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 SummaryBackgroundPlatelet 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.ObjectiveTo 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.ResultsUpon 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 α2β1 was localized to collagen fibers close to GPVI dimer clusters. GPVI clustering depends on a dynamic actin cytoskeleton.ConclusionsPlatelet 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.

Soluble GPVI is elevated in injured patients: shedding is mediated by fibrin activation of GPVI

“…14 On resting platelets, GPVI is predominantly monomeric, but clusters and dimerizes on activation, [15][16][17] triggering ITAM signaling involving Src family kinases. 17,18 In hemostasis and thrombosis, GPVI supports platelet adhesion and aggregation.…”

“…17,18 In hemostasis and thrombosis, GPVI supports platelet adhesion and aggregation. GPVI is critical for the maintenance of vessel wall integrity in inflammation 19 by inhibiting neutrophil-induced vascular damage.…”

Activation‐induced changes in platelet surface receptor expression and the contribution of the large‐platelet subpopulation to activation

“…Platelet suspensions were aliquoted on poly‐ l ‐lysine–coated MatTek dishes and centrifuged (2000 rpm, 5 minutes) in a Mistral 2000. Dishes were washed with PBS, blocked with 0.5% bovine serum albumin (BSA)/PBS, reacted with Alexa Fluor‐647–labeled M148 (anti‐CD41 mAb) for 60 minutes, and processed for imaging with an FV300 IX81 laser‐scanning confocal microscope with a 60 oil immersion objective (Olympus UK) …”

Glycoprotein VI in securing vascular integrity in inflamed vessels

“…Those tools helped define GPVI as the primary receptor for platelet/collagen interactions and subsequent platelet activation . They have also made possible the demonstration that GPVI forms highly competent dimers at the platelet surface whose clustering upon adhesion to collagen further increases its avidity and downstream signaling . The signaling activity of GPVI depends on its physical association with the homodimeric γ chain common to Fc receptors (FcRγ), whose cytoplasmic domains each bears a signaling motif known as immunoreceptor tyrosine‐based activation motif (ITAM) .…”