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Critical Role of FcR γ-Chain, LAT, PLCγ2 and Thrombinin Arteriolar Thrombus Formation upon Mild,Laser-Induced Endothelial Injury In VivoNeena Kalia a; Jocelyn M. Auger a; Ben Atkinson a; Steve P. Watson aa Centre for Cardiovascular Sciences, Institute of Biomedical Research, The MedicalSchool, University of Birmingham, Birmingham, United Kingdom

Online Publication Date: 01 May 2008

To cite this Article: Kalia, Neena, Auger, Jocelyn M., Atkinson, Ben and Watson,Steve P. (2008) 'Critical Role of FcR γ-Chain, LAT, PLCγ2 and Thrombin inArteriolar Thrombus Formation upon Mild, Laser-Induced Endothelial Injury InVivo', Microcirculation, 15:4, 325 — 335

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Microcirculation, 15: 325–335, 2008Copyright c© 2008 Informa Healthcare USA, Inc.ISSN: 1073-9688 print / 1549-8719 onlineDOI: 10.1080/10739680701728822

Critical Role of FcR γ-Chain, LAT, PLCγ2 andThrombin in Arteriolar Thrombus Formation upon

Mild, Laser-Induced Endothelial Injury In VivoNEENA KALIA, JOCELYN M. AUGER, BEN ATKINSON, AND STEVE P. WATSON

Centre for Cardiovascular Sciences, Institute of Biomedical Research, The Medical School,University of Birmingham, Birmingham, United Kingdom

ABSTRACT

Objective: The role of collagen receptor complex GPVI-FcR γ -chain, PLCγ 2 and LAT in laser-induced thrombosis is unclear. Controversy surrounds whether collagen is exposed in this model orwhether thrombosis is dependent on thrombin. This study hypothesized that collagen exposure playsa critical role in thrombus formation in this model, which was tested by investigating contributions ofFcR γ -chain, LAT, PLCγ 2 and thrombin.

Methods: Thrombi were monitored using intravital microscopy in anesthetized wild-type and FcR γ -chain, LAT and PLCγ 2 knockout mice. Hirudin (thrombin inhibitor) was administered to wild-typeand FcR γ -chain knockout mice.

Results: Significantly reduced thrombus formation was observed in FcR γ -chain and PLCγ 2 knock-outs with a greater decrease observed in LAT knockouts. Dramatic reduction was observed in wild-types treated with hirudin, with abolished thrombus formation only observed in FcR γ -chain knockoutstreated with hirudin.

Conclusions: GPVI-FcR γ -chain, LAT and PLCγ 2 are essential for thrombus generation and stabilityin this laser-induced model of injury. More importantly, a greater role for LAT was identified, whichmay reflect a role for it downstream of a second matrix protein receptor. However, inhibition ofplatelet activation by matrix proteins and thrombin generation are both required to maximally preventthrombus formation.Microcirculation (2008) 15, 325–335. doi:10.1080/10739680701728822

KEY WORDS: platelets, collagen, signalling molecules, thrombin, intravital

INTRODUCTION

Platelet-dependent thrombus formation is a keyevent in the pathogenesis of a number of clinical con-ditions, including acute myocardial infarction andthrombotic stroke [1–3]. Critical to the onset of thesemajor health problems is the activation of platelets byextracellular matrix proteins, which are exposed af-ter vessel injury and after a rupture of an atheroscle-rotic plaque. Among the constituents of the suben-dothelial matrix, collagen has been proposed to bethe most powerful in inducing platelet adhesion andaggregation [4]. Collagen interacts directly with the

This work was supported by the Wellcome Trust and British HeartFoundation SPW holds a BHF Chair.Dr Neena Kalia, Centre for Cardiovascular Sciences, Instituteof Biomedical Research, The Medical School, Edgbaston, Uni-versity of Birmingham, Birmingham, B15 2TT, UK. E-mail:n.kalia@bham.ac.uk

platelet immunoglobulin receptor GPVI (glycopro-tein VI). GPVI is a well characterized signaling re-ceptor which forms a complex with the FcR γ -chain.Interaction of collagen with this complex initiates anintracellular signaling cascade via tyrosine phospho-rylation of an ITAM (immunoreceptor tyrosine-basedactivation motif) present on the FcR γ -chain. TheFcR γ -chain is the critical signaling element of thecomplex and is also essential for GPVI expressionon the platelet surface [5, 6]. Numerous downstreamproteins form key elements of the GPVI-FcR γ -chainsignaling pathway, including the adapter protein LAT(linker for activation of T cells) [6–7]. LAT is re-quired for maximum tyrosine phosphorylation andactivation of the effector enzyme PLCγ 2 (phospho-lipase Cγ 2) by collagen, although a limited degree ofplatelet activation can occur in its absence. PLCγ 2mediates a number of events, including activationof platelet integrins such as α2β1 (GPIa-IIa) andαIIbβ3 (GPIIb-IIIa). These integrins promote firm

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adhesive platelet-platelet interactions, which are es-sential for thrombus growth and stability.

Although in vitro studies have attributed varyingdegrees of importance to the above signaling path-ways, little attention has focused upon their relativeimportance in vivo. Several murine models of vas-cular injury and subsequent thrombus developmenthave been described. We have utilized a laser-inducedinjury model as this can be programmed to induce amild injury to the endothelial layer. Since the laserbeam is focused on a small area of a pre-selected ar-teriole, it does not injure large areas of tissue and,therefore, subsequent thrombi are spatially and tem-porally defined [8]. In this model, it was originallybelieved that one or more endothelial cells were ab-lated leading to exposure of collagen and other suben-dothelial matrix proteins. However, a recent studyreported that thrombus formation in this model wasunaltered in the absence of the GPVI-FcR γ -chaincomplex, arguing against a role for collagen in thisresponse [9]. As an alternative, it was proposed thatthe laser injury causes endothelial damage similar tothat which occurs during an inflammatory response[10, 11]. This leads to exposure of P-selectin and thecapture of plasma-derived microparticles through P-selectin glycoprotein ligand-1 (PSGL-1), which alsocontain tissue factor [11]. On the other hand, usingthe same model, the group of Jackson et al. [12] havereported a role for thrombin generation and the colla-gen receptor GPVI in thrombus formation in responseto laser injury [12], which is consistent with the ob-servation that PLCγ 2 also contributes to thrombusgrowth in this model [13].

In the present study, we focus on the laser injurymodel and hypothesize that collagen exposure doesindeed occur in this model of thrombosis and, there-fore, the GPVI-FcR γ -chain, and two of its signalingproteins have a contributory role to play. We com-pared the role of the GPVI-FcR γ -chain complex inmediating thrombus formation alongside that of thesignaling proteins LAT, whose contribution in vivo iscurrently not known, and PLCγ 2, and that of throm-bin. Although the role of GPVI-FcRγ -chain [9, 12]or PLCγ 2 [13] has been compared with wild-typesin separate studies using a laser induced model, thisstudy is novel in that it allows the relative contribu-tion of GPVI-FcRγ -chain, PLCγ 2 as well as LAT tobe assessed in the same tissue and under the sameexperimental conditions. In addition, we have useda second model of thrombus growth, namely the tailbleed assay, which we have adapted slightly, to assessthe contributory roles of these proteins. Interestingly,

novel results from the present study have identifieda greater role for the adapter LAT in mediating arte-riolar thrombus formation than compared with thatof the GPVI-FcR γ -chain complex and of PLCγ 2,suggesting that this may reflect a role for the adapterdownstream of a second matrix protein receptor, suchas CLEC-2 [14].

MATERIALS AND METHODS

Animals

Intravital microscopy experiments were conducted on25–30 g male C57BL/6 wild-type mice, and PLCγ 2,LAT and FcR γ -chain knockouts [15–17]. Thereis no difference in platelet counts in the knockoutsthat were used or when compared to controls (datanot presented). All procedures were undertaken withUnited Kingdom Home Office approval in accor-dance with the Animals (Scientific Procedures) Act of1986 (Project License No’s: 40/2212 and 40/2749).

Surgical procedure for IVM

Anesthesia was induced with an intraperitoneal in-jection of ketamine (100 mg/kg Vetalar; Pharma-cia and Upjohn Ltd, Northants, USA) and 2% xy-lazine (20 mg/kg; Millpledge Pharmaceuticals, Not-tinghamshire, UK). Cannula were inserted in the tra-chea to facilitate spontaneous respiration and in theleft carotid artery to provide a route for administra-tion of additional anesthesia as required. The carotidcannula also provided access for administration offluorescently labeled antibody and saline. The leftcremaster, a transparent muscle surrounding the tes-ticle, was exteriorized and spread flat over an op-tically clear coverslip on a pedestal and continu-ously superfused with a bicarbonate-buffered saline(pH 7.4; 36◦C) gassed with 5% CO2/95% N2. Ani-mals were allowed to stabilize for 30 min followingthe induction of anesthesia and the establishment ofmonitoring.

The effect of a direct thrombin inhibitor, hirudin,was also assessed in wild-type and FcR γ -chainknockout mice. Hirudin (10 mg/kg in saline; Reflu-dan, Pharmion, Berkshire, UK) was administered viathe carotid artery as a single bolus dose [12]. Pre-treatment thrombi were generated in each animal andalso 5 minutes post-hirudin treatment.

Intravital Microscopy

High speed intravital microscopy experiments wereperformed as previously described by Falati et al. [18,

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19]. Experiments were conducted using a motorizedOlympus BX-61WI microscope (Middlesex, UK). Si-multaneous capture of a brightfield and fluorescentimage was achievable by rapidly alternating the il-lumination of the specimen between transmissionlight using a Uniblitz shutter (Vincent Associates,Rochester, NY, USA) and epi-fluorescent light using aLambda DG-4 high speed wavelength changer (Sut-ter Instrument Company, Novato, CA, USA). Digitalimages were captured using a high capture rate Sen-sicam CCD camera (640 × 480 pixels; 30 images/swithout binning; The Cooke Corporation, Michigan,USA). A high performance GEN III image intensifier(Videoscope Int Ltd, VA, USA) incorporated betweenthe microscope and camera, amplified light 1000-fold, allowing visualization of low fluorescent inten-sities. All these components were controlled by a Dellworkstation and imaging software (Slidebook, Intel-ligent Imaging Innovations, Denver, CO, USA) andstored as permanent digital images for subsequentoff-line analysis.

Laser Induced Vessel Wall Injury

To generate laser-induced thrombi, the microscopewas fitted with a nitrogen ablation laser (Microp-oint, Photonic Instruments, St. Charles, IL, USA),which introduced the laser through the microscopeobjectives. The output of the laser was 337 nm butwas subsequently tuned through a coumarin (440nm) dye cell interposed in its path. The laser deliv-ered 4 nsec energy pulses (3 pulses/sec) over a sur-face of 1 µm in diameter. The beam was directedthrough a water immersion objective lens (× 40; NA0.8 W; working distance 3.3 mm) onto the tissueand focused at the focal point of the microscope.It was, thus, possible to induce injury only at theluminal surface of the selected arteriole and simul-taneously monitor thrombogenesis. The laser powerat the objective lens is a key determinant of the ki-netics of the reaction and is inextricably linked tothe degree of damage to the endothelium [8, 20].However, in the current study, laser with enoughenergy to minimally ablate tissue in order to initi-ate thrombotic pathways with the fewest number ofperturbations was used. Although the actual laserpower energy at the objective was not measured, itcould not have been too varied as the kinetics ofthe thrombi between intra and inter-experimentationwithin control and individual knockout groups ofanimals were similar, and all experimental settingsunder which experiments were performed were keptconstant.

Only arterioles with a diameter of 25–35 µm wereselected for study. These were identified by followingthe branches of the main supplying arteriole (whichhad flow away from the animal) until a tertiary arte-riole of appropriate dimension was identified. Thesevessels were further confirmed as arterioles by the ab-sence of rolling leukocytes. This site of ablation wasidentified by pointing and clicking the mouse cursorat that position. Using Slidebook software, the powerof the laser beam was adjusted to between 70–75% ofthe maximum by appropriately positioning a rotat-ing neutral density filter in the path of the beam. Thenumber of times the laser beam repeatedly hit thesame place was also software controlled and was setto between 5–7 times. In addition, by increasing thenumber of different adjacent areas hit by the laser, thesize of the ablatory event could also be adjusted andwas also set to between 5–7 times. Our preliminaryexperiments suggested that these 3 values within theranges outlined were sufficient to induce an ablatoryevent that did not burst the vessel. These values werekept constant and used to induce a mild endothelialinjury in all four groups of animals. The ability tocontrol the nature of the laser pulse in this manner isan improvement on other similar studies where suchprecise control was not afforded. Using these values,the injury to vessels was not severe in that it did notresult in occlusive thrombi. It has previously been de-scribed that severe laser injury leads a near occlusivethrombus forming that can be up to 12 x larger thanthose observed after a mild injury and do not erodewith time [13].

Data Acquisition and Analysis

To fluorescently label platelets, 20 µL Alexa Fluor488 conjugated to goat anti-rat antibody (2 mg/mL;Molecular Probes, Eugene, OR, USA) and 5µL ratanti-murine GPIIb (CD41) antibody (0.5 mg/mL;BD Biosciences Pharmingen, San Diego, CA, USA)were added to 70 µL saline and infused via thecarotid cannula. This labeling procedure does notinterfere with platelet aggregation [21] as thrombiformed readily in control animals and were similar innature to the brightfield images obtained of thrombiin animals without the antibody. Critically, the anti-body is used at a concentration that has no discern-able effect on platelet aggregation in vitro [unpub-lished data]. All platelets are labeled as the amountof antibody added per mouse is in excess of the num-ber of platelets (assuming a platelet concentration ofabout 1 × 109 per mL blood) by around 1,000-fold.The brightness of individual platelets should be the

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same for each experiment as the same preparation oflabeled antibody, and the same amount of antibodyper gram of mouse is introduced. The antibody willdissolve in the blood as expected and since the Alexa-488 fluorophore is strongly conjugated to the primaryCD41 antibody, no free fluorescent label should beavailable to dissolve in the blood [22].

The cremaster microvasculature was identified usingbrightfield light, and 25–35 µm diameter arterioleswere selected for laser injury. Brightfield and fluores-cent images were captured simultaneously for a fewframes, and then laser injury was induced. Imageswere recorded of the developing thrombus for a fur-ther 4–5 minutes. Multiple thrombi in each cremasterpreparation were generated with a distance of at least200 µm between them and upstream to previous in-juries in the same or similar sized arterioles. Further-more, new ones were only generated once ‘old’ oneshad flattened to leave a remnant mural thrombus.These did not appear to affect flow as they did not ob-struct the vessel to any significant degree. The back-ground fluorescence intensity, predominantly due tofreely circulating platelets, was determined and sub-tracted from the fluorescence intensity of the devel-oping thrombus. The resulting value was multipliedby the sum of all pixels above background to give avalue for integrated intensity at each time point. Thisintegrated intensity value was directly proportional tothe size of the developing thrombus and, when plot-ted against time, provided a graph that illustratedthe dynamic kinetics of platelet accumulation. Addi-tional values that were determined included the peaksize of the thrombus and the time taken to reach thispeak size for each group of animals.

Tail Bleeds

Experiments were conducted on 25–35 g male and fe-male C57BL/6 wild-type mice (n = 32), and PLCγ 2(n = 7), LAT (n = 10) and FcR γ -chain (n = 16)knockouts. Mice were anesthetized with isofluoranethrough the use of a face mask throughout the exper-iment and subsequently injected with the analgesicbuprenorphine (ip). The animal was laid flat on abox of height 15 cm. The tail was laid horizontallyalong the box with the tip (1 cm) hanging horizon-tally over the edge. Three millimeters were removedusing a sharp razor blade, and blood was collectedin a graduated 3 mL blood tube containing 1.5 mLH2O. Mice were allowed to bleed until they lost ei-ther 15% blood volume (which was calculated priorto the experiment based on the animal weight andassuming a blood volume of 70 mL/kg) or for 20

min. The volume (µL) of blood lost in 10 min wasmeasured. The more commonly used technique formeasuring bleeding time is the immersion of the cuttail tip in saline and expressing the tail bleed dataas “time to stop bleeding.” However, we found thistest to be highly variable as has been encountered byothers [22].

Statistical Analysis

Intravital data from individual mice were collated,and statistics were conducted on N = 5. Since the val-ues for peak thrombus size for all groups observed anormal distribution, they were tested for significanceusing an ANOVA followed by a Bonferroni’s multi-ple comparison test. Non-parametric tail bleed datawere tested for significance using a 1-tailed Mann-Whitney test. Differences were considered to be ofstatistical significance when p < 0.05.

RESULTS

Thrombus Formation in C57BL/6 Wild-Type Miceis Inhibited by Hirudin

The dynamic profile of platelet accumulation af-ter laser-induced arteriolar injury was examined bymonitoring the fluorescent intensity of the develop-ing thrombus. In this model of mild injury, plateletaccumulation occurs in a specific temporal pattern. Akinetics curve was constructed based on the medianvalue of the integrated fluorescent intensity over aperiod of time for 20 independent thrombi from eachgroup of 5 animals (Fig. 1a). In wild-type mice, dur-ing the initial phase of this dynamic process, plateletsrapidly accumulated until a maximum thrombus sizewas obtained at 94 ± 10 s. Subsequently, a lossof platelets occurred, predominantly due to gradualstreaming from the leading edge of the thrombus inthe direction of the flow. This led to a diminishingin size over the course of several minutes until theplatelet content was stabilized within the thrombi,leaving a flattened mural thrombus at the end ofthe recording period. This final stage was identifiedas a plateau in the kinetics curve. This pattern ofthrombus growth, as shown in Fig. 1a, was typi-cal for approximately 80% of the thrombi analyzedfrom 5 wild-type mice. In the remainder, two peaks ofthrombus formation were sometimes observed, sug-gesting detachment of a large embolus from the mainbody, or there was no residual plateau indicatingthe absence of a mural thrombus. Interestingly, thispattern was more typical of the smaller thrombi thatformed.

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Figure 1. Kinetics of platelet accumulation in developing thrombi in wild-type and knockout mice (a) and in wild-type and FcR γ -chain−/− mice before and after hirudin treatment (b). Platelets were labelled in vivo with Alexa 488-conjugated goat anti-rat antibody bound to rat anti-CD41 antibody. Each curve represents the median integrated plateletfluorescence (i.e., fluorescence of platelets contributing to the thrombus minus background fluorescence) for 20 thrombiinduced in 5 mice for each group with data from individual mice collated first (a) or 2 thrombi pre-hirudin and 5thrombi post-hirudin for each group (b). Fluorescent intensity of platelets in arbitrary units is presented as a functionof time.

Strikingly, a dramatic reduction in thrombus forma-tion was observed at all time points in the presenceof the thrombin inhibitor, hirudin, although the buildup of a small mural thrombus could be seen over thecourse of the recording (Fig. 1b). The peak size ofthe mural thrombus was less than 10% of the peaksize of the thrombus in control platelets.

Thrombus Formation in PLCγ2, LAT and FcRγ-Chain Knockout Mice

Despite the dependence on thrombin generation fol-lowing laser injury, a marked reduction in throm-bus formation was also observed in the absence of

GPVI-FcR γ -chain complex, LAT and PLCγ 2 at alltime points (Fig. 1a). Figure 1 depicts the throm-bus kinetics as fluorescence intensity, which incor-porates the size of the thrombus in pixels. However,we found that even when the kinetics curves (Figs. 1and 4) were presented as thrombus size only againsttime using the pixel data alone, the graphs generatedwere similar in both kinetics and magnitude (data notpresented). The decrease in thrombus size as mea-sured in pixels was more marked for the LAT−/−(peak thrombus size only 33.3% of wild-type peakthrombus size) than for FcR γ -chain−/− (51.8%)and PLCγ 2−/− (55.6%) mice (Fig. 2). The peak inthrombus formation in the LAT−/− mice was less than

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Figure 2. Mean peak thrombus size in wild-type and knockout mice. Data are presented as mean ± SEM from 20thrombi induced in 5 mice for each group, with data from individual mice initially collated. Values for the area of thethrombus in pixels has been plotted (pixel size was equal to 9.9 µm × 9.9 µm for the camera used). A significantreduction in thrombus size was only observed when knockout mice were compared to wild-type controls, but not whencompared to each other. Time taken to reach a peak in thrombus size did not significantly differ from wild-type controlsfor any of the genetically modified groups of mice. Data from knockout mice were compared to C57BL/6 wild-typecontrols (*p < 0.05, **p < 0.01).

that in the other two knockouts, although this didnot reach statistical significance. The time to reachthis peak thrombus size, however, was not signifi-cantly different in wild-types compared to the threeknockouts (FcR γ -chain 108 ± 11.9 s; LAT 123.5 ±17.0 s; PLCγ 2 108.1 ± 16.6 s). The three phases ofthrombus formation, namely platelet accumulation,platelet loss and a plateau phase, could only be clearlyobserved in 50%, 26% and 47% of the thrombi fromFcR γ -chain, LAT and PLCγ 2-deficient mice, re-spectively, in comparison to a value of 80% in con-trols. Atypical patterns included a reduced rate ofplatelet accumulation (Fig. 3a), numerous peaks in-dicative of several emboli detaching from the mainthrombus body (Fig. 3b, 4a), the detection of largethrombi and the absence of a final mural thrombus(Fig. 4b). This atypical pattern of thrombus growthwas most apparent in the absence of LAT, consis-tent with the fact that these mice appeared to havethe greatest reduction in thrombus growth. Interest-ingly, the formation of a late stage mural thrombuswas absent in FcR γ -chain−/− mice pre-treated withhirudin (Fig. 1b).

Tail Bleeds

The volume of blood lost from FcR γ -chain and LATdeficient mice was significantly greater than that lost

in wild-type mice over a 10 minute period (p < 0.05).However, no differences between PLCγ 2 and wild-type mice were observed (Fig. 5).

DISCUSSION

Laser-induced endothelial cell injury is becoming afrequently used model for studying the events thatunderlie thrombus formation in vivo. However, thereis controversy surrounding whether thrombus forma-tion in this model is mediated by exposure to suben-dothelial collagen and other matrix proteins or byendothelial cell damage leading to exposure of P-selectin and recruitment of tissue factor-bearing mi-croparticles [9–11]. Emerging studies are suggestingthat the severity of injury is a key factor in deter-mining the relative contribution of certain signalingmolecules to thrombus formation [9, 12–13].

Dubois et al. [9], using FcR γ -chain−/− mice, demon-strated that GPVI was involved only following a se-vere FeCl3 injury in the mesentery and not in alaser induced injury model [9]. Similarly, a role forGPVI has been demonstrated by Massberg et al. [23,24] in three in vivo models of severe vascular in-jury [23, 24]. Blocking of the collagen binding siteon murine GPVI by the monoclonal anti-GPVI anti-body JAQ1 or by a soluble GPVI dimer completelyabolished stable platelet adhesion and aggregation

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Figure 3. Images of thrombus formation over time in wild-type and FcR γ -chain−/− mice (A) and of emboli detachingfrom the main body of thrombus in FcR γ -chain−/−and PLCγ 2−/− mice (B). Similar images to these were obtained forall three knockout mice following laser induced endothelial injury. Platelets (green) composited with brightfield images(black/white) of the cremaster arteriole. Blood flow is from right to left in each image. The dashed boxes representemboli detaching from the main body of the thrombus. Platelets were detected with purified rat anti-mouse CD41 andAlexa 488 goat-anti rat IgG. All microvessels were viewed at x400 magnification and images were acquired using adigital CCD camera (SensiCam II, Cooke Corporation) with a 640 x 480 pixel array. Vessels in figure B are 120% moremagnified than those in figure A.

following ligation-induced endothelial denudation ofthe carotid artery, FeCl3 injury and wire-induced en-dothelial disruption. The role of GPVI in a severeFeCl3 injury has again been supported by Munnix etal. [25] by utilizing FcR γ -chain−/− mice and the

JAQ1 antibody, with a role for this complex observedin both arterioles and venules [25]. In contrast, Man-gin et al. demonstrated that GPVI was not essentialin a severe Folts-type injury or when laser injury wassevere enough to form an occlusive thrombus [12].

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Figure 4. A typical kinetics of platelet accumulation inthrombi in FcR γ -chain−/− (A) and LAT−/− (B) mice.Two profiles of individual thrombi illustrating numerouspeaks indicative of emboli detaching from the main bodyof the thrombus (A) and lack of a plateau phase indica-tive of no mural thrombus remaining attached to the ves-sel wall (B). Platelets were labelled in vivo with Alexa488-conjugated goat anti-rat antibody bound to rat anti-CD41 antibody. Each curve represents integrated plateletfluorescence (ie. fluorescence of platelets contributing tothe thrombus minus background fluorescence). Fluores-cent intensity of platelets in arbitrary units is presented asa function of time.

However, when the laser was utilized to generate asub-occlusive thrombus, they did observe a key rolefor GPVI using FcR γ -chain−/− mice.

In the present study, our results using the FcR γ -chaindeficient mice are in broad agreement with the latterstudy by Mangin et al. [12], demonstrating a key butpartial role for the GPVI-FcR γ -chain complex in amild laser induced injury. Nonne et al. also demon-strated a role for PLCγ 2 following mild laser-inducedinjury, although surprisingly in light of the above ob-servations, not in response to a more severe level oflaser injury [13]. These previous studies have pre-dominantly focused on the role of one protein, while

part of the novelty of the current study lies in the eval-uation of all three proteins of the collagen-dependentthrombus pathway under the same set of experimen-tal conditions. This has allowed the relative contri-bution of FcR γ -chain, PLCγ 2 and LAT to be com-pared. Indeed, our study is the first to recognize thatLAT is also essential in vivo, which is especially im-portant since earlier work has suggested that LATis dispensable for collagen- and convulxin-inducedplatelet aggregation [26]. Although we did not com-pare shear rates/velocities, our results are probablynot explained by differences in these parameters asthere is nothing to suggest that these will be differentbetween the strains, given the hematopoietic-specificexpression of the proteins in the knockouts that havebeen investigated.

It is important to consider the explanation for thediffering conclusions from our study, and those ofDubois et al. using a similar laser injury model [9].The latter study emphasized the importance of tis-sue factor as the main contributor to thrombus de-velopment following laser injury. However, they wereunable to detect exposure of type I collagen follow-ing laser ablation using a fluorescently labeled anti-murine collagen antibody. It is possible that the ex-tremely rapid attachment of platelets to any exposedcollagen precludes contact by the collagen antibodyor that the tip of the laser ablates an area that is toosmall to be readily detected by the camera, possibil-ities that were acknowledged by Dubois et al. [9].We determined whether basement membrane colla-gen was exposed following laser injury using an al-ternative method. Rhodamine 6G, a small molecularweight fluorescent dye, was injected systemically andwas expected to leak into the interstitium followingendothelial ablation despite the presence of an un-derlying basement membrane. However, attachmentof platelets to the exposed site was either too rapid,thereby, preventing leakage or our camera system wasnot sensitive enough to detect a very localized leakageof a small amount of dye into the interstitium.

Despite identifying a key role for intracellular signal-ing proteins, our present findings are also consistentwith the involvement of multiple pathways mediatingthrombus formation following laser injury, includ-ing thrombin generation. We always observed a sig-nificant, albeit reduced thrombus formation, in theabsence of the GPVI-FcR γ -chain complex and thetwo signaling proteins following endothelial injury,demonstrating the presence of a GPVI-independentpathway of thrombus formation. The demonstrationthat the thrombin inhibitor, hirudin, reduced, but

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Figure 5. Volume of blood loss following tail bleeds in wild-type and knockout mice. Data for each individual animalis presented for each group. The volume of blood lost in 10 minutes was significantly higher for FcR γ -chain−/− (p <0.05) and LAT−/− (p < 0.05) mice compared to WT animals, with no difference between WTs and PLCγ 2−/− mice.

did not completely abolished thrombus formation inwild-type mice and that this residual response waslost in the FcR γ -chain knockout mouse providesstrong support for the interplay of GPVI and throm-bin generation in supporting thrombus formation.Mangin et al. [12] also presented similar findings, butunlike our results, they did not demonstrate a role forthombin in the formation of wild-type thrombi, butonly defined a major role for it in maintaining a ro-bust thrombotic response in FcR γ -chain−/− mice.It is interesting that in a recent study, Van Gestelet al. [27] did not demonstrate an important role forthrombin in the initial hemostatic reaction when rab-bit mesenteric arterioles were punctured. However,they concluded that thrombin was essential in pre-venting re-bleeding as reduced thrombus stability inthe long term was observed in its absence [27].

Although our data demonstrate important roles forthe FcR γ -chain, the signaling proteins LAT andPLCγ 2, and thrombin generation in response to mildendothelial injury, the significance of this in the con-text of bleeding is unclear as even a small build-upof platelets is predicted to be sufficient to preventleakage from the vasculature. Previous studies haveidentified that a defect in thrombin formation or theabsence of the two thrombin receptors present onmouse platelets, PAR3 and PAR4, gives rise to in-creased bleeding following removal of a small portion

of the mouse tail [28]. Similarly, a defect in bleed-ing in the absence of the FcR γ -chain has also beenreported in this assay [29]. Consistent with our in-travital observations, we have observed an increasedvolume of blood loss in the absence of FcR γ -chainand also in the absence of LAT in the tail assay. How-ever, we were surprised to observe that the PLCγ 2mice did not demonstrate a similar increase in bloodloss in the tail bleeding assay. It is possible that this isdue to the presence of a low level of PLCγ 1 in mouseplatelets [30], although it is perhaps more likely be-cause of the mixing of the blood with the lymphaticcirculation as a consequence of the developmentalproblems that are seen in this mouse, which would,therefore, bring about a lowering of blood pressure[31]. Importantly, a similar defect in the vasculatureand lymphatics is not seen in the FcR γ -chain orLAT-deficient animals.

In conclusion, the present study has confirmed a rolefor the GPVI-FcR γ -chain complex and two of itsdownstream signaling proteins, LAT and PLCγ 2, insupporting thrombus formation in vivo in response tomild laser injury of the cremaster endothelium, butalso demonstrated a significant role for thrombin inthis response. This study is the first to assess all fourproteins under the same in vivo conditions, allowingthe relative contribution of each to be determined.Importantly, ablation of both GPVI and thrombin

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formation is required to cause complete blockade ofmural thrombus formation demonstrating the impor-tance of the interplay of collagen-mediated plateletactivation and thrombin generation in mediating ar-teriolar thrombus formation in vivo. It is possible thatdifferences in current and previously published re-sults are due to slight alterations in the criteria setfor the laser prior to ablation. This study has, there-fore, provided detailed methodological informationon this which should allow greater ease of compari-son with future studies. These results reconcile manyof the contrasting observations in the literature andalso, more interestingly, raise the possibility that anadditional receptor might contribute to thrombus for-mation in this model in view of the greater defectobserved in the absence of LAT relative to that ofGPVI.

REFERENCES

1. Tan KT, Watson SP, Lip GY. (2004). The endothe-lium and platelets in cardiovascular disease: potentialtargets for therapeutic intervention. Curr Med ChemCardiovasc Hematol Agents 2:169–178.

2. Gawaz M. (2004). Role of platelets in coronary throm-bosis and reperfusion of ischemic myocardium. Car-diovasc Res 61:498–511.

3. Massberg S, Brand K, Gruner S, Page S, Muller E,Muller I, Bergmeier W, Richter T, Lorenz M, KonradI, Nieswandt B, Gawaz Ml. (2002). A critical role ofplatelet adhesion in the initiation of atherosclerotic le-sion formation. J Exp Med 196:887–896.

4. Farndale RW, Sixma JJ, Barnes MJ, de Groot PG.(2004). The role of collagen in thrombosis andhemostasis. J Thromb Haemost 2:561–573.

5. Nieswandt B, Watson SP. (2003). Platelet-collageninteraction: is GPVI the central receptor? Blood102:449–461.

6. Nieswandt B, Bergmeier W, Schulte V, RackebrandtK, Gessner JE, Zirngibl H. (2000). Expression andfunction of the mouse collagen receptor glycoproteinVI is strictly dependent on its association with the FcRγ -chain. J Biol Chem 275:23998–4002.

7. Andrews RK, Berndt MC. (2004). Platelet physiologyand thrombosis. Thromb Res 114:447–453.

8. Rosen ED, Raymond S, Zollman A, Noria F, Sandoval-Cooper M, Shulman A, Merz JL, Castellino FJ. (2001).Laser-induced noninvasive vascular injury models inmice generate platelet- and coagulation-dependentthrombi. Am J Pathol 158:1613–1622.

9. Dubois C, Panicot-Dubois L, Merrill-Skoloff G, FurieB, Furie BC. (2006). Glycoprotein VI-dependent andindependent pathways of thrombus formation in vivo.Blood 107:3902–3906.

10. Chou J, Mackman N, Merrill-Skoloff G, Pedersen B,Furie BC, Furie B. (2004). Hematopoietic cell-derived

microparticle tissue factor contributes to fibrin forma-tion during thrombus propagation. Blood 104:3190–3197.

11. Falati S, Liu Q, Gross P, Merrill-Skoloff G, Chou J,Vandendries E, Celi A, Croce K, Furie BC, Furie B.(2003). Accumulation of tissue factor into developingthrombi in vivo is dependent upon microparticle P-selectin glycoprotein ligand 1 and platelet P-selectin.J Exp Med 197:1585–1598.

12. Mangin P, Yap CL, Nonne C, Sturgeon SA, Goncalves I,Yaun Y, Schoenwaelder SM, Wright CE, Lanza F, Jack-son SP. (2006). Thrombin overcomes the thrombosisdefect associated with platelet GPVI/FcR γ deficiency.Blood 107:4346–4353.

13. Nonne C, Lenain N, Hechler B, Mangin P, CazenaveJP, Gachet C, Lanza F. (2005). Importance of plateletphospholipase Cγ 2 signaling in arterial thrombosis asa function of lesion severity. Arterioscler Thromb VascBiol 25:1293–1298

14. Suzuki-Inoue K, Fuller GLJ, Garcia A, Eble JA,Pöhlmann S, Inoue O, Gartner TK, Hughan SC,Pearce AC, Laing GD, Theakston RD, SchweighofferE, Zitzmann N, Morita T, Tybulewicz VL, Ozaki Y,Watson SP. (2006). A novel Syk-dependent mecha-nism of platelet activation by the C-type lectin recep-tor CLEC-2. Blood 107: 542–549.

15. Wang D, Feng J, Wen R, Marine JC, Sangster MY,Parganas E, Hoffmeyer A, Jackson CW, Cleveland JL,Murray PJ, Ihle JN. (2000). Phospholipase Cγ 2 is es-sential in the functions of B cell and several Fc recep-tors. Immunity 13: 25–35.

16. Zhang W, Sommers CL, Burshtyn DN, Stebbins CC,DeJarnette JB, Trible RP, Grinberg A, Tsay HC, JacobsHM, Kessler CM, Long EO, Love PE, Samelson LE.(1999). Essential role of LAT in T cell development.Immunity 10:323–332

17. Park SY, Ueda S, Ohno H, Hamano Y, Tanaka M, Shi-ratori T, Yamazaki T, Arase H, Arase N, KarasawaA, Sato S, Ledermann B, Kondo Y, Okumura K, RaC, Saito T. (1998). Resistance of Fc receptor- defi-cient mice to fatal glomerulonephritis. J Clin Invest102:1229–1238.

18. Falati S, Gross P, Merrill-Skoloff G, Furie BC, Furie B.(2002). Real-time in vivo imaging of platelets, tissuefactor and fibrin during arterial thrombus formationin the mouse. Nat Med 8:1175–1181.

19. Falati S, Gross PL, Merrill-Skoloff G, Sim D, Flau-menhaft R, Celi A, Furie BC, Furie B. (2004). Invivo models of platelet function and thrombosis: studyof real-time thrombus formation. Methods Mol Biol272:187–97.

20. Rumbaut RE, Slaaf DW, Burns AR. Microvascularthrombosis models in venules and arterioles in vivo(2005). Microcirculation 12:259–274.

21. Celi A, Merrill-Skoloff G, Gross P, Falati S, Sim DS,Flaumenhaft R, Furie BC, Furie B. (2003). Throm-bus formation: direct real-time observation and digi-tal analysis of thrombus assembly in a living mouse

Dow

nloa

ded

By:

[Hal

ford

, Gay

le] A

t: 10

:32

20 J

une

2008

Signaling molecules in laser-induced thrombosisN Kalia et al. 335

by confocal and widefield intravital microscopy.J Thromb Haemost 1:60–68.

22. Talmachova T, Abrink M, Futter CE, Authi KS, SeabraMC. (2007). Rab27b regulates number and secretionof platelet dense granules. PNAS 104:5872–5877.

23. Massberg S, Gawaz M, Gruner S, Schulte V, Konrad I,Zohlnhofer D, Heinzmann U, Nieswandt B. (2003). Acrucial role of glycoprotein VI for platelet recruitmentto the injured arterial wall in vivo. J Exp Med 197:41–49.

24. Massberg S, Konrad I, Bultmann A, Schulz C, MunchG, Peluso M, Lorenz M, Schneider S, Besta F, Muller I,Hu B, Langer H, Kremmer E, Rudelius M, HeinzmannU, Ungerer M, Gawaz M. (2004). Soluble glycoproteinVI dimer inhibits platelet adhesion and aggregation tothe injured vessel wall in vivo. FASEB J 18:397–399.

25. Munnix ICA, Strehl A, Kuijpers MJE, Auger JM, vander Meijden PEJ, van Zandvoort MAM, oude EgbrinkMGA, Nieswandt B, Heemskerk JWM (2005). TheGlycoprotein VI-Phospholipase Cγ 2 Signaling Path-way Controls Thrombus Formation Induced by Colla-gen and Tissue Factor In Vitro and In Vivo. Arterioscle-rosis, Thrombosis & Vascular Biology. 25:2673–2678.

26. Judd BA, Myung PS, Obergfell A, Myers EE, ChengAM, Watson SP, Pear WS, Allman D, Shattil SJ, Ko-retzky GA. (2002). Differential requirement for LATand SLP-76 in GPVI versus T cell receptor signaling.J Exp Med 195:705–717.

27. van Gestel MA, Reitsma S, Slaaf DW, Heijnen VV,Feijge MA, Lindhout T, van Zandvoort MA, Elg M,Reneman RS, Heemskerk JW, Egbrink MG. (2007)Both ADP and thrombin regulate arteriolar thrombusstabilization and embolization, but are not involved ininitial hemostasis as induced by micropuncture. Mi-crocirculation 14:193–205.

28. Weiss EJ, Hamilton JR, Lease KE, Cough-lin SR. (2002). Protection against thrombo-sis in mice lacking PAR3. Blood 100: 3240–3244.

29. Grüner S, Prostredna M, Aktas B, Moers A, Schulte V,Krieg T, Offermanns S, Eckes B, Nieswandt B. (2004).Anti–Glycoprotein VI treatment severely compro-mises hemostasis in mice with reduced α2β1 levels orconcomitant aspirin therapy. Circulation 110:2946–2951.

30. Suzuki-Inoue K, Inoue O, Frampton J, Watson SP.(2003). Murine GPVI stimulates weak integrin acti-vation in PLCγ 2−/− platelets: involvement of PLCγ 1and PI3-kinase. Blood 102:1367–1373.

31. Abtahian F, Guerriero A, Sebzda E, Lu M, ZhouR, Mocsai A, Myers EE, Huang B, Jackson DG,Ferrari VA, Tybulewicz V, Lowell CA, Lepore JJ,Koretzky GA, Kahn ML. (2003). Regulation ofBlood and Lymphatic Vascular Separation by Sig-naling Proteins SLP-76 and Syk. Science 299:247–251.