/g mouse weight) and Alexa Fluor 647 anti–mouse CD54 (ICAM-1)antibody (clone YN1/1.7.4; 0.5 �g/g mouse weight) were from Biolegend(Ozyme Biolegend). PE rat anti–mouse Ly-6G antibody (clone 1A8;0.1 �g/g mouse weight), purified rat anti–mouse Ly-6G and Ly-6Cantibody (clone RB6-8C5), Alexa Fluor 647 rat anti–mouse CD11b (Mac-1)antibody (clone M1/70; 1 �g/g mouse weight), blocking rat monoclonalanti–mouse P-selectin antibody (clone RB40.34), purified hamster anti–mouse ICAM-1 antibody (clone 3E2), rat anti–mouse CD11a (leukocytefunction antigen-1 [LFA-1]) antibody (clone M17/4), allophycocyanin ratanti–mouse CD45 antibody (clone 30-F11), rat anti–mouse TER-119antibody (clone TER-119; 0.59 �g/g mouse weight), and rat IgG2a isotypecontrol were from BD Biosciences. Blocking anti–mouse ICAM-1 andLFA-1 antibodies were, respectively, used at 4 �g/g and 2 �g/g of mouseweight. Isolectin B4 used to label the endothelial wall,19 DIO [iodide of(dodecyl-4 aminostyrile)-4 N-methylpyridium] was obtained from LifeTechnologies. The mouse mAb directed against human, rat, and mouse TF(ab104513) was from Abcam. 1H1 antibody directed against mouse TF wasobtained from Dr Daniel Kirchhofer (University of North Carolina, ChapelHill, NC).20 A mouse anti–human fibrin II �-chain antibody (cloneNYBT2G1) was from Accurate Chemical.

Fab fragments from the anti-CD41 antibody were generated using theImmuno-Pure Fab Preparation Kit (Pierce Thermofisher). Fab fragments,antibodies, and control isotypes were conjugated to dye light Fluor 488 or647 according to the manufacturer’s instruction (Pierce Thermofisher).Prostaglandin I2 was obtained from Merck. Apyrase grade VI, saponin, andBSA were purchased from Sigma-Aldrich. Corn trypsin inhibitor wasobtained from Cryopep.

Flow cytometry

Blood was collected from mice in a citrate solution (ACD: 85mM trisodiumcitrate, 67mM citric acid, 111.5mM glucose, pH 4.5) in the presence of0.5mM prostacyclin and 0.02 U/mL apyrase as previously described.21

Analyses of blood and platelet-rich plasma samples were performed on aflow cytometer (Gallios; Beckman Coulter) using appropriate antibodies tolabel white blood cells.22

Isolation of leukocytes

Leukocytes were extracted from spleen of wild-type mice as previouslydescribed by StemCell Technologies. Isolated leukocytes were countedin a Malassez cell and labeled with DIO according to the manufacturer’sinstruction (Vybrant Kit; Life Technologies). Purified labeled cells wereresuspended in saline buffer at a concentration of 1 � 109 cells/mL andwere infused in the blood of a recipient mouse at a concentration of2 � 106 cells/mouse.

Isolation of neutrophils

Neutrophils from bone marrow or peripheral blood were isolated aspreviously described23 using GR-1–coupled magnetic beads (anti–Ly-6GMicrobead kit; Milteny Biotec).

Depletion of neutrophils

Depletion of neutrophils from wild-type mice was performed by intrave-nous injection of 5 �g/g mouse of rat anti–Ly-6G/C (RB6-8C5; eBiosci-ence) as previously described.24 Control mice were injected with PBS.Neutropenia was evaluated 24 hours after injection by flow cytometry usinga PE-conjugated anti–mouse neutrophil mAb.

Fluorescence microscopy

Purified neutrophils were washed with PBS and fixed for 30 minutes at4°C in PBS containing 2% paraformaldehyde. Once fixed, cell suspensions

were cytocentrifugated for 10 minutes at 125g in a shandom cytospin 4(Thermo Fisher Scientific). Cells were permeabilized using PBS containing0.3% saponin for 20 minutes. Then, cells were blocked for 20 minutes at4°C with BSA 1%, saponin 0.3% in PBS buffer. Cells were incubated at4°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% saponinand were observed on a microscope (Nikon Eclipse TE2000-U).

Western blotting

Western blotting was performed after SDS polyacrylamide (11%) gelelectrophoresis. Proteins were transferred onto a nitrocellulose membraneusing an iBlot system (Life Technologies) according to the manufacturer’sinstruction. Transfer was verified by staining membranes with Ponceau red.Detection of the labeled proteins was performed by chemiluminescenceusing ECL from GE Healthcare.

Intravital microscopy

Intravital videomicroscopy of the cremaster muscle microcirculation wasperformed as previously described8 using an Intelligent Imaging Innova-tions system. Briefly, mice were preanesthetized with intraperitonealketamine (125 mg/kg; Panpharma), xylazine (12.5 mg/kg; Bayer), andatropine (0.25 mg/kg; Lavoisier). A tracheal tube was inserted and themouse maintained at 37°C on a thermo-controlled rodent blanket. Tomaintain anesthesia, pentobarbital (CEVA) was administered through acannula placed in the jugular vein. After the scrotum was incised, thetesticle and surrounding cremaster muscle were exteriorized onto anintravital microscopy tray. The cremaster preparation was superfused with athermo-controlled (37°C) bicarbonate-buffered saline throughout the experi-ment. Microvessel data were obtained using an Olympus AX microscopewith a 60� or 100� 0.9 NA water-immersion objective. The fluorescencemicroscopy system has previously been described.25 Digital images werecaptured with a Cooke Sensicam CCD camera in 640 � 480–pixel formator with a CoolSnapEZ HD camera in 1392 � 1040–pixel format.

Laser-induced injury

Antibodies or exogenously labeled mouse leukocytes were infused throughthe jugular vein into the circulation of an anesthetized mouse. Vessel wallinjury was induced with a nitrogen dye laser (Micropoint; PhotonicsInstruments) focused through the microscope objective, parafocal with thefocal plane, and aimed at the vessel wall.9 Typically, 1 or 2 pulses wererequired to induce vessel wall injury. For all the experiments performedinvolving injection through the jugular vein of antibodies or cells, 3 to5 mice were studied for each condition and a minimum of 10 thrombiinduced per mouse. In all experiments, new thrombi were formed upstreamof earlier thrombi to avoid any contribution from thrombi generated earlier.There were no characteristic trends in thrombus size or thrombus composi-tion in sequential thrombi generated in a single mouse during an experi-ment. Image analysis was performed using Slidebook (Intelligent ImagingInnovations). Fluorescence data were captured digitally at up to 50 framesper second and analyzed as previously described to determine the median offluorescent intensity signal overtime.8

Statistics

Significance was determined by the Wilcoxon rank-sum test for the in vivoexperiments.8 Differences were considered significant at P � .05.

Results

Leukocytes accumulate at the site of arterial injuryindependent of platelets

In the second after vessel injury, cells of 10 to 20 �m in diameterwere seen to accumulate at the injury site. These cells were also

2134 DARBOUSSET et al BLOOD, 6 SEPTEMBER 2012 � VOLUME 120, NUMBER 10




detectable 3 to 5 seconds after the injury when platelets started toaccumulate, as well as 80 to 120 seconds postinjury when thethrombus was at its maximal size and 3 minutes later when it wasstabilized (Figure 1A). Fluorescently labeled leukocytes infusedinto mice were detected at the site of injury with similar kinetics(Figure 1B). In addition, a fluorescent signal was observed in thesecond after the injury when a Mac-1 (macrophage 1 antigen,CD11b) but not an irrelevant antibody (Figure 1C) was used. Inaddition, when the red blood cell–specific (RBC) TER-119 anti-

body was infused, RBCs were detected in the circulating blood butnot at the site of thrombus (supplemental Figure 1A, available onthe Blood Web site; see the Supplemental Materials link at the topof the online article). The kinetics of Mac-1–positive cells indicatesthat leukocytes rapidly accumulate at the site of laser-inducedinjury in vivo (supplemental Figure 1B). After the depletion of thecirculating platelets, no signal corresponding to platelet accumula-tion was detected at the site of injury. However, the kinetics ofleukocyte accumulation were identical in the presence or the absence of

Figure 1. Real-time imaging of leukocyte accumulation at the site of injury in wild-type mice. (A) Representative images of cells (white arrows) accumulating at the site ofinjury (n � 32; 3 mice). (B) Exogenously-labeled leukocytes (blue) were detected at the site of injury (top panel). (C) Accumulation of Mac-1 (blue), but not the irrelevantantibody, 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 (top panel) or after (bottom panel) depletion of platelets. The graphs depict the median of the maximal integrated fluorescence intensity ofCD41 (left panel) and Mac-1 (right panel) corresponding to platelets and leukocytes accumulation, respectively (31 thrombi; 3 mice); *P � .05.

NEUTROPHILS INITIATE THROMBUS FORMATION IN VIVO 2135BLOOD, 6 SEPTEMBER 2012 � VOLUME 120, NUMBER 10




platelets (Figure 1D). We concluded that leukocytes accumulatedbefore platelets at the site of laser-induced injury by interactingwith activated endothelial cells.

LFA-1–expressing leukocytes interact with ICAM-1 expressedon injured vascular wall

Recently, Atkinson et al determined that endothelial cells wereactivated after the use of a dye laser.26 We confirmed these resultsand found that both LAMP-1 (CD107a) and ICAM-1 (CD54) weredetected on endothelial cells immediately after the injury (supple-mental Figure 1C). This laser-induced activation of the endothe-

lium was a prerequisite for the interaction of leukocytes because thefluorescently labeled circulating leukocytes did not interact with restingendothelium in vivo. Blocking ICAM-1 abolished the accumula-tion of leukocytes at the site of laser-induced injury (Figure 2A).Similar results were observed after blocking the ICAM-1 ligand,LFA-1 (CD11a), expressed by leukocytes (Figure 2B).

Neutrophils but not monocytes accumulate at the site of injury

To determine whether monocytes were rapidly binding to theinjured endothelium, thrombus formation was studied inCX3CR1GFP mice. CX3CR1GFP mice coexpress green fluorescent

Figure 2. Leukocyte accumulation is dependent on LFA-1/ ICAM-1 interactions. Leukocyte (blue) accumulation at site of injury (white arrow) in absence (top panel) orpresence (bottom panel) of (B) ICAM-1 or (C) LFA-1 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 (B) ICAM-1 or (C) LFA-1–blocking antibody.





protein (GFP) with CX3CR1 so that their monocytes, monocyte-derived microparticles, macrophages, and dendritic cells (DCs) areall fluorescently labeled.27 Exogenous monocytes and monocyte-derived microparticles were previously shown to play a role inthrombus formation.13 The kinetics of thrombus formation wassimilar in CX3CR1GFP and wild-type mice (Figure 3A). Althoughmacrophages and monocytes were detected in the cremaster tissueand circulating in veins, no GFP signal was detected at the site ofthrombus formation during the first 3 minutes after a laser-inducedinjury (Figure 3B, supplemental Video 1). Three to 5 minutes afterthe injury, GFP-expressing cells were detected rolling on thethrombus and bound to it 25 minutes later (Figure 3B). Altogether,these results indicate that monocytes are not required for theformation of the platelet thrombus in arteries.

Neutrophils are the main population of granulocytes andconstitute approximately 20% of total leukocytes circulating in theblood of mice. The 1A8 antibody is directed against Ly-6G, whichis mainly expressed in circulation by neutrophils, although approxi-mately 20% of circulating monocytes, identified as inflammatorymonocytes, also expressed this marker (supplemental Figure 2A).After infusion of the 1A8 antibody into wild-type mice, a fluores-cent signal was detected at the site of injury (Figure 4A). The signalappeared before any platelets could be detected and it increasedover time (Figure 4B). To confirm that neutrophils were thesubpopulation of leukocytes accumulating at the site of injury,experiments in CX3CR1GFP mice were performed. Neutrophilswere detected by infusion of 1A8 antibody; monocytes, monocyte-derived microparticles, DCs, and macrophages were detected bythe expression of GFP. Platelets were labeled by infusion of ananti-CD41 antibody; inflammatory monocytes expressed GFP andwere labeled by 1A8. We observed that neutrophils, but neithermonocytes nor inflammatory monocytes, accumulated at the site ofinjury in the seconds after the injury (Figure 4C, supplementalVideo 2). Three to 5 minutes after the injury, inflammatorymonocytes were detected rolling over the thrombus (Figure 4D).

We conclude that neutrophils were the exclusive circulating cellsthat bound to the activated endothelium in the second after thearterial injury before platelets. Previous studies have shown thatleukocytes participate in the activation of the blood coagulationcascade.2,28,29 After a laser-induced injury, thrombin is the mainplatelet agonist involved in thrombus formation.10,15,25 We con-firmed that thrombus formation and fibrin generation were largelyreduced in low TF mice (supplemental Figure 2B). PMNs assemblethe FXII-driven contact pathway that initiates the intrinsic pathwayof coagulation.30 We compared the kinetics of platelet accumula-tion and fibrin generation in wild-type and FXII�/� mice.16 Ourresults indicate that both thrombus formation and fibrin generationwere similar in wild-type mice and FXII�/� mice (Figure 4E).These data were confirmed in wild-type mice infused with corntrypsin inhibitor (CTI; an inhibitor of activated FXII). CTI affectedneither the kinetics of thrombus formation nor fibrin generationafter laser-induced injury in wild-type mice (supplemental Figure3). Altogether, these results indicated that after a laser-inducedinjury, TF was the main trigger leading to the generation ofthrombin and the formation of a thrombus.

The presence of PMNs is required for the activation ofcoagulation by the TF pathway

To determine whether PMNs are required for activation of coagula-tion, we compared thrombus formation and fibrin generation beforeand after blocking of ICAM-1 to prevent neutrophil binding toactivated endothelial cells. Thrombus formation and fibrin genera-tion were both significantly reduced when the binding of neutro-phils to the endothelium was inhibited (Figure 5). These resultswere confirmed in wild-type mice after a pretreatment with theGR-1 antibody. In such mice, 99% of circulating PMNs weredepleted in comparison with wild-type mice (supplemental Figure4A). After a laser-induced injury, thrombus formation and fibringeneration were significantly reduced (supplemental Figure 4B-C).

Figure 3. Kinetics of monocyte 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-monocyte (green) and platelet (red) accumulation was observed at the site of injury.





Altogether, these results indicate that the interaction of PMNs withthe injured endothelial wall is required for the activation ofcoagulation by the TF pathway.

PMNs are the main source of TF present at the site of injuredvessel wall

Previous studies have shown that in vitro neutrophils contain TFand may express it at the cell surface.3,16,31 In the present study,using 2 different anti-TF antibodies, a band corresponding to TF

was observed by Western blot in PMNs purified from the bonemarrow and from blood (Figure 6A left panel). The presence of TFin purified PMNs was confirmed in vitro by immunofluorescenceonly after permeabilization of the cells (Figure 6A right panel),indicating that naive PMNs do not express TF at the cell surface. Invivo, TF was detected at the site of injury in the blood circulationimmediately after a laser-induced injury using 2 different antibod-ies directed against mouse TF (Figure 6B, supplemental Figure 5).When PMN binding was prevented by the use of ICAM-1–

Figure 4. In vivo imaging of platelets, neutrophils, and monocytes during thrombus formation. (A-B) Representative images of (A) Ly-6G or (B) platelets and Ly-6Gneutrophils 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 athrombus. The monocyte is depicted in white (composite of blue plus green plus red) or in light blue (composite of dark blue and green). Platelets are in red, neutrophils are indark blue, monocytes, macrophages and DC are depicted in green. (E) Platelets and fibrin generation in wild-type mice and FXII�/� mice. Graphs represent medians offibrin-integrated fluorescence intensity in wild-type mice (29 thrombi, 3 mice) and FXII�/� mice (30 thrombi, 3 mice) over time.





blocking antibody, the signal corresponding to TF was significantlyreduced (Figure 6B, supplemental Figure 5), indicating that in vivo,after the interaction with endothelial cells, PMNs expressed TF atthe cell surface. To determine whether the interaction of PMNswith the endothelial cells is required and sufficient for thrombusformation and fibrin generation, purified PMNs from wild-typemice were infused into low TF mice. After infusion of PMNs, fibringeneration and platelet accumulation were significantly increasedafter a laser-induced injury (Figure 6C-D). These results indicatethat PMNs expressing TF are the main source of TF needed forplatelet thrombus formation and fibrin generation after a laser-induced injury in vivo.

Discussion

Our results demonstrated that the interaction of neutrophils withthe injured endothelial cells, through LFA-1/ICAM-1 interactions,is the first step leading to the generation of fibrin and thrombusformation in the laser-induced injury model of cremaster arterioles.Altogether, these data identify neutrophils as a key partner involvedin thrombosis by providing TF before platelet accumulation. Inaddition to this new key role in hemostasis, blood neutrophils arecrucial players in innate immunity, participating in antimicrobialmachinery. This crosstalk between innate immunity and blood

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 inabsence or presence of ICAM-1–blocking antibody (32 thrombi, 3 mice for each condition). Graph represents medians of platelet-integrated fluorescence intensity in presenceor 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 offibrin-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.





coagulation may represent an efficient strategy to prevent bothinfection and bleeding.

The traffic signals for neutrophil and monocyte localizationduring inflammation function in a 3-step process. This molecularmodel was described according to the observations that at sites of

inflammation, leukocytes first roll on the vessel wall and then becomefirmly adhered at a single location on the vessel wall before the last-stepdiapedesis.32 Tethering brings leukocytes into proximity with chemoat-tractants that are present at sites of injury/inflammation. Chemoattrac-tants activate integrins. Activated integrins can then interact with

Figure 6. Neutrophils contain TF 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 ofprotein extracts from purified Panc02 mouse pancreatic cancer cells, bone marrow neutrophils, circulating neutrophils, and washed platelets with 2 anti–mouse TF antibodies(left panel). Immunofluorescence microscopy of neutrophils (PMNs) isolated from mice with an antibody directed against mouse TF with an Alexa 488–conjugated secondaryantibody. Negative control was performed with an irrelevant antibody and the secondary antibody. Original magnification, �200; n � 3 (right panel). (B) Representative picturesshowing 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 mouseanti-TF 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 in3 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 andafter infusion of isolated neutrophils (PMNs). Fibrin is depicted in green; platelets are in red. (D) Graph depicts medians of fibrin and platelet-integrated fluorescence intensity inlow TF mice (32 thrombi, 3 mice) and low TF mice after infusion of PMNs (31 thrombi, 3 mice).





their ligands and induce the firm adhesion of leukocytes at the siteof injury. The immediate adhesion of neutrophils to the vessel wallwas mainly dependent on interactions between LFA-1 expressed byneutrophils and ICAM-1 expressed by activated endothelial cells.This interaction between an integrin and its ligand may occurspontaneously as is the case for the firm adhesion of platelets toimmobilized fibrinogen. Indeed, different studies have shown thatchemoattractants stimulate strong, integrin-mediated adhesion tobilayers containing ICAM-1 under static conditions but not in shearflow conditions.33 In addition, it is still unknown whether chemoat-tractants can act in the bloodstream, where they would be rapidlydiluted and swept downstream by the blood flow.34 The selectiveand varied responses of PMNs and monocytes to injury can beexplained by their receptivity to the distinct combinations ofmolecular signals such as LFA-1/ICAM-1 and P-selectin/PSGL-1.Our observations indicate that neutrophils, under arteriole flowconditions, stably attach to the vessel wall in the seconds after theinjury. Different publications have shown that neutrophils have theability to roll at 10-fold higher shear stress in vivo than in vitro.35,36

This may occur via the formation of slings, recently described asthe essential mechanism involved in high-shear rolling of neutro-phils.37 Lastly, it is possible that only a subpopulation of neutro-phils that have, for instance, performed a reverse transmigration areable to adhere rapidly at the site of injury.

The use of real-time digital intravital microscopy has helped thecomprehension of cellular mechanisms involved in thrombusformation. However, how the blood coagulation cascade is acti-vated after an injury has remained unclear. Falati et al proposed thatmonocyte-derived microparticles that expose TF accumulate at thesite of injury via interactions between P-selectin expressed onactivated platelets and PSGL-1 expressed on monocyte-derivedmicroparticles.13 However, these experiments were performed byinfusion of exogenously isolated monocyte-derived microparticlesand failed to show that such endogenous microparticles exist andindeed interact with a platelet thrombus in vivo. When we studiedthrombus formation in CX3CR1GFP mice, we did not observe anysignal corresponding to an accumulation of endogenous monocyte-derived microparticles at the site of injury. We concluded that theaccumulation of monocyte-derived microparticles was not essen-tial for thrombus formation and fibrin generation. This discrepancymay be explained by the fact that Falati et al used exogenouspurified, labeled monocyte-derived microparticles.13 We concludedthat neutrophils are the main source of blood-borne TF in the modelof laser-induced injury. Infusion of neutrophils only partiallyrestores thrombus formation in low TF mice. This may indicate thatother sources of TF are involved after a laser-induced injury,including the vascular wall.15 Thus, after a laser-induced injury,neutrophils may represent the initial source of blood-borne TFneeded to generate thrombin. Vascular TF could be involved foroptimal thrombus growth. Additional studies are needed to discrimi-nate the contribution of blood-borne versus vascular TF in thismodel of thrombosis.

A candidate identified as a possible activator of TF in vivo wasthe enzyme protein disulfide isomerase (PDI). Indeed, inhibition ofPDI reduced fibrin generation and thrombus formation.38,39 How-ever, PDI plays roles in platelet activation that may explain thisresult. For instance, PDI is needed to maintain the platelet integrin�IIb�3 in the right configuration, leading to inside-out andoutside-in signaling.40 PDI may also participate with neutrophils inTF activation. Indeed, PMNs may express inactive TF that could beactivated by PDI secreted by activated endothelial cells andplatelets.39 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 thelaser model of thrombosis. However, the cellular source ofblood-borne TF remains controversial.41 It is possible, as previ-ously suggested, that TF was transferred from monocytes ormonocyte-derived microparticles to PMNs.6

Massberg et al have recently shown that blood -activatedneutrophils can inactivate TFPI via the release of proteases at thesite of injury.2 In this model, the subendothelial matrix is exposedto blood leading to the formation of a platelet thrombus. Here, wedemonstrate, using the laser model of injury, that neutrophilinteraction with endothelial cells is required for thrombus forma-tion. This interaction may not occur when the subendothelialmatrix is exposed to the blood circulation. Neutrophils may thusnot be able to support the first steps of thrombus formation in thismodel. However, their presence was described to be important forformation of an occlusive thrombus.3 In the present study, wefocused on the initial steps leading to thrombus formation after alaser-induced injury. We observed a rapid and a constant recruit-ment of neutrophils. Thus, interactions of neutrophils with plateletsmay occur independently of the model of injury. This interactionmay be involved for the optimal growth of a thrombus incardiovascular diseases, such as myocardial infarction, as well as ininfectious disease. Alternatively, in the ferric chloride model,neutrophils may also bind to the endothelial cells surrounding thesite of injury. Hypothetically, this may lead to the formation ofmultiple microthrombi, similar to the one observed after a laser-induced injury, and will ultimately induce the occlusion of theartery observed in this model of injury.

Apart from the TF pathway, activation of coagulation may alsooccur through the FXII-dependent contact pathway.16 Using FXII�/�

mice, we observed that the extrinsic pathway was the exclusivepathway involved in the first steps leading to the initiation phase ofcoagulation after a dye-laser injury. Previous publications havereported that neutrophils may serve as a matrix for the activation ofthe contact phase.7 It is, therefore, possible that in models ofthrombus formation that are dependent on FXII activation, neutro-phils may also play a critical role. In our model, which is strictlydependent on TF activation, PMNs played a major role as theunique source of TF. In addition, PMNs may also inactivate TFPI.It is also possible that neutrophils, by producing NETs,42 coulddirectly activate TF. Further studies are needed to identify the exactmolecular mechanisms involved in TF activation and TFPI inacti-vation by PMNs.

Although neutrophils seem to be involved in both arterial andvenous injuries, their role may not be the same in different vesselbeds. Indeed, in a mouse model of DVT, von Bruhl et aldemonstrate by intravital 2-photon microscopy that blood mono-cytes and neutrophils crawling along and adhering to the venousendothelium provide the initiating stimulus for DVT development.3

The authors show that thrombus-resident neutrophils are indispens-able for subsequent DVT propagation by binding FXII and bysupporting its activation through the release of NETs. In this modelof venous thrombosis, platelets promote leukocyte recruitment andstimulating neutrophil-dependent coagulation. In our model ofarteriole thrombosis, neutrophils participate in the activation of theextrinsic pathway of coagulation via TF, independent of FXII andthe presence of platelets and monocytes at the site of injury.Inhibiting PMN from interacting with the endothelium at the site ofinjury prevents both accumulation of platelets and activation of theblood coagulation cascade. Our results identified that the interac-tion of neutrophils with the injured endothelial cells, through





LFA-1/ICAM-1 interactions, is the first step leading to the genera-tion of fibrin and thrombus formation.

Several vascular risk factors are associated with proinflamma-tory alterations, including leukocyte activation, and predisposecerebral vasculature to thrombogenesis on inflammatory stimula-tion.4 For example, accumulation of inflammatory cells within thevascular wall starts early during atherogenesis. During later diseasestages, their activation can lead to plaque rupture and thrombusformation, increasing the risk of stroke and acute coronarysyndromes. Moreover, the association of an acute inflammatoryreaction with unstable angina has been previously established and avariety of experimental studies suggested that neutrophil-inducedendothelial dysfunction occurred near the site of coronary obstruc-tion.5,43 Our model of injury is strictly dependent on endothelialactivation.26 We now show that participation of neutrophils isessential for thrombus formation. This may indicate that the lasermodel mimics what may occur after thrombus formation inducedby—or in relation with—inflammatory events, such as after lunginflammation,44 involvement of Shiga toxin thrombotic mecha-nisms in hemolytic uremic syndrome,45 or thrombus formation incerebral microvessels involved in sickle cell disease.46 Our resultsindicate that preventing the binding of PMNs to the vessel wallcould potentially represent a therapeutic strategy to prevent arterialthrombosis and inflammation. Targeting neutrophil adhesion inter-acting with endothelial cells may thus efficiently reduce the risks ofa stroke and prevent acute coronary syndromes or acute cerebralischemia.

Acknowledgments

The authors are indebted to Stephane Robert (Inserm UMR-S1076)for technical assistance. They also thank Dr Markus Riederer(Actelion Switzerland) for critical scientific review of the work,and Ben Atkinson (Intelligent Imaging Innovation) for technicalsupport of the intravital system.

This work was supported by the Fondation pour la RechercheMedicale (FRM) Association (C.D.), and by grants from institu-tional funding from Inserm, the Aix-Marseille University, theAgence Nationale pour la Recherche (ANR; grant ANR-09-JCJC-0053; C.D.), and the Agence pour la Recherche sur le Cancer(ARC) Association (C.D.).

Authorship

Contribution: R.D., S.M., C.F., and R.B. contributed to the designand performance of the research, analysis of data, and writing ofthe manuscript; and G.M.T., N.M., T.R., F.D.-G., C.D., and L.P.-D.contributed to the experimental design, analysis of data, andwriting of the manuscript.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Christophe Dubois, Faculty of Pharmacy,Inserm UMR-S1076, 27 Blvd Jean Moulin, 13385 Marseille,France; e-mail: [email protected].

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Tissue factor-positive neutrophils bind to injured endothelial wall and initiate thrombus formation

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