Platelet collagen receptor Glycoprotein VI‐dimer recognizes fibrinogen and fibrin through their D‐domains, contributing to platelet adhesion and activation during thrombus formation

Abstract

Essentials Glycoprotein VI (GPVI) binds collagen, starting thrombogenesis, and fibrin, stabilizing thrombi.GPVI‐dimers, not monomers, recognize immobilized fibrinogen and fibrin through their D‐domains.Collagen, D‐fragment and D‐dimer may share a common or proximate binding site(s) on GPVI‐dimer.GPVI‐dimer–fibrin interaction supports spreading, activation and adhesion involving αIIbβ3. SummaryBackgroundPlatelet collagen receptor Glycoprotein VI (GPVI) binds collagen, initiating thrombogenesis, and stabilizes thrombi by binding fibrin.ObjectivesTo determine if GPVI‐dimer, GPVI‐monomer, or both bind to fibrinogen substrates, and which region common to these substrates contains the interaction site.MethodsRecombinant GPVI monomeric extracellular domain (GPVI ex) or dimeric Fc‐fusion protein (GPVI‐Fc2) binding to immobilized fibrinogen derivatives was measured by ELISA, including competition assays involving collagenous substrates and fibrinogen derivatives. Flow adhesion was performed with normal or Glanzmann thrombasthenic (GT) platelets over immobilized fibrinogen, with or without anti‐GPVI‐dimer or anti‐αIIbβ3.ResultsUnder static conditions, GPVI ex did not bind to any fibrinogen substrate. GPVI‐Fc2 exhibited specific, saturable binding to both D‐fragment and D‐dimer, which was inhibited by mFab‐F (anti‐GPVI‐dimer), but showed low binding to fibrinogen and fibrin under our conditions. GPVI‐Fc2 binding to D‐fragment or D‐dimer was abrogated by collagen type III, Horm collagen or CRP‐XL (crosslinked collagen‐related peptide), suggesting proximity between the D‐domain and collagen binding sites on GPVI‐dimer. Under low shear, adhesion of normal platelets to D‐fragment, D‐dimer, fibrinogen and fibrin was inhibited by mFab‐F (inhibitor of GPVI‐dimer) and abolished by Eptifibatide (inhibitor of αIIbβ3), suggesting that both receptors contribute to thrombus formation on these substrates, but αIIbβ3 makes a greater contribution. Notably, thrombasthenic platelets showed limited adhesion to fibrinogen substrates under flow, which was further reduced by mFab‐F, supporting some independent GPVI‐dimer involvement in this interaction.ConclusionOnly dimeric GPVI interacts with fibrinogen D‐domain, at a site proximate to its collagen binding site, to support platelet adhesion/activation/aggregate formation on immobilized fibrinogen and polymerized fibrin.

Proteolytic processing of platelet receptors

“…Similarly, the fibrin‐binding site within GPVI is contentious. In one study, the GPVI‐fibrin interaction occurred only with GPVI in dimeric form and could be abrogated by pretreatment with collagen or CRP, implying at least partial overlap of the binding site for these ligands, however, fibrin‐GPVI monomer interactions and separate CRP and fibrin binding sites were proposed in another study . Under certain experimental conditions and in collaboration with αIIbβ3, the fibrin monomer component fibrinogen also can engage GPVI .…”

“…Fibrin is a more recently described ligand for GPVI and studies have demonstrated that while fibrin–GPVI interaction will generate intracellular signals, this signalling is not required for fibrin‐induced GPVI shedding . The fibrin interaction with GPVI is mediated by the D‐dimer region of fibrin and for GPVI shedding to occur, fibrin must be polymerized . Whether fibrin can bind platelet GPVI monomer or dimer remains a matter for debate; however, dimeric GPVI‐Fc fusion proteins do not engage fibrin .…”

“…The fibrin interaction with GPVI is mediated by the D‐dimer region of fibrin and for GPVI shedding to occur, fibrin must be polymerized . Whether fibrin can bind platelet GPVI monomer or dimer remains a matter for debate; however, dimeric GPVI‐Fc fusion proteins do not engage fibrin . Similarly, the fibrin‐binding site within GPVI is contentious.…”

“…In one study, the GPVI‐fibrin interaction occurred only with GPVI in dimeric form and could be abrogated by pretreatment with collagen or CRP, implying at least partial overlap of the binding site for these ligands, however, fibrin‐GPVI monomer interactions and separate CRP and fibrin binding sites were proposed in another study . Under certain experimental conditions and in collaboration with αIIbβ3, the fibrin monomer component fibrinogen also can engage GPVI . Understanding how these two GPVI ligands intersect and contribute to GPVI function is important, as selective disruption of one type of GPVI‐ligand interaction, either through competitive inhibition at the ligand‐binding site, or at the level of GPVI dimerization represents an enticing new approach to develop antiplatelet agents with minimal effects on hemostasis.…”

“…The cytoplasmic domain of FcRγ contains an immunoreceptor tyrosine activation motif (ITAM) and together the GPVI/FcRγ complex transmits ligand‐induced signalling events into the platelet by triggering phosphorylation of two tyrosine residues with the ITAM and subsequent activation of p72‐spleen tyrosine kinase (Syk) . Along with collagen, GPVI can bind laminin, fibrin, fibrinogen, histones, adiponectin, and the extracellular matrix metalloproteinase inducer (EMMPRIN) expressed on monocytes and leukocytes. Intact GPVI is also essential for efficient generation of thrombin at the platelet surface .…”