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dc.contributor.authorPoulter, NSen
dc.contributor.authorPollitt, AYen
dc.contributor.authorOwen, DMen
dc.contributor.authorGardiner, EEen
dc.contributor.authorAndrews, RKen
dc.contributor.authorShimizu, Hen
dc.contributor.authorIshikawa, Den
dc.contributor.authorBihan, Den
dc.contributor.authorFarndale, Richarden
dc.contributor.authorMoroi, Men
dc.contributor.authorWatson, SPen
dc.contributor.authorJung, Stephanieen
dc.date.accessioned2017-03-16T12:21:36Z
dc.date.available2017-03-16T12:21:36Z
dc.date.issued2017-03-02en
dc.identifier.issn1538-7933
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/263117
dc.description.abstractEssentials - Dimeric high-affinity collagen receptor glycoprotein VI (GPVI) is present on resting platelets. - Spatio-temporal organization of platelet GPVI-dimers was evaluated using advanced microscopy. - Upon platelet adhesion to collagenous substrates, GPVI-dimers coalesce to form clusters. - Clustering of GPVI-dimers may increase avidity and facilitate platelet activation SUMMARY: Background Platelet glycoprotein VI (GPVI) binding to subendothelial collagen exposed upon blood vessel injury initiates thrombus formation. Dimeric GPVI has high affinity for collagen, and occurs constitutively on resting platelets. Objective To identify higher-order oligomerization (clustering) of pre-existing GPVI dimers upon interaction with collagen as a mechanism to initiate GPVI-mediated signaling. Methods GPVI was located by use of fluorophore-conjugated GPVI dimer-specific Fab (antigen-binding fragment). The tested substrates include Horm collagen I fibers, soluble collagen III, GPVI-specific collagen peptides, and fibrinogen. GPVI dimer clusters on the platelet surface interacting with these substrates were visualized with complementary imaging techniques: total internal reflection fluorescence microscopy to monitor real-time interactions, and direct stochastic optical reconstruction microscopy (dSTORM), providing relative quantification of GPVI cluster size and density. Confocal microscopy was used to locate GPVI dimer clusters, glycoprotein Ib, integrin α$_{2}$β$_{1}$ , and phosphotyrosine. Results Upon platelet adhesion to all collagenous substrates, GPVI dimers coalesced to form clusters; notably clusters formed along the fibers of Horm collagen. dSTORM revealed that GPVI density within clusters depended on the substrate, collagen III being the most effective. Clusters on fibrinogen-adhered platelets were much smaller and more numerous; whether these are pre-existing oligomers of GPVI dimers or fibrinogen-induced is not clear. Some GPVI dimer clusters colocalized with areas of phosphotyrosine, indicative of signaling activity. Integrin α$_{2}$β$_{1}$ was localized to collagen fibers close to GPVI dimer clusters. GPVI clustering depends on a dynamic actin cytoskeleton. Conclusions Platelet adhesion to collagen induces GPVI dimer clustering. GPVI clustering increases both avidity for collagen and the proximity of GPVI-associated signaling molecules, which may be crucial for the initiation and persistence of signaling.
dc.description.sponsorshipThese studies were supported by a Project Grant (PG/10/011/28199, to S. M. Jung, M. Moroi, R. W. Farndale, and S. P. Watson) and a Special Project Grant (SP/13/7/30575, to S. M. Jung) from the British Heart Foundation and a Wellcome Trust Biomedical Resource Grant (09440/Z/10/Z, to R. W. Farndale). S. P. Watson and N. S. Poulter are supported by the British Heart Foundation (CH/03/003). A. Y. Pollitt was funded by Wellcome Trust Grant 088410 (to S. P. Watson).
dc.languageengen
dc.language.isoenen
dc.publisherWiley
dc.rightsAttribution 4.0 Internationalen
dc.rightsAttribution 4.0 Internationalen
dc.rightsAttribution 4.0 Internationalen
dc.rightsAttribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectglycoproteinen
dc.subjectplatelet activationen
dc.subjectplatelet adhesivenessen
dc.subjectplatelet membrane glycoproteinsen
dc.subjectreceptors, collagenen
dc.titleClustering of glycoprotein VI (GPVI) dimers upon adhesion to collagen as a mechanism to regulate GPVI signaling in platelets.en
dc.typeArticle
prism.endingPage564
prism.issueIdentifier3en
prism.publicationDate2017en
prism.publicationNameJournal of Thrombosis and Haemostasisen
prism.startingPage549
prism.volume15en
dc.identifier.doi10.17863/CAM.8422
dcterms.dateAccepted2016-12-29en
rioxxterms.versionofrecord10.1111/jth.13613en
rioxxterms.versionVoRen
rioxxterms.licenseref.urihttp://creativecommons.org/licenses/by/4.0/en
rioxxterms.licenseref.startdate2017-03-02en
dc.contributor.orcidFarndale, Richard [0000-0001-6130-8808]
dc.identifier.eissn1538-7836
rioxxterms.typeJournal Article/Reviewen
pubs.funder-project-idBritish Heart Foundation (RG/15/4/31268)
pubs.funder-project-idBritish Heart Foundation (PG/10/011/28199)
cam.issuedOnline2017-01-06en


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Attribution 4.0 International
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