Accepted Article Scientific category: Title:Runningan endocytic CLEC4MFVIII receptor is for 51References: Tables: 0 6Figures: Text: 4167 Abstract: 250 Email: [email protected] +1 Fax: 548-1356(613) Tel: +1 (613) 548-1304 CanadaK7L 3N6, OntarioKingston, Laboratory,Richardson University Queen’s Street Stuart 88 Dr. David Lillicrap Correspondence: †Department Canada Queen's Medicine, of Kingston, University, ON, Canada *Department Pathologyof Medicine, Queen's Molecular and ON, Kingston, University, Authors: This by is protectedarticle reserved. Allrights copyright. doi: 10.1111/jth.14404 lead to differences between this version and the throughbeen the copyediting, pagination typesetting, which process, may and proofreading hasThis article been accepted publicationfor andundergone peerfull but review not has Biology Vascular - Article :Original type Article Theendothelial clearancelectin receptorCLEC4Mbinds and internalizes Factor VIII in Swystun LL*, Notley C*, Georgescu I*, Lai JD*, Nesbitt K*, James PD†, Lillicrap D* NesbittK*,I*, Lillicrap PD†, JamesSwystun LaiJD*, LL*, Georgescu Notley C*,

Thrombosis and Hemostasis Thrombosis DR DEREK LAI JESSE : (Orcid ID 0000-0003-2916-2954) VWF-dependent and -independent manner a Version of Record. Please Version as this cite ofRecord. article Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. byendosomes catabolism for lysosomes. coated pit-dependent mechanism, inresulting transportFVIII of earlyfrom andlate mannose-exposed glycans. N-linked FVIIImediated CLEC4M internalization viaa clathrin- -independent FVIII CLEC4M-binding was on dependent to recombinant mechanisms. bound andinternalized and plasma-derived recombinant FVIII VWF-dependent through and evaluated assays. between Interactions andCLEC4M FVIII orliver sinusoidal endothelialwere cells CLEC4M with interactions were cell evaluatedby basedandsolid aninvitro phase binding and CLEC4M was characterized in the presence or absence of VWF. Methods: FVIII althoughVWF, itsinfluence is unknown. onFVIII Objectives: The interaction between FVIII endothelial CLEC4M hasbeen previously characterized as a clearancereceptor for FVIIIboundand VWF-freeare incompletely liver The expressed uncharacterized. human FVIIIquantitative Thedeficiencies. pathwaysreceptor regulate that clearancethe VWF- of some of pathological basis rapidand underlies the is FVIII VWF-free pathways.of Clearance likely byVWF-dependent is cleared FVIII non-covalent complex, andthemajority of Background: von (VWF) factor Willebrand and VIII (FVIII)factor circulate plasma in the as a Abstract: Essentials: glycansFVIIIpresence intheofabsence on or VWF. with interacts receptorthat mannose-exposed clearance novel is a CLEC4Mthat suggest TheseConclusions: VWF.absence or of inthe presence findings sinusoidal endothelialcells while FVIII:Cmurine withnormal in human recombinant associated FVIII infused mice, endogenous levelsof plasma decrease in a with hydrodynamic transferwas liver associated • • • • CLEC4Minternalization FVIIIinvolves clathrin pits of coated CLEC4M binds to mannose-containing glycans on FVIII CLEC4Minteractsin a with FVIII VWF-dependentand -independent manner CLEC4M is an endocytic receptor for factor FVIII in vivo byimmunohistochemistry. CLEC4M-expressing Results: HEK cells 293 In vivo In hepatic expression of CLEC4M expressionof hepatic after

Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. thrombosis. levels pathwaysincreased andan plasmaFVIII elevated to risk for could contribute clearance these and dysregulationof levels, FVIII physiological in important maintaining for thrombosis, the rapid VWF-freeof innormal clearance FVIII individuals may becrucially VWF-free FVIII are largely FVIII isa Furthermore, plasma aselevated unknown. risk factor [10]. In cases,both the pathways thatunderlie thispathological enhanced clearance of offorms hemophilia A arethe result of to FVIII,VWF deficiency Conversely inisolatedFVIII resulting some [9]. mild/moderate D’D3 variants inthe FVIII-binding the regionof accelerated clearanceof FVIII. Typeischaracterized VWF-free pathogenic 2NVWD by deficiencyfrom can result FVIII involvingquantitative inheritedbleeding disorders cleared FVIIIthe proportionMoreover, inaVWF-independent substantial. of isthus manner relatively hasclearance a6-8-fold VWF-bound low,rate it faster than FVIII, suggesting that VWF-freepathophysiologic in is FVIII of Althoughtheamount thecirculation relevance. likelycleared throughVWF-dependent receptor-ligand interactions. is thus circulating FVIII Theof majority levels. plasma FVIII andFVIII betweenregulate VWF plasmafrom the and [8] theconcentration thus circulating of andVWF, binding the affinity VWF VWF protects proteolysis[4–6]. clearance accelerated FVIII aswell from asfrom [7], Approximatelyplasma inadynamiccirculates plasmaof equilibrium FVIIIwith 95-97% inthe interaction (VWF).glycoproteinwith Factor its the plasma, and von Willebrand multimeric the clearance its of fromrate andsecreted, issynthesized whichby FVIII influenced at therate are levels Plasma (>150%) FVIII venous thrombosis [1–3]. andarterial increased risk for have andanimal elevatedplasmaepidemiological levelstoanmodels studies linked FVIII hemophiliadisorders and A von Willebrand disease (VWD) (<1 –50%), while the normal population (50-200%). Low levels FVIIIof associate with the inherited bleeding Introduction However,VWF-independent clearance pathwaysFVIII physiologic haveboth for and Plasmathe glycoproteincoagulation levelsof VIII factor (FVIII) are highly variable in F8 variants impair that FVIII binding to VWF VWF gene that result binding inimpaired genethat of

Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. ratherabsence thanan biological a of and CLEC4M.between interaction FVIII be relatedto genome-wide cut-off the reportingthreshold significance for thisassociation, FVIIIwas this reported, levels may andplasma association between genevariants CLEC4M infectionmediating previously described as an adhesivereceptor pathogens suchasfor HIV,capable of the onliver sinusoidal and the endothelial lymphoid tissues cellsof [16]. CLEC4M hadbeen L-SIGN orDC-SIGNR) termed expressed also M, 4 CLEC4Mmember family lectin (C-type (rs868875), which calcium-dependent atransmembrane (encoding receptor lectin encodes withinvariantcommon the but plasma with a FVIII not VWF levels associated Interestingly, [13–15]. FVIIIand VWF of levels plasma both with associating as clearance and and secretion receptor-mediated variantsinvolved inbiosynthesis ingenes identified analyses association. GWAS have andstatistical decreased influence of magnitude that the loci that but quantitativetrait VWF also majority with of plasma modify a FVIII modify VWF is levelsisassociatedwithplasma FVIII:C a~0.54%thought change [12],it inplasma for approximately the variability 50% of FVIII levels inplasma [11]. As every 1% changein investigate the ability of CLEC4M to act as an endocytic receptor in FVIII CLEC4Mfor receptor presence the investigate actasanendocytic ability the to of internalization its glycoprotein ligands of has not been demonstrated. In these studies, we able facilitate to which CLEC4Mbasis is by characterized. Additionally, themechanistic CLEC4M interact withFVIII in to orabsencethe presence of VWF beenhas not and the internalizationmediate ofVWF earlyto endosomes [18]. However, the ability of demonstrated CLEC4M that with N-linkedcan bind VWF glycans,through interactions its havealso the rs868875SNV, [18,19]. can phenotype typeof VWD We modify the 1 (VNTR) in theneck polymorphism regionof CLEC4M with which isinlinkage disequilibrium including theGWAS-identified SNV rs868875, or a variable number of tandem repeat internalizes and describesubsequent FVIII endocytic the ligand. pathway this of CLEC4M and bycharacterizewhich binds further We VWF. themechanism absence of and Variants inthe We and others have previously found that variants withinthe and that havepreviously others found We in trans VWF gene VWF-modifying andthe ABO account blood grouplocus inan ICAM-3-dependent [17]. manner Importantly while no

CLEC4M CLEC4M gene, gene, gene Accepted Article Immunocytochemistry: Germany). (Dianova, anti-LAMP1 anti-murineHamburg,andTechnology), CD31 rat (Abcam), mouse anti-EEA1 anti-Rab9 Technology, (CellSignalling MA, Beverly, rabbit (CellSignaling USA), (DAKO), anti-CLEC4Mrabbitmouse (R&D), anti- Antibodies: described [18]. as maintained the 7copy stable VNTR allele (pCIneo cells)weregenerated polymorphic andcontrol and methyl- some For(NIH). were experiments, cells preincubatedwith DMSO (0.2%, 30minutes), OrcaHamamastsu high resolutioncamera. Images were analyzed using software ImageJ and FX Disc confocal microscope Spinning with Slidesa Quorum wereimaged Wave This article is protectedThisarticle is byAll rightsreserved. copyright. bufferbinding (10 buffer HEPESmM KCl,NaCl, 10 135 pH7.4, CaCl 5 mM mM mM Imaging Studies: performedwithAdvate (rFVIII) Humate or P (pdVWF-FVIII). All tocontainhadbeenpurified 1% experimentswere unlessotherwise VWF. specified (Octapharma). Plasma-derived(pdFVIII) FVIII (Haemocetin) (Biotest, Dreieich, Germay) and Kovaltry (Bayer, Leverkusen, Germany), (Pfizer,Xyntha New York, andUSA), Nuwiq recombinant FVIII (rFVIII) products includedAdvate (Baxter, Deerfield, USA), Kogenate-FS Prussia,of USA), and (1:1, was Octapharma, from Lachen, Switzerland.Wilate) Human FVIII Products: Materials and Methods Plus Software (Media Cybernetics, (MediaRockville, USA).Plus Software (Sigma Aldrich). Quantification of immunofluorescent images was performed using ImagePro 10 µM, Cambridge,minutes) (Abcam, England), or dynasore hydrate 30 (80 µM, minutes) MgSO 4 ) for 15 – 120 minutes. werewashed and 15–120 ) for Cells prepared aspreviouslyminutes. described. β -cyclodextrin (5 mM, 60 minutes) (Sigma Aldrich, St. Lousi, MO.(20 60 Lousi, USA), (Sigma Aldrich, pitstop-2 minutes) (5 mM, St. -cyclodextrin

Antibodies used included: sheep anti-F8 (Affinity rabbitanti-VWFBiologicals), Plasma-derived VWF-FVIII (2.4:1, Humate-P) was CSLBehringfrom (King Cell Culture:

CLEC4M-expressing cellswere rFVIII exposed to or pdVWF-FVIII in

A HEK 293 stable cell lineexpressing CLEC4M possessing CLEC4M (Novus, Littleton, USA), rabbit Littleton, CLEC4M (Novus,

2 , 2 mM Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. anti-VWF (DAKO),monoclonal or mannan or polyclonalor anti-FVIII (Affinity Biologicals) experiments,some FVIII and/or -Fc chimera were preincubatedwith 1 mg/mL Fora BSA-coated control. bindingwithwas negative binding to FVIII compared background Mouse Ig-HRP(Southern Biotech, Birmingham, For CLEC4M-FcUSA). allexperiments, or anti-FVIII A1 antibody (GreenMountain8002 Antibodies, Burlington, USA) with agoat-anti ora withmonoclonal bindingwas ananti-FVIII-HRP antibody detected Biologicals) (Affinity Fc antibody Alternatively, (AbCam). CLEC4M-Fc was coated incarbonate and buffer FVIII Aldrich),µM) (Sigma MG132 inDMSO) (Sigma(50 µM Aldrich), NH preincubated with rFVIII with 5hours (250 for incubation followed chloroquine disulfide by (BioRad, concentration USA). experiments, cellswere Forsome Hercules, werenormalized FVIIIAldrich). total(Sigmalysates, To levels incell to quantify FVIII:Ag HRP Scientific,with Rockford, (Pierce/Thermo USA) anddeveloped IL o-Phenylenediamine poly- streptavidin with incubation Biologicals)by (Affinity anti-Factor followedVIII antibody Canada) was according performed protocolsto using biotinylatedmanufacturer’s a sheep Enhanced sensitivity human FVIII:AgELISA: objective. Imageswere analyzed FIJIusing ImageJ or Bethesda, (NIH, software USA). performed using aLeica oil SP8 scanning laser using63X immersionconfocal microscope paraffin-embeddedformalin fixed 7 on IHCanalysiswas performed were andformaldehyde prepared perfusion. by saline human rFVIII in accordance with previousstudies[20]. 30 minutes post-infusion, tissues Immunohistochemistry: Na modifications. FVIII wascoated onMaxisorp (Nunc, Rochester,USA)plates in50 mM withseveral[18] as described CLEC4M-Fc USA) (R&D chimera wasmeasured Minneapolis, phaseSolid immunosorbent assay: orAldrich), DMSO vehiclecontrol (0.2%) 2 for hours prior tolysis. 2 HCO 3 andFc-chimerawas binding detected an HRP-conjugated with anti-humangoat

FVIII KO or VWF/FVIII of FVIII received tail veininjections DKO mice μ M tissue sections as described [20]. Imaging was Imaging asdescribed[20]. sections M tissue The between interaction FVIII and recombinant FVIII ELISAFVIII (Affinity Ancaster, Biologicals,

4 Cl (20 mM) (SigmaCl (20mM) Accepted Article was CLEC4M-expressing [18]. >90%CLEC4M-positive and pCIneo (stablevector observations). therefore generatedWe CLEC4M-expressing astable HEK 293cellline that primary lymphaticsinusoidal endothelialcellsdonotexpress liveror CLEC4M (unpublished commerciallyand available [24] culture their phenotype in lose which[16] rapidly tissues and/or FVIII and VWF-FVIII complex is internalized by CLEC4M-expressing cells Results with * and P <.001 with **. ± asmean are expressed denote P<0.05Figureserror. ValuesArmonk,USA). NY,standard (IBM, 16 SPSS version or USA) CA, Jolla, (La 3.06 version software InStat GraphPad using This article is protectedThisarticle is byAll rightsreserved. copyright. Statistical analysis: were 10and centrifuged at10,000xg for untilminutes at maintained FVIII:Ag ELISA.-80˚C injection andbloodwas collectedby retro-orbital into 10% plexus buffered Samples citrate. hoursAldrich) 24 priorto Human rFVI study[23]. (LSEC) cytotoxicity, mice received an IP injection 200 of cyclophosphamidemg/kg (Sigma cell endothelial sinusoidal induce liver Tobackground [21,22]. C57BL/6 ona generated were with salineVWF/FVIIIafter perfused 30 andformalinwere minutes. DKO mice FVIII human Recombinant was by administered (100vein infusion and U/mouse) tail tissues pool.plasma C56Bl/6normal mouse against a directions manufacturer’s accordingthe to measured usingthe Coatest Chromogenix theCLEC4Mgene was [18].FVIII:C thepLIVE of transfer liver expressionvector cDNAin Animal models: experiments were from SigmaAldrich. (100 antibodies GMA-8002, -8016, -8011, or -8008 (Green Mountain Antibodies, Burlington, USA) μ g/mL) 30 for atroomminutes temperature. Mannan, CLEC4M liver andthe isexpressed onthesinusoidal lymphoid cellsof endothelial in vivo Hepatic expression CLEC4M of Hepatic was in via mice hydrodynamicinduced T-tests or T-tests one-way was onexperiments ANOVA with performed n assay Chester, USA) (Diapharma, West II was administered at 200 IU/kg by 200 was at II administered tail vein IU/kg

α -mannosidase used for some in vitro ≥ 4 Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. assays,FVIII internalized both as (VWF-free) for Additionally,a standard rFVIII wasused as complexFVIII larger is rFVIIIthan much alone which internalization. theratemay alter of accounts a theVWF- internalized,material total the of sizefraction for of andtherelative internalizedobserved FVIII of amount the two adifferencein We the total between products. As the ratio of VWF to FVIII in plasma-derivedthe complex FVIIIis 2.4:1, only internalization by LRP-1 [26]. withproteoglycans interactions sulfate canenhancethe cell surface onthe heparan endocyticsuch receptors as CLEC4M, reports have as FVIII that previous also indicated by FVIII andendocytosis of capture enhance the interactions if these is currently unknownIt interactionsdomain with phospholipid orthe bymembranes, sulfateheparanproteoglycans. C2 viaFVIII surface HEKcell293 the CLEC4M did express boundto not that be may cells lysate inFVIII of observed the VWF. with complex a of part aloneorinternalize as FVIII bindand/or able to CLEC4Mexpress is suggestingthat CLEC4M,not did cells that expressing CLEC4M approximately had two-fold FVIII detected inmore cell thanlysates Cells usinganenhancedsensitivity ELISA. waslysed, incell FVIII:Ag and lysates measured physiological ortherapeutic FVIII concentrations asdescribed, and cells werewashed, The binding rFVIII and internalization of (Figures 1Dand1E) orpdVWF-FVIII (Figures 1F and 1G) by CLEC4M-expressing cells was Cells quantified. were exposed to Figure 1). imaging conditions, CLEC4M-negative and isotypewere controls (Supplementalperformed Forall andFVIII as acomplex. CLEC4Mandinternalize VWF alsosuggesting that bind can exposed VWF-FVIII the to complex,and VWF partially FVIII colocalized (Figure1C), (overlaycomplex shown). not Additionally,we onCLEC4M-expressing that observed cells CLEC4M torFVIII (Figure exposed 1A), pdFVIII incells (Figure 1B) orthe VWF-FVIII CLEC4M express that didnot Figure (Supplement and1E). 1D FVIII co-localized with internalizedby cellsafter CLEC4M-expressing 1hour (Figure 1A-C),andnot bycells control Immunofluorescence that rFVIII, demonstrated pdFVIII, andpdVWF-FVIII complex were atphysiological products backbone) [25]. controlcells were concentrations to FVIII exposed

Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. pdVWF-FVIII, rFVIII or (Figure2D). Immobilized CLEC4M-Fc2 boundto partially byblocked with pre-incubation the mannose polymer or withmannan soluble environmentplasma ±VWF reversible, 2C),was(Figure and calcium-dependent, could be nM 2A (Figures and2B). that binding demonstrated rFVIIIfurther occurred to We ina <0.1 Kd of hasanapparent thisinteraction is=[27], ng/mL 100 product 1IU activity this of recombinant CLEC4M-Fc protein infusion dose-dependent assumingmanner; the specific We next confirmed that CLEC4M with usinga interacts FVIII solidphase bindingassaywith CLEC4M bindsmannoseglycans exposed on FVIII FVIII detectedby polyclonal antibody the anti-FVIII Figure (Supplemental 5). some ofpresence VWF-bound in FVIII theselysates the may decreaseapparent amount of theHowever, ability. loseits VWF-binding and thusmay degraded, is rapidlydenaturedand N-linked, N-linked, O-linked, N -and highO-linked, mannose glycans, and CLEC4M interact with to N-andO-linked glycans was onrFVIII Removal characterized. of domains each contain asingle theability high glycanHere, [27,29,30]. mannose of N-linkedof glycansonFVIII are complex alsoof type, theA1(Asn-258) and C1 (Asn-2137) the highmajority While evidenceof structure [28]. mannose trace with onlylinked glycans glycomic analysis human plasma-derived of VWF identified predominantly N- complex-type a describedas lectin, mannose-binding [18].Interestingly,whileVWF onCLEC4M been has CLEC4M-Fc, whileanti-FVIIIantibody an not (Figure 2H). did PHumate with a polyclonal anti-VWF antibody (Affinity Biologicals)binding attenuate to 2E)domains (Figure couldbindingPre-incubation CLEC4M-Fc. partially to of attenuate Preincubation of immobilized with FVIII monoclonal to antibodies the A2, C1, andC2 antibody(±VWF) Figure5). (Supplemental detection anti-FVIII by polyclonal products the FVIII andthevariablerecognition of product, activity each glycanof orspecific content productsto interaction between differences berelated inthe Ofmay note, (Figure FVIII 2G). generation rFVIII products (Figure 2E), andpdFVIII (<1% VWF) andpdVWF-FVIII complex We We have previously that demonstrated CLEC4M-Fc interacts with N-linked glycans

α (1 → 2,3,6)-linked mannose nd and 3 and rd

Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. glycans,mannose or FVIII:Ag in cell lysates with wasELISA. ofRemoval measured N-linked glycans, high andhours 2 rFVIII or control with for deglycosylated incubated cells were expressing the interaction between rFVIII andCLEC4M-Fc respectively 3B). CLEC4M- (Figure of high glycansmannose greatly and rFVIII N-but notO-linked bi glycans attenuated the of Figure (SupplementalUSA) Removal Burlingame, 2). Labs, (Vector lectin binding by USA)England andconfirmed Ipswich, (SigmaBiolabs, New Aldrich or bysugars was performed digestion asdirected the using endoglycosidase manufacturer We have VWFthat internalized previously HEK We by CLEC4M-expressing observed 293 cells toearly istransported To [18]. better endosomes characterize thisendocytic CLEC4M internalizesVWF FVIIIand through clathrin-coated pits FVIII (Figure 3D). the binding/internalization attenuated of (100 µg/mL) polymer with mannose the mannan CLEC4M-expressingcells of Additionally,preincubation (Figure3C). controls relative to exposed to rFVIII 15 for weminutes, observed colocalization FVIII andearly between incubated with or60 [30]. rFVIII CLEC4M-expressingminutes 15, 30, werefor cells When endocytic pathway - methyl- the clathrin-coatedwas pit-mediatedreagents used thethree todisrupt reducedby eachof demonstrated that binding/internalization of rFVIII or pdVWF by cells CLEC4M-expressing Internalized 4AwasVWF or and4B). byrFVIII analysis quantified and ImagePro pdVWF-FVIII for 1hour visualized and internalization (Figurewasby immunofluorescence were incubatedwith then rFVIIIpits.human Cells internalization clathrin-coated or of GTPase bindsthe that clathrin of andpitstop-2 domain terminal activity, andprevents dynasoredynamin hydrate inhibit lipidrafts, disrupt to cell cholesterol and membrane pathway, CLEC4M-expressingcells werepretreated with methyl-

To visualize the endocytic pathway of FVIII,endocyticTo pathway HEKvisualize the CLEC4M-expressing 293were of cells α (1-2,3,6) linked mannose sugars decreased internalization rFVIII of β -cyclodextrin, dynasore hydrate,and-cyclodextrin, pitstop-2 (Figure 4A-D). α (1-2,3,6) linked mannose sugars modestly decreased sugars modestly linked mannose (1-2,3,6) nding to CLEC4M-Fcnding (Figure to 3A).Removal

β -cyclodextrin to deplete to -cyclodextrin Galanthus nivalis Galanthus

Accepted Article immunohistochemistry),FVIII:Cplasma levelsofwere decreasedcompared with control where 10-30% ofhepatocytescharacterized expressed CLEC4M by (as Inmice FVIII:C. endogenous levels of characterized examine weresamples further to then had gene levels VWF:Ag.of a 46% with effective Plasmatransfer inplasma decrease daysFour [18].mice genetransfer post-injection, CLEC4M hydrodynamic expression using This article is protectedThisarticle is byAll rightsreserved. copyright. chloroquine disulfide (CLQ) and NH lysosomeinhibitors tothe then exposed werewashed, cells 5hours, with rFVIII and for proteasomes FVIII,in thecatabolism of CLEC4M-expressing HEKcells were treated 293 293-based HEK in (Figure protein 5C).this Toand confirm model lysosomes roleof the the internalizationFVIII by catabolism lysosomal-mediated of theFVIII of leadsto CLEC4M wehour, FVIIIcolocalization with observed of LAMP1, suggesting alysosomal thatmarker, endosomes (Figure 5B). CLEC4M-expressing were cells When incubatedwith rFVIII for 1 late for a Rab-9, marker with we colocalization30 observed minutes, with rFVIII for trafficked early to endosomes (Figure 5A). CLEC4M-expressingWhen cellswereincubated endosomal antigen1(EEA1), isupon internalization that confirming CLEC4M, by FVIII Associationof FVIII CLEC4M-expressingwith cells VWFcolocalization of with LAMP1 (Figure 5F). FVIII for 1 hour, and in contrast to what we had seen with FVIII, we observedlittle with whenwere cells pdVWF- However, (Figure CLEC4M-expressing incubated 5E).Rab-9 VWF with with pdVWF-FVIIIco-localized 30minutes, wereincubated expressing cells for internalization by cells[18]. CLEC4M-expressing Herewe demonstratewhen that CLEC4M- FVIIIdetectable in the lysates of CLEC4M-expressing cells (Figure 5D). inhibitors (CLQandNH inFVIII celllysates by was measured ELISA. Exposure of the cellsto lysosomalboth the As mice doAs notexpress hepatic wehave a CLEC4M mice induced previously ortholog, We have previouslydemonstrated VWF that colocalizeswithEEA1 post- 4 Cl), and the proteasome inhibitor MG132 significantly increased 4 Cl, and the proteasome inhibitor MG132 for 2 hours. 2 MG132 inhibitor for proteasome the and Cl, in vivo

Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. t human DKO context LSEC of (VWF/FVIII rFVIIIinfused cytotoxicity inthe mice observed anextended cyclophosphamide toinduce LSECof half-life We cytotoxicity. post-injection (Figures VWF/FVIII 6Cand6D). DKO were mice with pre-treated described, and FVIII localization with CD31-expressing LSECs was observed30 minutes endocytosing FVIII. FVIII KO orVWF-FVIII DKO weremice infused with human rFVIII as demonstrate LSECs, that evenin the absence CLEC4M of are expression, capable of expressing CLEC4M as well didnoexpress CLEC4M ascells that (Figure 6B). Finally,we anti-human FVIII and CLEC4M observed that was internalizedantibodies.rFVIII We by cells post-FVIIIminutes wereprepared injection, tissues the andimaged IHC usinganti-humanfor received ahydrodynamicdays genetransfer CLEC4Mthe of previously. 30 cDNAfour human FVIII notcontrols shown).(data To that demonstrate CLEC4M-expressing cells caninteract with mice that received gene hydrodynamic transfer with CLEC4Mthe of compared cDNA Supplemental Figure 3forcontrolexperiments. of See (Figure received emptythe 6A). backbone hydrodynamic mice injections that vector FVIII has a short hasa FVIII both and VWF FVIII such orSIGLEC-5 asLRP1, stabilin-2, However,[31–34]. VWF-free that the VWF-bound majority of FVIII is clearedby VWF, thatbindonly toor to receptors VWF-bound FVIII has a prolonged half-life compared with FVIII, and VWF-free itis thought Discussion min; cyclophosphamide-treatedVWF/FVIIIDKO = 25.79 p=0.0075)mice 6E). (Figuremin; both the absence and absence both VWFof presence the [18]. show CLEC4M that can regulate the binding, internalization and plasma levels ofFVIII in work tohere we this VWF, extended clearancereceptor for a CLEC4M demonstrated is that asialoglycoprotein (Supplemental receptor Figure 4)[35–39]. we havepreviously While SIGLEC5,LRP1, andthe such as FVIII binding capableof VWF-free receptors We were unable to observe a change in endogenous plasma FVIII:C in VWF KO FVIII:Cendogenous inVWF wereplasma change in observea unableto We

in vivo in vivo , human we, rFVIII infused (10 half life, and up to ~24% of plasma FVIII may be cleared by FVIII may ~24%cleared plasma andupto be of life, half μ g) into VWF/FVIII DKO mice that had that DKO mice VWF/FVIII g) into

1/2 =17.03 Accepted Article demonstrated using a solid phase assay that CLEC4M is capable of binding to humansoliddemonstrated bindingto capableof usinga CLEC4Mphase assayrFVIII is that levels not half-lifethis In [34]. studyLSECs,weplasma have doesmodify VWF-FVIII or the CLEC4M homologuemurine acididentity) SIGNR1(~55% amino isexpressed on which internalizationviral particles by of DC-SIGN[44]. exist withinlipid and rafts, depletion of cholesterol attenuatedmembrane binding and lipid with Consistent rafts. these findings, theCLEC4M-homolog of microdomains DC-SIGN itsligandsthroughaclathrin-coatedendocytosis pit-dependent involves of that mechanism CLEC4M This receptor the that mediates 4). CLEC4M suggests activity (Figure of cholesterol, andinhibition of clathrinand dynamin GTPase activity impaired the endocytic depletion ligands,of observedthat these fate membraneis uncharacterized. of We Thus, the bymechanism whichCLEC4M VWF internalizes and FVIII, and the subsequent previouslybeen to endocytose proteins shown expressing high other glycans[43]. mannose mediating viral infection viralinfection mediating capable havecharacterizedadhesiveCLEC4M receptor CLEC4M. studies asan of Previous vitro byrecognitionFVIII of receptor (CD206)mannose the ondendriticand cells macrophages lectin clearance receptor FVIII,for althoughhigh glycansmannose areinvolvedin the productsthe first CLEC4Mmannose-binding To [27,41]. with is date, to interact able allFVIII wasand CLEC4M conserved glycans isrelatively high of glycan expression mannose sites, andoccupancy of sialic acidcontent, determinants, group ABOincluding blood preparations, Importantly,while exist between glycosylation differences plasma derived andrFVIII complex typeexpressed glycans are N-linked that FVIII throughout the (Figure 3). molecule vivo [37,39,40], suggesting that glycans expressedon FVIII areimportant determinants its of receptors SIGLEC-5, the asialoglycoprotein receptor and the CLEC4M homolog DC-SIGN This article is protectedThisarticle is byAll rightsreserved. copyright. half-life. In theabsencehalf-life. VWF, of CLEC4M with interacts both highthe and mannose [42]. [42]. Mice express donot aCLEC4M and ortholog, we previously have demonstrated that VWF and FVIII areidentifiedand FVIIIVWF among endogenousfirst the glycoprotein ligandsfor The glycans N-linked have beenFVIII of shown interactions thelectinto mediate with in trans in an ICAM-3-dependent manner [17] and not inanICAM-3-dependent CLEC4M [17] has manner

in in Accepted Article contribute to the clearanceFVIIIcontribute to in thepresence VWF.or absenceof of the supra- While rFVIII can be taken up by CD31-expressing bymediated CLEC4M. We did confirm, however, using VWF/FVIII DKO human that mice was hepatocyte FVIII of inthismodel that state uptake possible todefinitively it isnot beento bindVWF-free [39,45,46], has reported receptor hepatocyte FVIII asialoglycoprotein model (FigureHowever, 6B). asFVIIIby was alsotaken up inthe liver, and othercells the rFVIII associated with CLEC4M-expressing gene hepatocytes in ourhydrodynamic transfer infused observedthat expression. We hydrodynamic CLEC4Mhepatic by expression of likely VWF, of the to VWF-freein of vivo half-life FVIIIshort due and variable the andartificial CLEC4M on plasmaFVIII levels inwasvivo challengingmore to demonstrate in theabsence when with compared a BSA (Figure control 2B). However, quantitative the influence of defibrinatedrecalcified, VWF-deficient interaction plasma occur in this that can confirmed andprolonghalf-life the of human rFVIII inFVIII KO mice [34]. can increaseplasma endogenous FVIII sinusoidallevels of innormal cells, endothelial mice previous observation treatmentthat of withmice cyclophosphamide, which is cytotoxic to the liverpromote the association between FVIIIwith andLSECs andisconsistent our the absence CLEC4M of expression, patternthe blood of flow andanatomical structure of whereKO VWF circulating mice murine is (Figure present 6C). This evenin suggeststhat that infused human rFVIII can associate with CD31-expressing LSECs in FVIIIliver of the theVWF-FVIIIclearance of endogenous complex. facilitate murine further demonstratedWe via hydrodynamic injection (Figure 6A), suggestingthat hepatic expression CLEC4M can of CLEC4M received hepatocytes 10-30% mice that theempty of relative on to pLIVE vector levels VWFof wea Here, [18]. decrease observed inendogenousFVIII in expressingmice plasma with decreased CLEC4Mwas associated cDNA the of hydrodynamicusing injection havepreviously We that transgenic demonstrated CLEC4M of expression by hepatocytes This article is protectedThisarticle is byAll rightsreserved. copyright. compared toaBSA control 2B) (Figure suggesting thatFVIII andCLEC4M interact in defibrinated, recalcified thepresenceof normal plasma (containing human when VWF) As CLEC4M can also directly interact with FVIII in the absencewithwe VWF, of alsoCLEC4MdirectlyAs inthe can FVIII interact LSECs, suggesting that these cells can these cells suggesting that LSECs,

in vivo . Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. acknowledge Mewburn The authors M.Gordonand J. from Chairin Hemostasis. Molecular SocietyHemophilia a D.Lillicrap grant. Canadianfellowship is therecipientof Research award,Council andaCanadian Advisory aQueen’sUniversity Senate fellowship, Research VWD Swystun (HL081588).Biology L. was of Fellowshipby supported Stroke aHeartand and theNational Institutes Health of Zimmerman Program the Molecularfor and Clinical andFoundationHeart 6881), Canada(NA Strokeof the (FDN154285), Health Research Acknowledgements: cycle. life FVIII the into and clearancepathways, includingwill CLEC4M, yield involving those novelinsights likely interactionsstudies on betweenthe FVIII VWF, the sinusoidalendothelial and cellsecretory catabolism, and are thus likely toimportant plasma regulators of be FVIII levels. Future proportion of FVIII production [49–51], aswell alocationof VWF-FVIII clearance and replacement products. LSECs arerecognizedbe to the cellular source a substantialfor improving insightsFVIIIlevelsof novel into the for andcontributes strategies half-life clearance provides new information concerning quantitative loci influencing FVIII plasma VWF-independent FVIII of the basis mechanistic Elucidationindependent of manner. receptorcan alsobindandinternalize forVWF, aVWF-dependent FVIII inboth human and free FVIII even in the absence of CLEC4M expression. VWF- of clearance the to LSEC-expressedthat contribute endocyticpathways confirming cyclophosphamideagent prolongs FVIII of thehalf-life intheabsenceof VWF (Figure 6E) intoVWF/FVIII(200 human rFVIII with U/kg) treated levels theLSECof DKO cytotoxic mice we Moreover,infusiontherapeutic demonstrateand that of [20,47,48]. fibrinogen VWF such as physiological rangeof largeplasma proteins other concentrationswith the antigen of thosethesestudies, dosesare to employed andarein similar line inpreviouspublications physiological rFVIII were levelsof facilitateFVIII utilizeddetection to intheseimaging Together, datathis suggestsCLEC4M, that a previouslyclearancecharacterized This research wassupportedThisCanadian by research the grants from of Institute

Accepted Article FayFJ. JV, Coumans PJ, Walker von protection factor Willebrand factorof mediates 7 Rawley Jenkins PV, OP, Smith O’DonnellJS. andElevated O, of VIII risk levels factor 2 This article is protectedThisarticle is byAll rightsreserved. copyright. Noe D.A mathematical ofmodel coagulation VIIIfactor kinetics. 6 BE, FischerKramer G, Mitterer A,Grillberger 5 Golder M, Mewburn J, Lillicrap D. In vitro and in vivo evaluation of effectthe of 3 ZimmermaninB,Valentino review. L.Hemophilia: 1 References: interests. financial competing no declare CSL Behring, Bayer, and Baxter educationalfor presentations. The remaining authors and honoraria CSLBehringresearchP.D.J. from fundingfrom receives andOctapharma. Conflict-of-interest disclosure: paper. D.L. wrote the paper; L.L.S., P.D.J., andD.L. designed theresearch study andedited the Contributions: providing plasma-derived FVIII. and Biotest technical Ungerer assistance, S.Kistnerfrom andC. Queen’sfor University for Leyte A, Verbeet MP, Brodniewicz-Proba T, Van Mourik JA, Mertens K. The 4 26 venous thrombosis. 1996; 1996; aggregation, collagen bindingto coagulation andbindingof VIII. factor ofEffectand human recombinant of multimerization von factor Willebrand on platelet VIIIelevated on factor thrombogenic the process. Biochem J interaction between human blood-coagulation factor VIII and . 60. : 289–303. : 84 : 55–66. : 1989; L.L.S., C.N., K.M., J.D.L., and I.G. performed the experiments; L.L.S., and theexperiments; L.L.S., andI.G. performed J.D.L., K.M., C.N., L.L.S., 257 Br J Haematol Br J : 679–83. D.L. receives grant receives Bayer, from Bioverativ,support grant CSL, D.L. 2012; 157 L, Mundt M, DornerEiblReiter F, J. W, : 653–63. :

Thromb Haemost Pediatr Rev 2013; Haemostasis 2013; 34 Thromb Res Thromb : 289–95. 109 1996; : 53–

Accepted Article 15 Huffman Huffman JE, Vries PS De, Morrison AC, Sabater-lleal M, Kacprowski T, Auer PL, 15 Oudot-MellakhA, AntoniP, G, T,Cohen M, Dimitromanolakis Germain Wells W, 14 This article is protectedThisarticle is byAll rightsreserved. copyright. 13 Smith NL, Chen M-H, DehghanChen M-H,NL,DP,A, Soranzo Smith Strachan Hayward S, Basu N, C, 13 F,Bolgiano Song CamposHouckD, J,Chen LE,Chambless M, K, Folsom KK, Wu 12 MorangeTregouet PE, Frere DA,C,Saut N, Pellegrina MC,TiretAlessi L, Visvikis S, 11 Brinkhous KM, Sandberg H,Garris MattssonJB, C, Palm M, Griggs T, Read MS. 8 10 Jacquemin M, Lavend’homme M, Jacquemin Benhida R, A, Vanzieleghem B, R, Lavergne D’Oiron 10 Mazurier C,Goudemand J, Hilbert L, Caron C, Fressinaud E, Meyer D.Type 2N von 9 2011; 2011; genome-wide association studies on vWF and FVIII plasma levels. Lathrop M, Gagnon F, Morange P-E, Tregouet D-A. Combined analysis of three frequency andvariants their association withplasma levels. Wiggins KL, Qi L, Sennblad B, Harris SE, Polasek O, Riess H,et al. Rare and low- DI,BrodyLin Guo L, Chasman M, ChenJA, M,MarioniX, Martina Cushman RE, M, Genome Epidemiology) Consortium. von and TheAging factor: CHARGE in Research Heart (Cohorts and Willebrand for Novel associations of genetic multiple loci with levels plasma of VII,factor VIII,factor FMK,Williams VitartH,Mälarstig V, Campbell A, KL, VanDuijn CM, etal. Wiggins RudanI, Sabater-LlealM, Bis JC, deMaat MPM, KongRumley A, Yang X, Q, study 11,673 subjects.of VIIIfactor anditsratio to willebrandnovelvon an observations factor, ARIC from Boerwinkle DongCouperAR,E, ABOof Quantitative D, JF. influence groups blood on 128 ina healthyStanislas Results from cohort. familythe population: Juhan-VagueL, Biological genetic factors I. and influencingplasma VIII levels factor after infusions into hemophilic and von disease Willebrand dogs. Purified human VIIIfactor procoagulant protein: comparative hemostatic response VIII activated from protein C-catalyzed inactivation. binding von to Willebrand factor. hemophilia A: inmutations scattered VIII the C1domain factor reducefactor VIII JG, Peerlinck K, VermylenJ, Saint-Remy AJM. novel of cause mild/moderate JM, Brackmann HH,Schwaab R,VandenDriessche T, Chuah MK, Hoylaerts M, Gilles biology.molecular Willebrand disease: clinical pathophysiology,manifestations, laboratory diagnosisand 1985; : 91–9. : 12 82 : 102.: : 8752–6. : Clin Haematol PLoS One Blood 2001; 2015; Circulation 2000; 14 : 337–47. 10 : 1–11. 96 2010; : 958–65.

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Accepted Article 25 Pipe SW, Montgomery Pipe SW, PrattinKP,PJ, Life shadowthe RR, Lenting a D. Lillicrap of 25 Elvevold K,SmedsrødB,MartinezThe sinusoidalliver I. endothelial cell: acelltype of 24 Swystun RB, CartierHartholt D,van Velzen LL,C,Haan JMM,denJD, Lai Hough AS, 20 This article is protectedThisarticle is byAll rightsreserved. copyright. R,vanDM, Vreeling vanTil KunneC, H, derRijt Markusic Hiralall JK, NP, Frederiks 23 N,FrenetteMethia M, DenisC, PS, Rayburn Ullman-Culleré Hynes H, RO, Wagner 22 Bi L, Lawler AM, Antonarakis SE, High KA, Gearhart JD, Kazazian HH.Targeted 21 Sanders Y V,vanderBom JG, IsaacsCnossende A, MPM, MH, Maat Laros-van 19 RydzSponagle SwystunNotleyPatersonK,Riches AD, N, JJ, Boonyawat LL, C, B, 18 Bashirova GCF AA, TBH, Geijtenbeek Vliet Duijnhoven Van, Van, SJ Eilering JBG, 17 Khoo ChanU-S, KYK,Chan LinVSF, DC-SIGN CLS. and L-SIGN: the SIGNs for 16 in human factor VIII-exposed hemophilia A mice. Voorberg EarlyLillicrapJ, D. cellular andimmune interactions transcriptome profiles 2015; hemophilia A. partner:dominant FVIII-VWF the itsclinicalimplications association and for controversial and confusing identity. cells prevent lentiviral transduction of hepatocytes.ofcells preventlentiviral transduction Oude-ElferinkWM, RPJ,J.Kupffer Seppen cells andnotliversinusoidal endothelial thrombosis. DD. A ofmouse severemodel von disease: defectsWillebrand in hemostasis and Genet disruption of the mouse factor VIII gene produces a of haemophiliamodel A. 45. von level in factor variation von Willebrand disease. Willebrand Leebeek Eikenboom J, FWG.STXBP5 CLEC4M and genevariations contribute to Gorkom BAP, Fijnvandraat K, Meijer K, van Duijn CM, Mauser-Bunschoten EP, levels. factor vonplasma Willebrand internalizes,clearsandvon and factor variation contributes in the to Willebrand TheMontgomeryC-type LillicrapCLEC4MD. PD, James lectinreceptor RR, binds, EndothelialCells andPromotes HIV-1 Infection. )– DC-SIGNProteinrelated Highly Is LiverSinusoidal Expressed onHuman Dendritic Cell – specific Intercellular Adhesion Molecule 3 – grabbing Nonintegrin ( MartinMP,L, Martin TD,Wu ViebigN, KnollePA, KewalramaniVN, Kooyk Y Van. A infection. 1995; 13 : 956–66. : J Mol Med ProcNatl Acad Sci 10 Blood : 196–201. 2016;1–42. : 2008; 86 : 861–74. 1998; Am J Physiol Blood 95 : 9524–9. 2013; J Exp Med J ThrombHaemost

121 2008; Mol Ther : 5228–37. 294 2001; 2005; : G391-400. J ThrombJ Haemost 193 11 2018; 2018; : 26–34. 26–34. : : 671–8. 16 Nat : 533–

Accepted Article 36 Lenting PJ, Neels JG, van den BergvanJG,PJ, Lenting den Neels BM,Meijerman DW, PP, Clijsters Pannekoek H, 36 HJ,Sussman Weiss Hoyer II, Stabilizationplasmainby LW. VIII factor of thevon 35 SaenkoYakhyaev EL, Mikhailenko Sarafanov I,StricklandA, D, A. lowRole of the 32 34 Swystun LL, Lai JD, Notley C, Georgescu I, PaineI, Georgescu Notley Lai AS, Mewburn SwystunK, C, JD, LL, J, Nesbitt 34 Kurdi PegonM, JN, Lenting OD, Christophe S,OdouardC, CasariDenisCV, PJ. 33 RastegarlariG, PegonJN,Casari C, Odouard S, A-M, Navarrete Saint-Luvan N, 31 0 Canis K, Anzengruber J, Garenaux E, Feichtinger M, Benamara K, Scheiflinger F, 30 Lenting P, Pegon J, Christophe O, Denis C. Factor VIII and von Willebrand factor--too 29 McKinnonSMM, Haslam TAJ,K, Canis Laffan A, Morris M, Panico HRR, Nowak 28 7 Lai J,Swystun L, Cartier Nesbitt K,D, Zhang C,Hough DennisC, LillicrapJ, N-D. 27 This article is protectedThisarticle is byAll rightsreserved. copyright. Ananyeva SarafanovG, M, ShimaNM, Saenko EL. heparan Cellsurfacesulfate 26 Chem comprisesa bindingsite forlow density lipoproteinreceptor-related protein. van Mourik JMertensa,van K, ZonneveldJ. Theof lightchain a factor VIII Willebrand factor. Biol Chem density lipoprotein-related protein receptor in mediation of factor VIII catabolism. complex half-life and half-life complex immunogenicity. James PD, Lillicrap D. The endothelial cell receptor stabilin-2 regulates VWF-FVIII KzhyshkowskaGéraudSchledzewskiK, C, J,Goerdt HopmanMontgomery S, W, R, 5. Factorandvon VIIIare ligands factor Willebrand carbohydrate-receptorfor the Siglec- contributesclearance the to von of factor. Willebrand Vlijmen BJ, Legendre P, OD,Christophe Denis C V, Lenting PJ. Macrophage LRP1 to clinical implications. human plasma-derived VIIIfactor anddifferent recombinant products: structure from Savoy LA, Reipert BM, Malisauskas M. In-depth comparison of N-glycosylation of theirsweet own good. for 2012; MAA,MappingDellA. N-glycome the von human of factor. Willebrand in hemophilia A mice. linked glycosylation the modulates immunogenicity recombinant of human factor VIII receptor-related protein. proteoglycans participate in VIIIfactor catabolism bymediated low density lipoprotein Haematologica 1999; 447 : 217–28. 1999; 1999; 274 : 23734–9. 274 2012; J Clin Invest : 37685–92. Haematologica J Thromb Haemost J Thromb 97 J Bol Chem Haemophilia : 1855–63. 1977; 2001; 2018; 2018; 60 J ClinInvestig J 2010; 2010; : 390–404. 2018; 276 103 16 : 11970–9. : 194–9. : 1925–36. 16

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Accepted Article 46 Pipe SW, Montgomery Pipe SW, PrattinKP,PJ, Life shadowthe RR, Lenting a D. Lillicrap of 46 Saenko EL. Regulation of Factor VIII Life-Cycle by Receptors from LDL Receptor 45 Guo Y, Feinberg H, Conroy E, Mitchell D a, Alvarez R, Blixt O, Taylor ME, Weis WI, 43 Navarrete DasguptaS, A, Bayry Delignat S,B,J, Andre Christophe S, Wootla O, 42 Hironaka T, Furukawa K, Esmon P, Fournel M, Sawada S, Kato M, Minaga T, Kobata 41 Herczenikvan E,A, SD, Kaijen Haren den P, van BiggelaarWroblewska Meijer M, 40 Bovenschen N, Rijken DC,Havekes LM, van Vlijmen BJM, Mertens K. The B domain 39 This article is protectedThisarticle is byAll rightsreserved. copyright. Cambi A, De Lange F, Van Maarseveen NM, Nijhuis M, Joosten B, Dijk EMHP Van, 44 Sarafanov AG, Ananyeva NM, Shima M, Saenko EL. Cell surface heparan sulfate 38 Kurdi PegonLentingM, OD, JN, Christophe S,OdouardC, DenisCV, Casari PJ. 37 Remy J,Kazatchkine M, Kaveri S, Lacroix-Desmazes S. A role exposedfor JacqueminNascimbeniMartinez-Pomares M, M, Moris Geijtenbeek T, L, Saint- A, 1992; and culture the from baby of mediarecombinant hamster kidney cells. ComparativeA.chains studysugar VIII the of of purified factor human from plasma 501–9, 509.e1-5. byisdomain. VIII cells viaits C1 mediated dendritic AB, Martinez-PomaresL, tenBrinke A, Voorberg Uptake of bloodJ. coagulation factor Haemost of coagulation VIII factor interacts with theasialoglycoprotein receptor. hemophilia A. partner:dominant FVIII-VWF the itsimplicationsclinical association and for Superfamily. DC-SIGN andDC-SIGNR. receptors Drickamer K. Structural basis distinctfor ligand-binding and targeting properties of the lymphocytes. human of mannosylationstherapeuticTin presentation self-proteins CD4+ to dendritic cells. portals are MicrodomainsFigdor C-typevirus the of for DC-SIGN CG. entry lectin into BI De,FransenBakker JAM, Bovee-geurts PHM, Leeuwen NF Van, Hulst FN Van, receptor-related protein. proteoglycans participate in VIIIfactor catabolism bymediated low density lipoprotein 5. Factorandvon VIIIare ligands factor Willebrand carbohydrate-receptorfor the Siglec- Haematologica 267 2005; : 8012–20. 36th Hemophilia36th 2005 Symposium Hamburg Proc Natl Acad Sci Acad Natl Proc Blood J Cell Biol 3 : 1257–65. 2012; 2016; 97 J Biol Chem J 2004; : 1855–63. 128 : 2007–16. : 2007–16. 164 2007; : 145–55. 2001; Nat Struct MolBiol 104 : 8965–8970. 276 : 11970–9.

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: Accepted Article (G). For all ELISA conditions, n=3-5 independent experiments. ± SEM, *p<0.05, **p<0.001. byexpressing cells FVIII:Ag ELISA; dose response(1 hour) (F),time course (1.5 U/mL FVIII) (E).time QuantificationU/mL course (2 FVIII) ofpd-VWF-FVIII internalization byCLEC4M- internalizationcellsdose by ELISA; by CLEC4M-expressing hour) response FVIII:Ag (D), (1 Imagesare representative independent of 5-6 experiments. Quantification of rFVIII FVIII, U/mL VWF, 0.83 1 hourincubation). (yellow)DAPI (blue), colocalization (2U/mL Internalization VWF-FVIII of complex by CLEC4M-expressing cells; VWF (red), FVIII (green), hour incubation); CLEC4M (red), FVIII (green), (blue),DAPI colocalization (yellow). (C) rFVIII, pdFVIII(B) byCLEC4M cells using expressing immunofluoresence FVIII, (2U/mL 1 internalizationimmunofluorescence using was ELISA. or measured (A)Internalization of expressing cellswere exposedtorecombinant (r) or plasma-derived (pd) FVIII and Figure Internalization 1. by ofFVIII CLEC4M-expressing 293 cells. HEK Figures. L, EverettA, Cleuren Khoriatysynthesized VIII GinsburgD.Coagulation is in factor R, 51 50 Fahs SA, HilleMT,Q, Fahs H,Montgomeryconditional Shi A knockout RR. Weiler mouse 50 HealeyP.Lollar E,Expression DoH, of VIII J, Waller factor by liversinusoidal murine 49 Van Der Flier A, Liu Z, Tan S, Chen K, Drager D, Liu T, Patarroyo-White S, Jiang H, 48 This article is protectedThisarticle is byAll rightsreserved. copyright. S,A, NavarreteCaligiuriBayry DasguptaS, O, Nicoletti DelignatA, C, Christophe J, 47 endothelial cells. plasma factor VIII. VIII. factor plasma revealsendothelialmodel principalcellspossiblyand asthe sourceof exclusive endothelial cells. 23. independentFVIIIclearance pathway hepatocytes. in mouse FcRn rescuesLight DR. recombinantFc VIII factor proteinfusion a from VWF J Thromb HaemostJ Thromb primary critical the for response allo-immune totherapeuticVIII factorin hemophilia A. Kaveri S, Lacroix-Desmazes S. Splenic marginal zone antigen-presenting cells are Blood J Biol ChemJ Blood 2009; 2014; 2014; 7 : 1816–23. 1999; 123 123 : 3697–706. : : 3706–13. 274 : 19587–93.

PLoS One 2015; 2015; CLEC4M- 10 : 1– Accepted Article cells. (D) Internalization rFVIII of U/mL,1-hour(2 incubation) by CLEC4M-expressing cells deglycosylated/demannosylated (2 FVIII U/mL, 1-hour incubation)by CLEC4M-expressing CLEC4M-Fc bindsto immobilized (30 demannosylated FVIII U/mL). (C) Internalization of CLEC4M-Fc (10 Figure 3. Glycan-dependent binding/internalization ofFVIII by CLEC4M SEM, *p<0.05, **p<0.001. Affinity ForBiologicals)not (Figure 2H). experiments,did allassays, n=3-5independent ± antibodyBiologicals) while bindingtoCLEC4M-Fc, anti-FVIII attenuate (100 an 2.4:1 VWF-FVIII complex (1 U/mL) with a polyclonal anti-VWF antibody (100 complex (1U/mL) (2.4 VWF: 1FVIII, or1VWF:1 FVIII). (H) Pre-incubation of immobilized (5 interaction betweenand soluble(10 CLEC4M-Fc FVIII immobilized rFVIII (30 U/mL) with domain specific antibodies (100 with specific domain (30U/mL) immobilized rFVIII using a A1 antibodyanti-FVIII monoclonal asdescribed in Methods.(F) Preincubation of (2deleted) bound U/mL) toimmobilized (5 CLEC4M-Fc recombinant products (E) (100 Various FVIII (full-length soluble rFVIII U/mL). andB-domain complex and VWF-FVIII with plasma-derived soluble (10U/mL) NaClwash, andcompeted with calcium-dependent, a500 mM partiallyreversible(1 with mg/mL), blocked mannan This article is protectedThisarticle is byAll rightsreserved. copyright. interaction between(10 soluble CLEC4M-Fc CLEC4M-Fc (10 with binding toa BSA (10 to immobilized (10 CLEC4M-Fc μ assayin (A)Methods. as described Soluble rFVIII immobilized binds CLEC4M-Fcto (10 CLEC4M-Fc protein fusion was FVIII to characterized immunosorbent usingasolidphase ofFigure Binding 2. to recombinantFVIII CLEC4M-Fc. g/mL) in a dose-dependent withmanner an apparent Kd < 0.1 nM. (B) Soluble rFVIII binds μ g/mL) bound plasma-derivedFVIIIto U/mL) containing(1 (<1%VWF), andpdVWF-FVIII μ μ g/mL) normal pooledin defibrinated andVWF-deficient (D) plasma. The g/mL) binds toimmobilized deglycosylated U/mL).(30 (B) FVIII Soluble μ g/mL) control. (C) Soluble rFVIII (5U/mL) binds immobilized to μ g/mL) at low (<0.2 concentrations U/mL) when compared μ g/mL) and immobilized FVIII (30 U/mL) was μ

g/mL). (G) Immobilized CLEC4M-Fc μ g/mL). FVIII binding was detected The binding recombinant of μ g/mL) attenuated the μ . (A) . (A) Soluble g/mL, Affinity μ g/mL, Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. (chloroquine inhibitors with two lysosomal and (CLQ), disulfide NH expressing cellswere preincubatedwithrFVIII 5hours, andincubated 2U/mL for washed, CLEC4M- bylysosomes/proteasomes. FVIII of Catabolism independentexperiments. (D) FVIII (green), DAPI (blue), colocalization are Images (yellow). representative of5-6 colocalization (yellow). (C)Colocalization Lysosomal with LAMP-1 lysosomes. (red),marker with late endosomes. Late endosomal FVIIIRab-9 (red), marker (green), DAPI (blue), antigen1 (EEA1) (red), (green),FVIII DAPI colocalization (blue), Colocalization(yellow). (B) endosomal early Early endosomes. Colocalizationwith immunofluorescence. (A) pdVWF(2U/mLor VWF, 2.4:1 VWF-FVIII complex) 15, 30, and60 andimagedfor minutes expressing cells. Figure 5. Intracellular trafficking and catabolism of FVIII and VWF by CLEC4M- ofpernumber CLEC4M view. of ± cells expressing field **. SEM,= *, p<0.05 p<0.001= wereusing ImageProsoftware quantifiedcell-bound VWF and or FVIII normalized was to the DAPI (blue), colocalization (yellow). (CandD) Images 4-5 independentfrom experiments experiments. wereCells with imaged CLEC4M immunofluorescence; (red),FVIII (green), hydrate (DH)as described Im in Methods. inhibitors of endocytic pathways, methyl- with VWF-FVIII 2.4:1 withVWF, pre-treatedcellsCLEC4M-expressing (1hour) complex) (AandB) inMethods. houras described Association rFVIIIof (2 U/mL pdVWF (2U/mL) or pathway. Figure 4. Internalization of FVIII by CLEC4M by clathrin-coated a pit-dependent SEM, *p<0.05, **p<0.001. withpreincubated For (100µg/mL). all assays,mannan experiments, ± n=3-5 independent endosomes. Late endosomal Rab-9(red), (green), VWFmarker DAPI colocalization (blue), independent experiments. ± SEM, **p<0.001. (E)*p<0.05, Colocalization withlate inhibitor FVIII (MG132) levelsfor 2 hours. werequantified by then n=3-5 ELISA, CLEC4M-expressing 1werewith cellsinhibitors preincubated endocytosis for CLEC4M-expressing HEK 293 cells were rFVIIIincubated with U/mL)(2 β -cyclodextrin -cyclodextrin pitstop-2(MBCD), (PS), dynasore ages are representative from 4-5 independent4-5 ages are representative from

4 Cl), Cl), and aproteasome Accepted Article This article is protectedThisarticle is byAll rightsreserved. copyright. DKOVWF/FVIII compared withmice VWF/FVIIIwith DKO cyclophosphamide. treated (200 expressing LSEC Half-life rFVIII human DKOof aVWF/FVIII in(E) U/kg) in mouse. humanexpressing (D)Association LSEC rFVIII with of KOCD31- inaFVIII mouse. a FVIII DKO (C) mouse. infused human Association of rFVIII (100 U/mouse) with a CD31- infused human rFVIII (100 with U/mouse) a CLEC4M-expressing hepatocyte aVWF- from alonereceiving pLIVEbackbone (n=16). vector ± of Association p<0.05 (B) = *. SEM, expressing CLEC4Mlevels hepatocytes inFVIII:C with on>10% of mice ascompared mice AssociationFigure 6. CLEC4M-expressing between cells and FVIII vivo. in experiments. DAPI colocalization (yellow).(blue), Images ofare representative 5-6 independent with(yellow). Colocalizationlysosomes.(F) Lysosomal LAMP-1 VWF (red), (green),marker

(A)

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