Receptor homodimerisation significantly prolongs the lifetime of ligand-induced crosslinking of CLEC-2 but not GPVI

Receptor homodimerisation significantly prolongs the lifetime of ligand-induced crosslinking of CLEC-2 but not GPVI | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Receptor homodimerisation significantly prolongs the lifetime of ligand-induced crosslinking of CLEC-2 but not GPVI Joanne Clark, Eleyna Martin, Alexandre Slater, Davide Calebiro, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7742583/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The ITAM receptors, GPVI and CLEC-2, are activated by a diversity of ligands and are targets for anti-platelet drugs. We used two-colour single-particle tracking to monitor GPVI and CLEC-2 diffusion and dimerisation in resting and stimulated CHO-K1 cells. GPVI and CLEC-2 were fluorescently labelled via SNAP and Halo tags. When expressed at ~10% of their level in platelets, GPVI and CLEC-2 are monomeric. Divalent and trivalent nanobody ligands induce GPVI and CLEC-2 homo-dimerisation, a reduction in free diffusion and an increase in immobile receptors proportionate to ligand valency. Dimers of CLEC-2 are longer-lived than those of GPVI despite a lower affinity of the monomeric nanobody that forms the backbone of the ligands. Recombinant monomeric CLEC-2 but not GPVI dimerises with an affinity (K D ) of 18.5 µM. Synergy between ligand-induced crosslinking and dimerisation underlies the longer lifetime of the CLEC-2 interactions. Targeting dimerisation may be an effective way to inhibit activation of CLEC-2 by all of its ligands. Biological sciences/Cell biology/Cellular imaging/Super-resolution microscopy Biological sciences/Cell biology/Cell signalling Health sciences/Cardiology/Cardiovascular biology/Platelets GPVI CLEC-2 ITAM hemITAM platelets nanobodies receptor clustering receptor kinetics single-molecule microscopy single-particle tracking Full Text Additional Declarations Yes there is potential Competing Interest. S.P.W. and A.S. have a patent for the anti-GPVI nanobody Nb2 (WO2022/136457). All other authors declare no conflicts of interest. Supplementary Files JO137GPVIbasaltracking.gif Supplementary Video GPVI basal tracking SupplementaryMaterialsv1.pdf Supplementary materials JO18CLEC2basaltracking.gif Supplementary Video CLEC-2 basal tracking JO591GPVINb22tracking.gif Supplementary Video GPVI Nb2-2 tracking JO18CLEC2basal.gif Supplementary Video CLEC-2 basal JO591GPVINb22.gif Supplementary Video GPVI Nb2-2 JO137GPVIbasal.gif Supplementary Video GPVI basal JO343CLEC2Nb42.gif Supplementary Video CLEC-2 Nb4-2 JO115CLEC2Nb43.gif Supplementary Video CLEC-2 Nb4-3 JO115CLEC2Nb43tracking.gif Supplementary Video CLEC-2 Nb4-3 tracking JO418GPVINb23.gif Supplementary Video GPVI Nb2-3 JO343CLEC2Nb42tracking.gif Supplementary Video CLEC-2 Nb4-2 tracking JO418GPVINb23tracking.gif Supplementary Video GPVI Nb2-3 tracking SupplementaryVideos.pptx Supplementary videos Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7742583","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":526004753,"identity":"7249259d-d6a8-4f64-a731-d80a0c0af8be","order_by":0,"name":"Joanne 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All other authors declare no conflicts of interest.","formattedTitle":"Receptor homodimerisation significantly prolongs the lifetime of ligand-induced crosslinking of CLEC-2 but not GPVI","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"GPVI, CLEC-2, ITAM, hemITAM platelets, nanobodies, receptor clustering, receptor kinetics, single-molecule microscopy, single-particle tracking","lastPublishedDoi":"10.21203/rs.3.rs-7742583/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7742583/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The ITAM receptors, GPVI and CLEC-2, are activated by a diversity of ligands and are targets for anti-platelet drugs. We used two-colour single-particle tracking to monitor GPVI and CLEC-2 diffusion and dimerisation in resting and stimulated CHO-K1 cells. GPVI and CLEC-2 were fluorescently labelled via SNAP and Halo tags. When expressed at ~10% of their level in platelets, GPVI and CLEC-2 are monomeric. Divalent and trivalent nanobody ligands induce GPVI and CLEC-2 homo-dimerisation, a reduction in free diffusion and an increase in immobile receptors proportionate to ligand valency. Dimers of CLEC-2 are longer-lived than those of GPVI despite a lower affinity of the monomeric nanobody that forms the backbone of the ligands. Recombinant monomeric CLEC-2 but not GPVI dimerises with an affinity (K\u003csub\u003eD\u003c/sub\u003e) of 18.5 µM. Synergy between ligand-induced crosslinking and dimerisation underlies the longer lifetime of the CLEC-2 interactions. 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