Accepted Article Científicas yCientíficas TécnicasArgentinaAires. (CONICET). Buenos Universidad Nacionalde InvestigacionesAires. Lanari. Alfredo deBuenosConsejo *Departamento Instituto deHematología deInvestigacionesInvestigación. Médicas This hasThis article been accepted for publication andundergone peer full review but has § ‡ † Oyarzun CP,*Lev PR,* Korin L,* LaguensRP, Glembotsky AC,* Bluteau D, potential role for candidate RUNX1-targets Mechanismsunderlying platelet function defect in a pedigree with FPD/AML: ArticlePlatelets :Original - Article type Accepted :19-Feb-2014Date Received: 09-Dec-2013 Date This This article is protected by copyright.reserved. Allrights cite this article as an 'AcceptedArticle', doi:10.1111/jth.12550 between versionPleasedifferencesVersion may to this the andleadwhich ofRecord. been not through pagination the typesetting,copyediting, andproofreading process Université Paris-Sud.Université France Villejuif. Combatientes deMalvinasCombatientes Aires 3150.Buenos1427. Argentina MédicasInvestigaciones Alfredo Lanari. Universidad de Buenos Aires. CONICET. de Investigación. Instituto deHematología Departamento PaulaG. Heller. Corresponding author: INSERM, UMR1009. Equipe UMR1009.INSERM, Labellisée LigueContreleCancer. Gustave Roussy. Departamento de Patología.de Universidad Favaloro. Buenos Aires.Argentina Ecole Pratique desHautesEtudes (EPHE), Paris,France †‡ EspasandinYR,* Goette NP,* MartaMarin RF,* § Molinas FC,* Raslova H, MolinasFC,* Raslova † Heller PG* Heller Accepted Article activation and combined andcombined activation Platelet function defectPlatelet function in FPD/AML Short title and Lumi-aggregometry, Methods defects. in these involved genes by usingacandidategene we approach, to identify sought potential RUNX1-regulated anddefectiveabnormalities activation pathways theplatelet phenotype. addition, In to aimed contribution platelet todetermine the structuralWe of incompletely clarified. underlying Theremain plateletpredisposition. dysfunction mechanisms FPD/AML characterized by thrombocytopenia, platelet defect function and leukemia FPD/AML Background Summary Severe decrease in platelet aggregation, together with defective Results NF-E2 regulation by RUNX1. reporter assayluciferase andchromatin immunoprecipitation were performed to study FPD/AML pedigree. Expression levels of candidate geneswere and measured Email address:[email protected] Phone/Fax:+5411 452389 47 This This article is protected by copyright.reserved. Allrights α IIb β 3 integrinoutside-inin activation signalingwere and one of assessed members

is an inherited platelet disorder caused by germline disorder caused by germline platelet an inherited is α - and dense- granule content and release, plateletgranule content andrelease, - anddense- ultrastructure α δ storage pool deficiency was found. However, whileHowever, dense found. was storagepooldeficiency

RUNX1 mutation and α IIb β 3 integrin Accepted Article heightened diagnosticawareness andthan more forty have been families reported to RUNX1 neoplasms.heterozygous Germlinehematologic in mutations transcription factor thrombocytopenia, platelet function anddefect a lifelong risk development of of (FPD/AML) is an inheritedplatelet disorder characterized by autosomal dominant myelogenous leukemia acute platelet disorderwith to Familial predisposition Introduction human;protein, blood platelet disorder function platelet protein; RUNX1 FPD/AML; Keywords biogenesisand platelet function. RUNX-1-target NF-E2 as part of the network bymolecular regulates whichRUNX1 This process. byaspectsstudy this platelet function regulatingdiverse highlights of andRUNX1 orchestrates acomplex represents trait defect function platelet FPD/AML Conclusions NF-E2 expression. not butactivatemegakaryocytesand wild-type FPD/AML was RUNX1, ableto mutants, andMYL9. wasRAB27B RUNX1 shown tobind andotherderegulatedgenes included lysates, RNAand in platelet NF-E2 was shown impaired, reflecting altered outside-in signaling. A decreaseintranscription p45 factor towards decreased platelet spreading was while found, were reduced, granules markedly This This article is protected by copyright.reserved. Allrights underlie thisdisease [ 1 ] , which isnowbeing increasingly recognized due to α -granule content washeterogeneous. Atrend-granule tests; Rab27B NF-E2transcription factor; β 3 NF-E2 integrin wasphosphorylation promoter in primary Accepted Article cells, it playslymphoid and it roleinthe cells, anessential lineages megakaryocytic stemAlthough adulthematopoietic of the development. maintenance dispensablefor duringembryonic definitiveestablishment of hematopoiesis the crucialfor is isa hematopoieticRUNX1 transcriptionmaster whichfactor with dimerizes CBF function plateletFPD/AML patient,providing defective analternativeone explanation for represents a useful model to unravel the role of RUNX1 inplatelet andmegakaryocyte RUNX1 the roleof tounravel model a useful represents in platelet and FPD/AML isinvolved megakaryocyte formation development, function. it platelet phenotype. In this regard, impaired phenotype.this regard, platelet In storage pooldisease incompletely understood. Although haverevealed studies several presence the of bywhich mechanisms whileplatelet secretion defect, complex showa phenotype.others more The reported patients, cases. some among the pattern beseems In predominant to a study in recruitedtoUK genotypingdescribed the patients phenotyping andplatelet (GAPP) platelet predominant function disorder without history malignancy, of as recently bleeding diathesis orno individuals andhave affected some condition. developmild leukemia While displaymutation thisabnormality. FPD/AML representsa clinically heterogeneous of thisdisorder, itispresentlyof unknown whether withgermline all individuals constant beanalmost feature platelet to seems dysfunction of Although thepresence thrombocytopenia,degree of due thepresenceto of associated platelet function defect. individuals affected andtends to be severemore thanexpected according the to casesbeen insome reported have counts patients, and,althoughmoderate orevennormal low-normal presentin plateletmost Thrombocytopenia,characterizeddate. by isusually normal-sized platelets, to mild This This article is protected by copyright.reserved. Allrights [ 3 [ ] 8 . Likewise,. and spectrum severitythe platelet of varyfunctional abnormalities ] . [ 2 [ 4-7 ] , ontheotherhand, patients other may with present a RUNX1 ] , it, isunclearwhether abnormalitythis accounts for the fully mutations lead to the platelet function defect remain defect plateletfunction to the lead mutations α IIb [ β 2 3 ] . Severity of bleeding varies among. bleeding Severity varies of integrin activation has been integrinactivation shown in [ 9 ] RUNX1 , where, β and and δ -

Accepted Article MPL C- ( 12-lipoxygenase includingplatelet profiling, expression have beenfunction found be decreasedto in one FPD/AML patientby platelet chain ( formation formation inproplatelet well a block polyploidization, as as and megakaryocyte maturation decreased thrombocytopenia FPD/AML-related recently is dueto that have shown RUNX1 inand shRNA-induced knockdown primary etal Bluteau megakaryocytes, biology. By using acomplementary approachincluding study the ofpatient samples This This article is protected by copyright.reserved. Allrights myelodysplastic syndromemyelodysplastic anddied after AML Platelettransformation. studies thisfor Patient are described features Duringfollow-up,in Table patient 1. (P) II.2 developed current nomenclature (www.hgvs.org/mutnomen) RUNX1 apreviously from pedigree harbouringp.Thr219Argfs*8Four the described members Patients Patients andMethods abnormalities. functional we tosought identifyfurther genes potential involved RUNX1-regulated in platelet the pathways theplatelet to phenotype. by Inaddition, usinga gene candidate approach, activationand defective structuralabnormalities contribution platelet the of determine RUNX1-targets incompletely involvedremain characterized, study, inthis weto aimed Considering the that underlyingmechanisms FPD/AML platelet function defect and θ ( PKC receptor MYL9 mutation, previously p.Pro218fsX225renamed as mutation, according to and reported θ [ ) 10 [ 12-16 ) ] . [ [

10 11 RUNX1-targets involved in decreased platelet productionRUNX1-targets platelet involved indecreased includethe ] ] ] . Besides , myosin IIA ( IIA , myosin . MYL9 MYH9 , other RUNX1-target genes with a role in platelet ) and IIB ( MYH10 [ 11 ] , were included in this study. this includedin, were ) ALOX12

and myosin regulatory light ) and protein kinase

Accepted Article modified Tyrode’s buffer (134mM NaCl,12mM Tyrode’s (134mM NaHCO modified buffer was calculated. For PAC-1 Forand PRP P-selectin was calculated. analysis, (1x10 between intensity ratio fluorescence mean obtainedwith (MFI) andwithoutmepacrine a and analysis cytometry by flow temperature (RT) room followed at 30 during min. Na analyzed. The platelet population was identified with phycoerythrin-conjugatedCD42b.identifiedThe analyzed. plateletpopulationwas (Biopool)thrombin during 5 atRT. Aftermin. platelets werestimulation, and fixed (Molecularfibrinogen Probes, Eugene, OR,USA) and withstimulated 0.25U/mL Tyrode’s werewithmodified 10µg/mL buffer preincubated during 5 min. at RT. Toat RT.binding,1x10 fibrinogen measure during 5min. (BD San Biosciences, anti-P-selectin José, CA, USA) with andstimulated ADP 10µM preincubated with isothiocyanatefluorescein (FITC)-conjugated PAC-1 antibody or To evaluate dense granule content, PRP (5x10 densegranulecontent, To evaluate cytometryFlow assays Log Corp). concurrently wasrelease (Chrono-Lume, determined Chrono usingluciferin-luciferase ATP (Biopool, Bray,Ireland). and1uMepinephrine ADP ristocetin, 2µM 1,5mg/mL Havertown, PA, USA) after stimulation with 1mM arachidonicacid, collagen, 4ug/mL aggregation wasplatelet evaluatedusing alumi-aggregometer (Chrono Log Corp, plasma Platelet-rich 200g(PRP)by wasprepared at during and centrifugation 10 min. Light transmission aggregometry and ATPrelease Committee Ethics andsubjectsinformedwritten gaveconsent.Institutional bythe was before were approved transformation The MDS.performed patient study to This This article is protected by copyright.reserved. Allrights 2 HPO 4 , 1mM MgCl 2 , 1mM CaCl 1mM , 2 , 10mM, 5mM glucose, Hepes, pH=7.4), 7 /mL) was/mL) with labelled 2mM mepacrine 3

8 , KCl,2.9mM 0.34mM Alexa Fluor 488human /mL washed in /mL platelets 8 /mL) was diluted 1:10 in/mL) was diluted1:10 Accepted Article platelets wereplatelets classified according to numberthe of TSP1-positive granules into Plus, Göttingen, Germany)Axiostar observers. For by two analysis,independent and atleast were200 platelets under examined a fluorescence microscope (Zeiss tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin (Sigma-Aldrich) goatantibody.conjugated by anti-mouse wereidentifiedPlatelets labeling F-actin with anti-TSP1 antibodyimmunostaining(Sigma-Aldrich) withmouse followed by FITC- Platelet thrombospondin-1 (TSP1)contentwas onblood assessed by smears Immunofluorescenceanalysis for thrombospondin-1 Germany). Aldrich) and examined inatransmission electron (Zeiss, EM 900,microscope stained uranylwith (Riedel-d acetate Germany) (Sigma-Haen, AG, and leadcitrate were(Sigma-Aldrich, Thin inAralditeLouis, embedded MO,USA). Stsections and buffer,phosphate pH7.2, postfixed in1%rinsed, intetroxide osmium the buffer same plateletPRP ultrastructure,Towas evaluate with 5% fixed glutaraldehyde in0.1M Electron microscopy of platelets as units(MFU).expressed fluorescence mean inwasdays, normalizeddifferent variation and to control samples according MFI Forthis positive for comparison betweenmarker. conducted on measurements percentage platelets as of expressed platelets,whereas P-selectin was activated in obtained MFI from unstimulatedwas plateletssubstracted to binding after MFI obtained performedMeasurements were induplicate. To determine PAC-1 or fibrinogen binding, This This article is protected by copyright.reserved. Allrights displayinggranules andthosewithplatelets >5 ≤ 5 granules,asdescribed [ 18,19 ] .

Accepted Article study To adherent cells wereadherent lysed in buffer RIPA (50mM Tris-HCl, NaCl, 1% 150mM pH=8, were 60 atRT.added andincubatedfor Nonadherent min. cells and were removed were incubated withrabbit polyclonalwere incubated integrin anti-phospho platingwere after After on3%BSA-coatedprepared SDS-PAGE, plates. membranes SDS, 0.1% deoxycholate). P-40, Lysatescells non-adherentNonidet 1%sodium of Tyrosine phosphorylation of extension.lamellipodia asround, with andclassified filopodia-bearingmicroscope orplatelets a fluorescence Atleast(Sigma-Aldrich). were 200platelets analyzed examinersby underdifferent two with permeabilized FITC-labelled were andstained phalloidinfixed, Adherent platelets withcoated human 100 ug/mL fibrinogen (Sigma-Aldrich) during 30 min. atRT. Gene expression analysis plateletsin USA). with on was assessed duplicate membranes rabbit anti- antibodydetection. Protein chemiluminescence (ECL)rabbit loading andenhanced Cambridge, UK) (Abcam, plateletTo spreading, assess 5x10 spreadingPlatelet This This article is protected by copyright.reserved. Allrights NY), described Co, as Products PL, Pall Biomedical To plateletprepare RNA, PRP was filtered through a leukocyte reduction filter (Purecell platelets (3x10 platelets human albumin ug/mL andblocked 1% (BSA).with fibrinogen bovine serum Washed β 3 integrin phosphorylation,3 integrin 6-well polystyrene 8 /mL) /mL) in Tyrode’sresuspended containingbuffer modified

followed by horseradish-peroxidase (HRP)-conjugated anti- (HRP)-conjugated followed by horseradish-peroxidase β 3 integrin inplateletsintegrin 7 /mL platelets/mL were platedon glass coverslips [ 20 β

] plates were coated withplates were 100 coated , red blood cells were lysed, redbloodcells 3 integrin (Santa Cruz, CA, β 3 (Tyr773) antibody

1mM CaCl 2

Accepted Article rabbit IgG (Santarabbit Cruz Biotechnologies), followed secondary byHRP-conjugated described for preparation of platelet RNA, and washed platelets (2 x 10 x (2 plateletand washedplatelets preparation RNA, of for plasmaPlatelet-rich was subjected leukocyteto blood depletionandredas celllysis, Western blot analysis of platelet NF-E2 content efficiencytargetthecontrolgenes.amplification andinternal of Supplementary Table. Serial cDNA weredilutions toassessed ensure similar using SYBR® Green (Life Technologies), primer sequences are listed in PCRby relativereal-time to target Expression geneswereTechnologies, levelsof USA). NY, assessed intriplicate were performed in HEL cells, as previouslydescribed as cells, inHEL were performed Twoduplicate. independentexperiments were performed. system using detection SYBR® Green (Applied Saint-Aubin, Biosystems, France) in described using chromatin prepared human previously from performed megakaryocytes, as Billerica, using antibodyUSA) MA, anti–RUNX1 Abcam). (ab23980, Assays were Biotechnology,(Millipore were Upstate kit with assays ChIP performed assay a ChIP Chromatin Immunoprecipitation (ChIP)and promoter activity assays as 100%.set Meanvalues two from different experiments were calculated. blot,normal same assayed ratio of the percentage controls mean of as on expressed Aldrich). A ratio between NF-E2 and anti- reprobed withand then mouse ECL detection andantibody with gr/Lammonium 8.6 chloride and 1x10 This This article is protected by copyright.reserved. Allrights in Supplementary Table. [ [ 21 11 ] ] . Immunoprecipitated DNAwas analyzed onaPRISM® 7700sequence , , followed by SDS-PAGE. wereMembranes probed with anti-NF-E2 GAPDH in an iCycler (Bio Rad Life Science, CA, USA) β -actin by calculated was densitometry 9 platelets were lysed Trizol in (Life [ 21 ] . Primer sequenceslisted . are

Promoter activity assays activity Promoter 9 /mL) were/mL) lysed, as β -actin (Sigma

Accepted Article in response to 1µM epinephrine (datanot shown).1µM in responseto Mepacrine uptake was reducedin andabsent in PII.2 PIII.3 and decreased in PII.1, who also showed ATP absent release arachidonic responsewas 1mM in acid to while absent (Table ATP secretion 2), or secondaryof was ADP ATPseverelydecreased by aggregation. release triggered 2µM severedecrease from ranging in primary wave primary to preserved withlack response affected among or members familyduring sequentialevaluation patient, inthesame aggregation in traces response to ADP and arachidonic acidwere more variable orabsent, while toepinephrine was impaired response severely andcollagen always As patient each shown, 2. inTable valuesaredepicted and mean occasions for response to ristocetin was largely Tests werepreserved. performed on two to three ADP, of concentrations epinephrine, and arachidoniccollagen acid(Fig.1), while aggregationin response standard showedabnormalitiesPlatelet tests to marked Light transmission aggregometry and dense granule content and release Results Prism 5 (La Jolla, CA, USA) software. performed with InfoStat (Universidad Nacional deCórdoba, Argentina) and GraphPad usingpairedpairs days,on different control and patient samples handled in parallel analyzed were as variability in experimental conditions among flow cytometry performedmeasurements variances andand equality usingShapiro-Wilks F-test, of Torespectively. accountfor Mann-Whitney-Wilcoxon test, asappropriate. Variableswere analyzed for normality using was andcontrols Student´s performed betweenpatients Comparison Statistical analysis This This article is protected by copyright.reserved. Allrights (n=4) comparedpatients 0.61±0.21 tocontrols(n=4), 1.00±0.26vs. MFU, paired t- test, indicating impaired besides that, dense granule secretion, is there a t- test. P valueswere< 0.05 was considered Analysissignificant. P = 0.028, = 0.028, t- test or Accepted Article (Fig. 2BTableand D (Fig. 2),further indicatingimpaired in patients compared to controls, 0.55±0.10 compared in patients vs.1.00±0.19 MFU, decreasedwas thrombin U/mL 0.25 Accordingly, fibrinogenshown). binding inducedby to besimilarly ADP(data was found impairedµM not 5µM and20 to response patients, Intwo results. twice,least withsimilar the at was performed measurement patient, expression of normal despite 0.58±0.1 vs. 1.00±0.17 MFU, in ADP decreased controls, compared stimulated with was 10µM patients to platelets bothPAC-1aggregation, andbinding fibrinogen were PAC-1 bindingto evaluated. in the number dense of in the while granuleswas found, Markedwithdistended opencanalicular appearance. vacuolated and decrease system heterogeneity in plateletsize and shape,disorganized submembrane microfilaments two electron revealing abnormalities, patients was performed microscopyfor whetherTo evaluate Electron microscopy of platelets showed normal ornormal nearshowed normal granules,of while others as some devoid completely were heterogeneous, platelets assess the impaired contribution assess of Assessment of of Assessment similar twice, with results. was performed indensegranule and/orcontent.reduction Forpatient, number eachthemeasurement This This article is protected by copyright.reserved. Allrights Agonistto binding their receptors triggersplatelet inside-out signaling lead events that to α IIb β 3 integrin conformational change and exposure of binding sites for fibrinogen.sites for binding To integrin conformationalexposure of and change α IIb β 3 integrin activation RUNX1 α IIb α P -granule content (Fig. 3). germline leads germline mutation to platelet = 0.01, paired = 0.01, β 3 on the platelet surface (data not shown). each (data not For surface platelet 3on the α IIb β 3 integrin defective platelet activation to t- test (Fig. 2A andC test (Fig.2A Table 2), α IIb β 3 integrin activation. α -granule content was content -granule P = 0.04, paired

structural t- test Accepted Article and downstream signaling substrates. signalingPhosphorylationand downstream substrates. of Fibrinogen to ligation tyrosineleads phosphorylation of each difference parameterfor was notstatistically (Fig. 5A significant andB). 29.4±10.2 vs. 12.4±3 and 53±16.3 vs.86.6±1.95%, respectively,although the showing in lamellipodia patients compared to 17.5±11.5 controls, 0.9±0.5,vs. with in together roundordisplaying adecrease remaining only platelets filopodia, adherentto platelets fibrinogen platelets increasedpercentage revealed of an of Morphologic analysis formation. andlamellipodia filopodia leading tocell spreading, stabilize that cascades platelet aggregate the and cytoskeletontrigger reorganization 5C). 5C). platelets adherent toimmobilizedpatient fibrinogencompared control to (Fig. platelets granule deficiency, as deficiency, reported granule Five orless than 5 ordertoquantifyIn expression Immunofluorescence for This This article is protected by copyright.reserved. Allrights compared to controls (n=4), 38.7±16.8vs.68.5±16%, induced was (n=4)P-selectin expression ADP by decreasedin patients 20µM for representingplatelet amarker surface, to the membranes 4A and leads Platelet B). activation P-selectinredistribution to of from compared controls, to 15±2.3% 3.1±1%, vs. Mann-Whitney-Wilcoxon p=0.03, test (Fig. percentage platelets of with severe hemostasis in these patients. Fibrinogen binding to patients. inthese hemostasis wewhetherdefective exploredcontribute outside-inNext, abnormal to mechanisms Outside-in signaling responses α α -granules, immunofluorescence TSP1was-granules, performed. labelingfor granuleswas perplatelet usedasa stringent cut-off value for α -granule marker Thrombospondin-1-granule marker P-selectin and [ 18,22 19, α -granule reductionwas in patientsfound ] . A mild but significant increase inthe increase butsignificant . A mild α IIb P β = 0.008, 3 triggers downstream signalingtriggers downstream β 3 subunitcytoplasmatic tail β 3 integrin was in impaired integrin α

-granule degranulation. paired t -test. α -granule α - Accepted Article E2-regulated genes implicated in inside-out trafficking andvesicle dense granule packaging atrendtowards reducedlevels of we found Concerning granule NF-E2 genes likely target biogenesis, indefective be involved to NF-E2 downstream with effectors apossibleinthe platelet phenotype.studied role control levels 6B), (Fig. confirming that NF-E2 is downregulated in FPD/AML.we Next, NF-E2 inwas found three evaluated FPD/AML patients, ranging 33 to62%from of NF-E2 assessed content by blotinplatelet lysates.Western Aconsistent reduction in we levels, protein NF-E2 decreased of ledto geneexpression if reduced determine did potentially biologically reachnot relevant, statistical significance (Fig. 6A). To promoters alternative 1F transcripts were measured, which lead tothe same protein but are regulated by signaling alternative NF-E2 candidate targets with a role in witha role NF-E2 candidatetargets alternative induced integrinactivation nucleotide exchange factor To address whether NF-E2 is a direct tr whetherTo NF-E2 is adirect address Chromatin Immunoprecipitation (ChIP)and promoter activity assays in platelets, (Fig. 6A), as already indescribed FPD/AML samples shown). not a profound Besides,(data weconfirmed reduction MYL9RUNX1-target in [ which playskeywhich a roleinplatelet function wedefects, assessedfirst gene expression levels transcriptionof p45 factor NF-E2, To with identify candidateRUNX1-targets apossibleroleintheplatelet functional Expression ofcandidateplatelets RUNX1-targets in This This article is protected by copyright.reserved. Allrights 31 ] and thiol isomerase and ERP5 [ 24 ] and development for of normal granule content [ 26 ] . . We a found reduction in both isoforms, which, although

[ CALDAG-GEFI, which step a represents in critical agonist- 29, 30 [ 23, 32 ] , were normal whileexpression (Fig.6A), platelet of ] anscriptional target of RUNX1, we performed RUNX1, anscriptional of target , was below thedetection limit ourof assay [ α 23 IIb RAB27B, intracellular which regulates ] β , beingessential for [ α 3 27,28 activation, levels of activation, Rap1 guanine IIb β 3 activation, including caspase 12 includingcaspase activation, ] , Fig. 6A. With regards to NF- [ 25 [ ] 10,12 . Both Both NF-E2. 1Aand α ] . IIb

β 3 inside-out Accepted Article sites 3, 4 and 5 localized in the A and B PCR amplified regions, butBandA PCRamplified withanda lower 5 localizedinthe 4 3, sites megakaryocytes, we tested by these ChIP sites and showed thatalso RUNX1 binds bleeding disorder. thatactivation may contribute, together with to mild thrombocytopenia,moderate to this defect function platelet germline In genes involvedinthisprocess. the present study, we of effects the have studied a andaggregation, clarify inof thefunction platelet activation as wellinvolved asto dissect themechanisms to provide usefultools disorders platelet function Inherited Discussion efficiency than it binds to site2 (Fig. 7B). transcription starttranscription site (numbered 4,5and 6inFig. 7A) relevance, weofregion upstream clonedthispromoter the relevance, sitebinds to 2localized in F PCRamplified the region (Fig.7B). To test its functional wereassays primary performed in human RUNX1 and megakaryocytes that showed bindingRUNX1 sites(numbered 1 and 2 in Fig. 7A). Chromatin immunoprecipitation NF-E2activated in potential megakaryocytes,promoter andidentified main is the which promoter, i.e.withinpromoter, the and verapatients the region polycythemia adifferent HEL 1A NF-E2 of cellsin shown that RUNX1 binds the NF-E2 binds RUNX1 promoter the shown that exhibit this not transactivation property (Fig. RUNX1 bindDNA able are not whichR139X contrast, R174Qorto mutants, in CBF gene reporterperformed assays. wild-typeof Over-expression its RUNX1 cofactorwith an This This article is protected by copyright.reserved. Allrights in silico β increased luciferase activity transfection assays increased while, intransient HEL cells, in RUNX1 conserved sequence analysisconserved sequence mutation in several members of a FPD/AMLpedigree of a with inseveral members mutation severe [ 11

segment bpat segment –3376,–3235,and–3085 ] . We have shown abnormalities in platelet have shownmorphology inplatelet abnormalities . and We [ 33 ] 7C). Furthermore,7C). ithasbeen previously of its 2Kb 1A upstream promoter region,1A itspromoter 2Kb upstream of in vitro and in vivo in luciferase

granulocytes from [ 34

from exon1Afrom ] .

gene and In primaryIn [ 10 ] , did Accepted Article maturation together maturation together with a striking granule deficiency displaymice thrombocytopeniaprofound and arrest megakaryocytes alate in show plays a roleinkey both granulebiogenesis and inside-out signaling which besides regulating terminal megakaryocyte maturation and proplatelet formation, impaired SPD and To RUNX1-targets searchfor potentially in abnormalities, i.e.involved specific these defect.function contribute activation this phenotypeto thecomplexity highlights FPD/AML of platelet decreased wascontent and platelet dysfunction was normalproposed torely granule solely on been previously reported anotherfor FPD/AML patient, although in this case, dense combinedalthough α heterogeneous revealed ultrastructure previous inFPD/AMLfindings other pedigrees bycontent bothgranule confirmingand electron mepacrine staining microscopy, Regarding underlyingmechanisms dysfunction, we platelet decreaseddense found This This article is protected by copyright.reserved. Allrights study platelet function of in More recently, this model. theessential roleNF-E2 of in agonists response to in additional mechanism involvedadditional inmechanism plateletdysfunction. Abnormal was indicating anactivation shown, inside-outsignaling impaired that represents contribution thepotential inhemostasis. defect to Inaddition, defective the of establish to significance required FPD/AML inanother reported pedigree our with findings, partial described patientsfor with combined reductionin decreased, the and uniformly markedly -granule -granule TSP1marker confirmed the partial in reduction α IIb β 3 function α α IIb and β [ α 3 activation, we focused in transcription factor p45NF-E2, factor intranscription weactivation, focused 3 24 granuleplus dense granule deficiency hasbeenpreviously ] δ . However, the virtual absenceof platelets precludes the [ granuledeficiency was while found, 8 ] . . The finding thatboth SPD and impaired α α δ -granuleimmunofluorescence contentand for storage pool deficiency In(SPD). agreement [ 35,36 α -granule deficiency inFPD/AML andits ] [ . Study of additional willStudy be patients . of 4-7 ] [ . In addition, analysisIn . platelet of 25 α ] and failure tobindfibrinogen andfailure granules was variable, as α -granules. Therefore, α IIb . β δ NF-E2 knockout 3 activation has granuleswere α IIb β 3 integrin α IIb β 3 Accepted Article for FPD/AML-associatedfor wewereabnormalities, able decrease asaplausibleexplanation toidentify RAB27B Regarding NF-E2 downstream effectors with a possiblerole in thesespecific platelet megakaryocyte lineage. the of on the effects RUNX1 of key as a mediator NF-E2point to data these Altogether, physiologicaland describe a setting, binding new RUNX1 siteonthis promoter. relevant a which megakaryocytes, represent more in may human also primary addition,we In showbinding to thatRUNX1 th RUNX1 mutants FPD/AML work, we wild-typethis NF-E2 two confirm that andregulation by RUNX1 show and reduced NF-E2 expression was found in conditional RUNX-1 deficient mice as a RUNX1-targetidentified in fetal livermouse m phenotype. addition, In decreased could NF-E2 also in participate altered platelet the platelet function to contribute could factor transcription inthis decrease the strongly samples, that downregulation NF-E2 in suggesting patient leads to FPD/AML has been shown been regulated byhas RUNX1 tobepositively expression vera andNF-E2 inpolycythemia HELand granulocytes cells demonstrated RUNX1biogenesis inthisdisorder. bindingthe to NF-E2 1A promoter was previously promoter hasbeenpromoter described in CMK cells Besides being regulatedbyformation. occupancy theRAB27B NF-E2, RUNX1 at biogenesisplatelet could alsoby bedirectlycandidate NF-E2-targetsConcerningcould with RUNX1. regulated a selectin expression selectin show thrombocytopeniawhich mild andreduced thrombin-induced aggregation andP- wasplatelet function revealed inexpressing ahypomorphicmice NF-E2 mutation, This This article is protected by copyright.reserved. Allrights [ 23 [ 28 ] . In .the current In study, we show that ] , possibly coordinatinggranuletransport and proplatelet δ -SPD. This small GTPase regulates GTPase This small -SPD. [ 10 ] , lose the ability to mediate NF-E2 promoter activation. NF-E2 lose theabilitymediate , to [ 38 e NF-E2 proximal 1Apromotere NF-E2proximal occurs ] , thepossibilityraising , that RAB27B egakaryocytes by ChIP-seq ChIP-seq by egakaryocytes [ 34 ] . RUNX1

More recently, NF-E2 was δ -granule number and number -granule germline mutation analysis [ 37 ] . In . In Accepted Article RUNX1 could contribute to defective inside-out signaling in this setting. In this regard, thisregard, In in thissetting. inside-outsignaling todefective couldcontribute RUNX1 Alternatively, notyet other, identified, NF-E2-targetsdirectly or genes by regulated genebe expressionanalysisrewarding.scenario, more inmegakaryocytes might [ fiber andoutside-infiber formation with demonstrated signaling, in as mice megakaryocyte- shapein plateletcontractile such change, asplatelet involved several functions, stress inrole platelet function plays akey biogenesis, MYL9 inplatelet itsrole Besides thisdefect. to contribute may only to [ RUNX1-target PKC incontrast to byand findings al Sun et RUNX1, recently, NF-E2 has been identified as a direct regulator of regulator recently, hasbeenidentified of asadirect RUNX1, NF-E2 and platelets in RUNX1-target MYL9reduction has been previously shown in megakaryocytes demonstrated in FPD/AML. Among genes be dysregulated,to found a profound genesDysregulation of involvedincytoskeleton rearrangementpreviouslyhas been megakaryocyte. helpmay the regulated gene expressionprogram dissect byin the RUNX1 for theobservedresponsible phenotypes. Further potential targetsstudy of RUNX-1 gene be onemay RUNX1-regulated than in more reduction plausible that is it beenshown behas to a regulatormaster of megakaryocyte program, geneexpression signalinginside-out triggered by GPVI and PAR agonists signalingout was of ruled caspase 12 while out, theinvolvement role in This This article is protected by copyright.reserved. Allrights 23 23,32 ] . The profound decrease in MYL9 in found FPD/AML may be thus notascribed, ] α couldnot beascertained because of their low plateletexpression. In this RUNX1 IIb β 3 activation, the potential implication of CALDAG-GEFI in defective inside- CALDAG-GEFI of defective potential implication in the 3 activation, [ mutation, but also, to also, but reduced NF-E2 to study, inthis levels whichmutation, found 12 ] and confirmed in platelet samples from this pedigree. pedigree. in Besidesthis from plateletsamples and confirmed θ , by, not PCR(data real-time shown),whichimplicated in hasbeen [ 40 ] , through , through activity. regulation of ismyosin motor Myosin [ 13 ] , we did not find decreased we not , find platelet did levelsof [ 39 ] . Considering that RUNX1 . Considering that MYL9 [ 31 ] andERP5 expression [ 10 ]

Accepted Article results and wrote the paper; D. Bluteau: designed and performed luciferase assay and D. Bluteau: designedandperformed wrote paper; and the results experiments, Glembotsky analyzed discusseddata, andperformed A. C. designed Addendum function. These to data currentmay contribute understanding the of platelet basismolecular of inthe platelet andeffects megakaryocyteformation lineageandregulates function. its exerts byasnetwork the of NF-E2 which RUNX1 molecular part RUNX1-target this study Inaddition, this by aspects process. highlights of function regulating diverse contributeabnormalities tothiscomplex andtrait orchestrates RUNX1 that platelet defectreveals that pathwaysplatelet FPD/AML function underlie multiple activation plateletstructuralboth abnormalities and in that defects finding conclusion,the In in platelet aggregation and spreading, in FPD/AML pathogenesis. of the points to key involvement defect this signalingoutside-in eventsin FPD/AML. Together with inside-out abnormal signaling, indicatingabnormalsamples, was inpatient but platelets responseimpaired this control Furthermore, adhesion to a fibrinogen triggeredmatrix integrin orround filopodia-bearing platelets and decreased lamellipodia extension. pattern an abnormal spreadingof platelets adherent to for fibrinogen, with increased andfound phenotypeFPD/AML patients in withisa similar associated deficiency MYL9 spreadingon fibrinogen cell impaired induces cells inHEL knock-down RUNX1 to secondary MYL9levels decreased addition, signaling thrombusoutside-in growth anddecreased under flow conditions restricted This This article is protected by copyright.reserved. Allrights experimentsperformed Lev R. andP. C.P. Korin MarinOyarzun, L. Marta, R.F. Goette, experiments, analyzedChIP dataand Y. results; N.P. R.Espasandin, discussed

myh9 inactivation, which stress inactivation, displayabnormal impairedfiber formation, α IIb β [ 3 integrin, the major receptor involved the 3 integrin, major 15 ] . We therefore whether assessed . We β 3 phosphorylationin [ 41 ] . In

Accepted Article Poon Leber B, MC, SinclairJohnson GD, PR, Macheta A, Yin MJ,Lister Barnett JA, Owen Koochin DL,Smith JM, Toze SC, A, CA, Stevens 2. Jackson CL, Forrest CJ, Genet 1999; 23: 166-75. Nat leukaemia. propensitydevelop acute plateletmyelogenous disorderwith to familial Bushweller Gewirtz LiFP. NA, Haploinsufficiency of C, JH, AM, Speck D, Willman L, Kurnit Busque DC, Roy Loh M,FelixC, R, Hock Haworth C, IC, Resende HutchingsSullivan D, Tan J, SongLegare RD, MG,S, X,Kufrin Ratajczak 1. WJ, References labelliséeRaslova). 2013,H. (équipe (ANR-jeunes Rasl Recherche chercheurs,H. Agence Nationalebyde la Heller);P. the grants and from French (PICT01931, Agencia the and from Tecnológica Nacionaly dePromoción (ANPCyT)Científica CientíficasyTécnicas Heller) (PIP11220090100213,P. (CONICET) Investigaciones workThis was supported by Argentine grants thefrom Consejo Nacional de Acknowledgements editing and final approval. contributed to analyzedmanuscript andwrote authors thepaper.All experiments, data and study,wrote thepaper; P. supervisedG. Hellerdesigned performed the study experiments, analyzed anddata Raslovacontributed to design,designed H. results; samples F.C. Molinasclinical anddiscussed and providedpatient results; data LaguensR. P. and discussed results; performed electron microscopy and interpreted This This article is protected by copyright.reserved. Allrights with disorder platelet propensity112: 2008;to myeloid Blood. malignancy. 4639-45. TA, Fitzgibbon Fivewith new J. causingpedigrees inherited RUNX1 mutations familial

ova) and from the Ligue contre le Cancer lethe Liguecontre ova) andfrom CBFA2 causes

Accepted Article impaired pleckstrin phosphorylation.impaired Blood1996; 1368-76.87: with patient IIb-IIIa ina glycoprotein of activation signaltransduction-dependent out Gabbeta J,Yang8. McLaneSunX, L, Niewiarowski MA, Rao S, AK. Abnormal inside- 33:e264-6.2011; thrombocytopenia with associated normal-sized platelets.JPediatr Hematol Oncol propensity toacute myeloid leukemia in caseof unexplained inherited Dysmegakaryopoiesis, an earlyclue for a diagnosisfamilial of plateletdisorderwith Latger-Cannard V,7. T, Lecompte Favier JonveauxP, Philippe R. C, withdisorder propensity todevelop myeloid malignancies. Blood. 2001; 98: 2856-8. Pearson P, Lokhorst H. A novel CBFA2 single-nucleotide mutation in plateletfamilial Solinge BuijsBorst PoddighevanA, P, R, van 6. W, E,VerdonckL, A, Hagenbeek Wijk Eur J Genet.Hum 2008;16: 1014-8. point haploinsufficiency:germline RUNX1 tolarge mutations from genomic deletions. LecompteGrégoire MJ, Bordigoni P, T, Jonveaux P.ClinicalphenotypeLeheup B, of Béri-Dexheimer M,Latger-Cannard RothV, Bonnet5. P, Philippe C,Chambon V, C, 246-55. with ahighincidenceassociated acute of leukaemia. myeloid Br J Haematol 1991;79: D,GreenbergWalz AH, Ray LG. M, Israels Inherited platelet-storage pool deficiency IsraelsED,SJ, SchroederAJ, BishopLL, McNicolA, Israels GerrardJM, ML, Beattie 4. doi:10.1182/blood-2013-06-506873. defects. densegranule bleeding excessive secretion Blood andplatelet with 2013; FLI1 in of andRUNX1 families M, SP,mutations DalyME. Enrichment Makris Watson JT,CollinsTalks Motwani K, Horner PW, LeoVC, J, Wilde K, K, Macfarlane MA, Stockley D,3. Lowe Bem J, Morgan NV, B, GC, Lordkipanidzé Simpson M, Dawood This This article is protected by copyright.reserved. Allrights Accepted Article Regulation platelet of lightmyosin chain (MYL9) by implications RUNX1: for Mao Jalagadugula Dhanasekaran RaoAK. G,KaurGoldfinger LE, G, 15. DN, ThrombRUNX1.2011;of Biol Arterioscler Vasc 921-7. 31: target a transcriptional is PRKCQ RUNX1 C-theta human mutation: deficiencywith kinase G, Jalagadugula DhanasekaranG,proteinG, Mao Kaur Platelet RaoAK. 14. DN, 103:948-54.2004; activation. Blood GPIIb-IIIa byphosphorylation:a rolein CBFA2proteins regulated play PKC-theta and impaired receptor-mediated activation of GPIIb-IIIa and pleckstrin plateletwith CBFA2decreased of Association mutation AK. Rao MaoG, SunL, 13. 2007; Haemost Thromb 146-54.5: and insights CBFA2/RUNX1 dysfunction mutation: platelet from expression profiling. J regulatorygenes chainmyosin polypeptide(MYL9) with andother light platelet Sun L,12. Gorospe JR, Hoffman EP, Rao AK. Decreased platelet expression of novel AML1 Bloodmutation. 2005; 105: 4664-70. anda leukemia with to acute myelogenous disorder predisposition platelet familial with Kornblihtt LI, Drachman JG,Molinas FC. Low Mpl expressionreceptor ina pedigree RF, Marta CL,Pirola Heller CJ, 11. Gandhi PG,Glembotsky MJ,Cummings AC, Blood to deregulated linked myosin expression. II 2708-18.120: 2012; isDysmegakaryopoiesis FPD/AML with pedigrees constitutional RUNX1 of mutations Heller R,RaslovaH. Vainchenker Favier PG,Debili N, Alessi MC, W, Nurden P,P, Rameau Gilles L, Balayn A, N, Glembotsky Bluteau D, Raimbault AC, 10. andfunction a role in adult hematopoiesis. Kurokawa9. M. This This article is protected by copyright.reserved. Allrights 6037-45.116: thrombocytopenia andplatelet dysfunction in haplodeficiency. Blood2010; RUNX1

AML1/Runx1 as a versatile regulator of hematopoiesis: regulation of itsof regulation hematopoiesis: of regulator aversatile as AML1/Runx1

Int J Int Hematol

2006; 84: 136-42. Accepted Article platelet phenotype and NBEAL2 genotype in patients withthrombocytopenia congenital genotype patients phenotype NBEAL2 in platelet and Podda M, AC, betweenGM, Glembotsky A.Correlation Balduini CL,Savoia Cattaneo De RoccoD, NorisP, Heller N, PG, Pujol-Moix Pecci A,DeCandiaE, R, Bottega 22. megakaryocyte andisregulated maturation byBlood 2008;111: AML-1. 4081-91. Debili N,Vainchenker RaslovaH. endomitotic P19INK4D W, links arrest and Bluteau C, D,Cordette-LagardeFavier Gilles Guièze F, Larbret R, V,Lacout 21. L, R, of possess thesoluble IL-6platelets 2005; form Cytokine receptor. 29:13-7. Normal FC. Molinas CJ, Pirola CD, Chazarreta PR, Lev NP, Goette RF, Marta 20. ofcountry. adeveloping setting J Thromb Haemost 10:1653-61. 2012; collaboration asatool for patients diagnosisof withinherited thrombocytopenia inthe Noris International P,SavoiaNegro F, BalduiniFC, Molinas HellerPG. A, NP, CL, YR,GoetteEspasandin GnanC, D, DeRocco Pecci A, RF, GlembotskyMarta AC, 19. and relatives. Jpatient four Thromb Haemost2007;5: 551-9. correlationsyndrome: between alpha-granule defect theplatelet andthe in content the ofprotease-activatednovel case agray and thrombin in agonists receptors platelet platelet responsiveness Defective BalduiniI, Landolfi to R,S, CoughlinLazzareschi CL. DeCandia18. E,Pecci R,A, Ciabattoni G,De Rutella Yao-WuCristofaro Z,S, 1530-7. 91:2006; Haematologica leukemia. myeloid with acute patients thrombocytopenic 17. K, Cook RJ, Webert Sigouin bleeding HeddleRebulla The CS, of P, in NM. risk Blood 2010;115: 3128-35. haplodeficiency.human RUNX1 (ALOX12): 12-lipoxygenasegene studiesin platelet A2regulates binding RUNX1/core factor Rao AK. G,JalagadugulaMao G, G, Kaur 16. This This article is protected by copyright.reserved. Allrights and α -granule deficiency. Haematologica 2013; 98: 868-74. Accepted Article 30. Crittenden JR, Bergmeier CrittendenJR, Y, Zhang30. LiangY, Piffath CL, DD,Housman W, Wagner integrin signaling. ProcNatl Acadin US Sci 12819–12824.99: A2002; Shattil SJ.Megakaryocytesembryonic derived from CalDAG-GEFI cellsimplicatestem 29. Eto K, Murphy R,KerriganSW, Bertoni A, Stuhlmann H,Nakano T, Leavitt AD, 5872-7. 104: A 2007; U S SciNatl Acad dense granules.Proc platelet of andsecretion number T, Tolmachova Abrink M,28. Authi Futter CE, Rab27b KS, Seabra MC. regulates Blood 2003;102: 3970-79 platelet formation. pathways of NF-E2–dependent inRab27b role for A Shivdasani RA. Tiwari27. S, JE, BarraItaliano DE, Mules EH, Novak EK, Swank RT, Seabra MC, alternative promoter.intronic 2000 JBiol 10567-76. Chem 275: ; Regulation of mouse p45 NF-E2 transcription by anerythroid-specific GATA-dependent Moi Ottolenghi Giglioni P, L, S, Mastrangelo B. Moroni Cairns E, 26. Razzini R, T, 695-704. the actions thrombopoietin/MGDFof in megakaryocyte development. Cell 1995;81: platelet independent SH.Transcription formation NF-E2Orkin factor for of isrequired Hunt CW,Rosenblatt MF,Zucker-Franklin P,SarisCJ, Jackson Shivdasani RA, D, 25. NF-E2 inintegrinalphaIIb beta3 signaling. CellBiol 147:1419-30. J 1999; ShattilA, S. Leavitt Primarya G, for transcription factor revealrole megakaryocytes G, Murphy Ybarrondo B, D, White V, Baron Wilcox S, AidoudiA, RitchieM, Shiraga 24. FunctionofNormal Platelets. MolCellBiol 2013;2659-70. 33: A, Bresnick Yamamoto EH, M, Motohashi H. NF-E2 p45 Is Important for Establishing Sarai S, Fujii R, Takayama-TsujimotoAburatani H, H, Satoh Fujita M, L, Gutiérrez 23. This This article is protected by copyright.reserved. Allrights Natthrombus formation. 2004; 10: 982-6. Med Graybielintegrates DE, signalingCalDAG-GEFI AM. platelet aggregationand for Accepted Article 38. Pencovich N, Jaschek R, Tanay interactionsof combinatorial Groner Y. R, A, Dynamic Jaschek PencovichN, 38. maturation. PLoS One2013; 8:e64248. Runx1transcriptionallymegakaryocytic of regulatesmouse function autonomous DickenAmit J,J,Tanay PencovichN,Lotem A, JaschekR, 37. Y. A,Groner Cell- pathogenesis. ofimplications Bloodfor mechanisms 1364-72. 2002;99: plateletdisorderwithfamilial dominant predispositiontoacute myelogenous leukemia: in analysesmutations In known andnovelRUNX1/AML1 vitro HS. of Horwitz M,Scott Benson KF, Raskind Rossier C,AntonarakisWH, SE, Israels S, McNicol A, H, Weiss Michaud Osato F,36. J, GM,Yanagida M,Cottles ShigesadaWu M,AsouN, Y, K, Ito storagealpha delta pool 919-29. 87:J ClinInvest deficiency. 1991; expression alpha-granuleof protein GMP-140membrane two plateletin oneof types of Lages 35. B, Shattil contentSJ, Bainton DF, Decreased HJ. and surfaceWeiss overexpression of NF-E2. Blood 2010; 116: 254-66. with myeloproliferative neoplasms and JAK2V617F-independentmediates overexpressed inpatients AML1 is HL. Pahl EO,Schwemmers Hexner S, 34. W, Wang BMC genomes. Bioinformatics 2005; 6: 79. computational identification putative transcriptionof bindingsitesfactor in multiple the for engine a search MAPPER:A. Riva Kohane IS, VD, Marinescu 33. forrole thethiolisomerase proteinERP5in platelet function. Blood2005; 105: 1500-7. Jordan32. PA, Stevens JM, Hubbard GP, Barrett NE, Sage T, Authi KS, Gibbins JM. A 2004; Bloodactivation. 104: 1327-34. between link G-protein-coupleddevelopmental andintegrinalphaIIbbeta3 receptors a Caspase-12: AD. Leavitt SJ, Shattil RP, Murphy Gaur M, Kerrigan SW, 31. This This article is protected by copyright.reserved. Allrights models. Blood 2011; 117: e1-14. RUNX1 and cooperating partners regulates megakaryocytic in differentiation cell line Accepted Article bleeding; 1, petechiae; 2,1, gross and 4, 3 bleeding; petechiae; and debilitating blood loss, respectively.mild, wasBleeding assessed according to bleedingWHO scale values Reference MPV, 6.2-10.4 for fL plateletMPV, M,female; F, volume; male; WHO, mean Organization. Health World Table 1. Patient clinical and laboratory features aggregation platelet preserving secretion. and Blood2007; 110:3183-91. Megakaryocyte-restrictedC. MYH9 inactivation dramatically hemostasis affects while Gratacap J,TiedtWeber R, MP, Cazenave SeverinS, JP,LanzaGachet F,Skoda R, LéonC,Eckly41. C, Jourdain B,Aleil Freund B, M,Ravanat M,NonneC, A,Hechler 2591984; 9826-31. phosphorylation in theinitiationof the platelet shape changeresponse. J Biol Chem. role myosin of a for G. Evidence Stewart M, Rigmaiden IR, Molish DanielJL, 40. Bloodisoform. 2009 113: 2557-2567. C-{theta} lacking protein activation platelets of kinase Impaired Kunapuli. and SatyaP. Yamini Todd S.Bynagari, Soochong Kamala Bhavaraju, Kim Getz, Jr, Bela Nagy, 39. This This article is protected by copyright.reserved. Allrights II32 agtro I29 . stable stable 2 2 7.5 6.6 98 137 II.2 of daughter II.1 of son F M 22 25 PIII.3 PIII.1 I. 7Fpooiu 2923MDS/AML stable 3 3 9.2 8 72 145 propositus II.2 of sister F F 37 45 PII.2 PII.1 (years) A g e Gender (F/M) member Famil y

Platelet count count Platelet (x10 9 /L) MPV (fL) [ 17 (WHO score) (WHO ] : grade: 0 indicates no Bleedin g

Disease status status Disease at follow-up

Accepted Article patients and patients controls) show decreased binding in patients compared tocontrols. Alexa Fluor488-conjugatedinduced washed fibrinogen (n=3 to binding platelets for (B) thrombin- andcontrols) and patients (n=4 for PRP PAC-1 bindingto conjugated x 10 shown. are Mean±SD in values healthy (n=40) weresubjects 0.72 ± 0.40 FPD/AML patients ATP release values normalized to platelet count plateletATP x10 normalized to releasevalues (1 75±9, 78±9.5, 77±11, 79±982±8.6, and respectively. ristocetin 1.5 mg/mL is shown. Mean±SD (%) in healthy values controls(n=60)were arachidonic acidand collagen,1mM ADP, epinephrine,4µg/mL 1µM to2µM response Mean±SDlight in aspercentage expressed platelettransmission, aggregation, maximal arachidonicacid; ND,AA, FBG, done; not fibrinogen. Table 2.Platelet aggregation tests, ATP release and This This article is protected by copyright.reserved. Allrights PAC-1 and fibrinogen PAC-1 andfibrinogen bindingwere of a percentage expressed as simultaneously Figure 2. (C)compared to acontrolsample. decreasesevere inplatelet aggregation patients (PII.1, PII.2,for PIII.1, PIII.3), as patients. Figure 1. Figure legends assayed control sample. II13 5±08±74±575 D5. 46.7 51.8 69.2 ND 58 50 ± 0 4± 5.7 0 8 ± 7 59.7 ± 23.3 33.7 ± 40.8 21.3 ± 5.5 15 ± 0 18.7 ± 1.5 34.3 ± 8.7 30± 3 PIII.3 PIII.1 I. 93±550±01 135. . 4ND 50.2 44 64.8 0 0.11 52.3 ± 9.2 8± 11.3 66 ± 0 28.5 ± 40.3 13 ± 4 20 ± 0 0 ± 0 14 ± 6.4 19.3 ± 5.5 31 ± 16 PII.2 PII.1 8 /mL platelets./mL

ADP Epinephrine Collagen AA Ristocetin Ristocetin AA Collagen Epinephrine ADP Representative traces in responsetodifferent asindicated, showagonists, Flow cytometry analysis of integrin Flow of analysis cytometry Platelet aggregation bytransmission aggregometry light in FPD/AML Platelet aggregationPlatelet (%)

α IIb β 3 activation. (A)activation. ATP release ATP release 8 /mL) in response to 2 µM ADPto 2µM in response /mL) ( η α mole) IIb β 3 integrin activation in (% of control) of (%

ADP-induced-FITC- PAC-1 η mole ATP/ ATP/ 1 mole (% of control) FBG-binding

Accepted Article Figure 5. TSP1-positive single Scalebar:granule. 5µm. on fibrinogen-coated coverslips andactin fibers were labelledwith FITC-phalloidin. TSP1 displaying normal platelets some with panel),(right patient inthe is heterogeneous pattern staining the while pattern, granular Representative image of platelets a from healthy individual panel) (left display a packed ± are expressed asmean SEM,(n=10).Data * tocontrols compared value cut-off for stringent of plateletspercentage harbouring wereidentified by TRITC-conjugated platelets phalloidin(red (A) fluorescence). The antibody secondary followed by FITC-conjugated antibodyfluorescence) and (green (TSP1). Blood were permeabilized, smears fixed, and stained withTSP1 primary Figure 4. granule content. normal bar:1.1µm. Scale (B)Representative granules a image from decrease avacuolatedappearance, with inalpha dense granulesandabsence of sample showingpatient roundplatelets, enlargement of the open canalicular system Figure 3. representsgraysolid binding unstimulated to platelets. trace healthystudied (left control panel) is depicted by the open black histogram, while the and asimultaneouslypanel) (right aFPD/AML patient from binding platelets induced to * andstimulation are data as expressed unitsfluorescence mean (MFU) (mean ± SEM), Binding was tounstimulated platelets values from substracted obtained agonist after This This article is protected by copyright.reserved. Allrights decreased while is extension tocompared lamellipodia displaying only filopodia, higher (n=3)showpercentageremaining Patients round or platelets for atrend of P < 0.05. Representative (C) PAC-1 and (D (C) PAC-1 Representative < 0.05. Outside-in signaling in events FPD/AML platelets. Immunofluorescence labeling for Electron microscopy of blood platelets. (A) Representative image from a (A)Representative bloodplatelets. Electron from image microscopy of α -granule deficiency, wasin increased patients (n=4) ≤ 5 TSP1-positive granules,which representsa5 TSP1-positive

healthy control showing a platelet with showingaplatelet healthy control content while another (arrow) shows a while (arrow) shows another content ) fibrinogen binding analysis. Agonist- analysis. binding) fibrinogen α -granule marker Thrombospondin-1 marker -granule

(A) Platelets were plated P < 0.05. (B) Accepted Article numbers site. start transcription region.The the promoter putative arrow represents human Figure 7. (PII.1, PIII.1,patients PIII.3) C2, compared controlsto C3). (C1, experimentsshows levelsin NF-E2 blot two of Representative decreased different anti- protein loading, reprobedwith were membranes To assess ECL detection. with anti-NF-E2probed secondaryantibody followed by antibody HRP-conjugated and NF-E2 content. ** SEM; profound reduction of RUNX1-target MYL9 is shown. addition, a In and controls), wasCalDAG-GEFI preserved. while expression patients NF-E2towards targets, (n=4 atrend reduced RAB27Band 1F.Among was for found Patients show decreased expression of transcription p45 NF-E2,factor 1A isoforms byPCR real-time triplicate (n=3) patients inFPD/AMLcompared tocontrols (n=3). genes levelstarget analysis. of Expression relativewereGAPDH to assessed in Figure 6. shown in(ii). is bluntedinresponse platelets. Densitometric analysispatient band intensity of is to immobilizedcomparedadherence platelets, non-adherent fibrinogen while to the integrin antibody. Controlplatelets show enhanced (Tyr773)anti-phospho with wereSDS-PAGE to bufferstopped andlysates probedwith and subjected lysis fibrinogen-coated plates 60for After min. removal of non-adherent cells, was reaction were platelets phosphorylation layeredover Washed panelinintegrin isshown (i). and patient(right panel)(C) Scale sample. bar= 5µm panel)a control (left platelets in fibrinogen adherentshowing to image of morphology ± (n=3). areexpressed as mean Data controls SEM, This This article is protected by copyright.reserved. Allrights designate genomic positions designate inUCSC genomic hg18assembly P NF-E2 (A)regulation by Schematic RUNX1. representation theNF-E2of < 0.01 for MYL9;< 0.01 for Analysis of inplatelets. candidate RUNX1-targets Analysis of

Platelet lysates wereto subjected SDS-PAGE were and membranes β 3 integrin antibody. Protein loading was assessedwith anti– P > 0.05 for allother genes. (B) blotWestern analysisof β 3 phosphorylationintegrin upon

Data depictedare by ±mean P

Western blot analysis of analysis blot of Western > 0.05.(B) Representative

(A) Gene expression β -actin. Italic β β 3 3 Accepted Article immunoprecipitation assay.immunoprecipitation analysis ChIP CD41 in mature and binding RUNX1 was site putative usedasanegative control. analysisChIP in used amplicons of positions genomic bars(A-F) binding (http://bio.chip.org/mapper/). designate Black sites RUNX1 putative represent SD of in *represent twothe each experiments, triplicate. showhistograms onerepresentative experiment of two, each intriplicate. Error bars Thevector. MPI empty with an by transfection constant DNA was kept transfected amount of total alone. NF-E2 The promoter Luc construct with the transfected CBFcontaining R174Q RUNX1 cDNAs andpEF6/V5-His-TOPO R139X typeor RUNX1 vector (WT), containing wild MPI vector RUNX1 cells with500ngof cotransfection HEL of transient by performed assaywas Luciferase N°2. containing bindingsite RUNX1 (NFE2-Luc) * triplicate. orIgG.immunoprecipitation usinganti-RUNX1 bars Error representQ-PCR SD of region.immunoprecipitated The thepercentage represents input after histogram of ableto sets areamplify this both EandFprimer DNA and fragments 200bp afterChIP experiment generatesabout Sonication hg18). nt, 982728 –52982733 52 predicted withsets primerwas performed (NF-E2_A (http://genome.ucsc.edu/). Gray boxes numbered from 1 to 6 designate1 Gray to (http://genome.ucsc.edu/). boxes numbered from This This article is protected by copyright.reserved. Allrights in silico P < 0.05. (C) NFE2-promoter luciferase assayNFE2-promoterwithwild-type promoter luciferase (C) 0.05. β . The binding. RUNX1 site is FN°2 localized inside (between amplicon . Luciferase levels are shown as fold change relative to cells changerelativeto fold levels are shownas Luciferase . to _F) directed toward RUNX1–binding sites . C amplicon does not contain doesnot amplicon C P < 0.05. + CD42 + megakaryocytes

(B) Chromatin in silico

Accepted Article

This This article is protected by copyright.reserved. Allrights

Accepted Article

This This article is protected by copyright.reserved. Allrights

Accepted Article

This This article is protected by copyright.reserved. Allrights

Accepted Article This This article is protected by copyright.reserved. Allrights