title: Clustering of glycoprotein VI (GPVI) dimers upon adhesion to collagen as a mechanism to regulate GPVI signaling in platelets.
abstract: Essentials 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 α2β1, 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 α2β1was 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.
authors.affiliation: Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.|Centre for Membrane Proteins and Receptors (COMPARE), College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.
authors.forename: N S
authors.initials: NS
authors.lastname: Poulter
authors.affiliation: Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.
authors.forename: A Y
authors.initials: AY
authors.lastname: Pollitt
authors.affiliation: Department of Physics and Randall Division of Cell and Molecular Biophysics, King's College London, London, UK.
authors.forename: D M
authors.initials: DM
authors.lastname: Owen
authors.affiliation: Department of Cancer Biology and Therapeutics, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia.
authors.forename: E E
authors.initials: EE
authors.lastname: Gardiner
authors.affiliation: Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria, Australia.
authors.forename: R K
authors.initials: RK
authors.lastname: Andrews
authors.affiliation: Research Department, Chemo-Sero-Therapeutic Research Institute, Kaketsuken, Kumamoto, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Shimizu
authors.affiliation: Research Department, Chemo-Sero-Therapeutic Research Institute, Kaketsuken, Kumamoto, Japan.
authors.forename: D
authors.initials: D
authors.lastname: Ishikawa
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: D
authors.initials: D
authors.lastname: Bihan
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: R W
authors.initials: RW
authors.lastname: Farndale
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: M
authors.initials: M
authors.lastname: Moroi
authors.affiliation: Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.|Centre for Membrane Proteins and Receptors (COMPARE), College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.
authors.forename: S P
authors.initials: SP
authors.lastname: Watson
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: S M
authors.initials: SM
authors.lastname: Jung
mesh_terms: D000199:Actins / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism*; D001808:Blood Vessels / Q000293:injuries; D002448:Cell Adhesion; D003094:Collagen / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D015539:Platelet Activation; D010973:Platelet Adhesiveness; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D055503:Protein Multimerization; D015398:Signal Transduction
chemical_list: D000199:Actins; D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI; D003094:Collagen
keywords: glycoprotein; platelet activation; platelet adhesiveness; platelet membrane glycoproteins; receptors, collagen