Binding Of Glycoprotein VI Research Articles

Targeting platelet GPVI with glenzocimab: a novel mechanism for inhibition.

Platelet glycoprotein VI (GPVI) is attracting interest as a potential target for the development of new antiplatelet molecules with a low bleeding risk. GPVI binding to vascular collagen initiates thrombus formation and GPVI interactions with fibrin promote the growth and stability of the thrombus. In this study, we show that glenzocimab, a clinical stage humanized antibody fragment (Fab) with a high affinity for GPVI, blocks the binding of both ligands through a combination of steric hindrance and structural change. A cocrystal of glenzocimab with an extracellular domain of monomeric GPVI was obtained and its structure determined to a resolution of 1.9 Å. The data revealed that (1) glenzocimab binds to the D2 domain of GPVI, GPVI dimerization was not observed in the crystal structure because glenzocimab prevented D2 homotypic interactions and the formation of dimers that have a high affinity for collagen and fibrin; and (2) the light variable domain of the GPVI-bound Fab causes steric hindrance that is predicted to prevent the collagen-related peptide (CRP)/collagen fibers from extending out of their binding site and preclude GPVI clustering and downstream signaling. Glenzocimab did not bind to a truncated GPVI missing loop residues 129 to 136, thus validating the epitope identified in the crystal structure. Overall, these findings demonstrate that the binding of glenzocimab to the D2 domain of GPVI induces steric hindrance and structural modifications that drive the inhibition of GPVI interactions with its major ligands.

Dimers of the platelet collagen receptor glycoprotein VI bind specifically to fibrin fibers during clot formation, but not to intact fibrinogen

ObjectiveThe platelet collagen receptor glycoprotein VI (GPVI) has an independent role as a receptor for fibrin produced via the coagulation cascade. However, various reports of GPVI binding to immobilized fibrin(ogen) are not consistent. As a collagen receptor, GPVI‐dimer is the functional form, but whether GPVI dimers or monomers bind to fibrin remains controversial. To resolve this, we analyzed GPVI binding to nascent fibrin clots, which more closely approximate physiological conditions. Methods and resultsELISA using biotinyl‐fibrinogen immobilized on streptavidin‐coated wells indicated that GPVI dimers do not bind intact fibrinogen. Clots were formed by adding thrombin to a mixture of near‐plasma level of fibrinogen and recombinant GPVI ectodomain: GPVI dimer (GPVI‐Fc2 or Revacept) or monomer (GPVI‐His: single chain of Revacept GPVI domain, with His tag). Clot‐bound proteins were analyzed by SDS‐PAGE/immunoblotting. GPVI‐dimer bound to noncrosslinked fibrin clots with classical one‐site binding kinetics, with µM‐level KD, and to crosslinked clots with higher affinity. Anti‐GPVI‐dimer (mFab‐F) inhibited the binding. However, GPVI‐His binding to either type of clot was nonsaturable and nearly linear, indicating very low affinity or nonspecific binding. In clots formed in the presence of platelets, clot‐bound platelet‐derived proteins were integrin αIIbβ3, present at high levels, and GPVI. ConclusionsWe conclude that dimeric GPVI is the receptor for fibrin, exhibiting a similar KD to those obtained for its binding to fibrinogen D‐fragment and D‐dimer, suggesting that fibrin(ogen)'s GPVI‐binding site becomes exposed after fibrin formation or cleavage to fragment D. Analysis of platelets bound to fibrin clots indicates that platelet GPVI binds to fibrin fibers comprising the clot.

GPVI (Glycoprotein VI) Interaction With Fibrinogen Is Mediated by Avidity and the Fibrinogen αC-Region.

GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth. Graphic Abstract: A graphic abstract is available for this article.

Computer prediction of paratope on antithrombotic antibody 10B12 and epitope on platelet glycoprotein VI via molecular dynamics simulation.

BackgroundInteraction between immunoglobulin-like receptor glycoprotein VI (GPVI) and collagen plays a central role in platelet activation and sequent firm adhesion. Of various antithrombotic agents targeting GPVI, antibody 10B12 is of great potential to block the GPVI-collagen interaction, but less is known about 10B12 paratope and GPVI epitope.MethodsAlong the pathway in the computer strategy presented in our previous work, the 10B12/GPVI complex model was constructed through homology modeling and rigid-body docking, and the molecular dynamics (MD) simulation was used to detect the paratope residues on 10B12 and their partners on GPVI. Quantified by free and steered MD simulations, the stabilities of hydrogen bonds and salt bridges were used to rank the contributions of interface residues to binding of 10B12 and GPVI.ResultsWe predicted 12 key and seven dispensable residues in interaction of 10B12 to GPVI with present computational procedure. Besides of the 12 key residues, two are epitope residues (LYS41 and LYS59) which had been identified by previous mutation experiments, and others, including four epitope residues (ARG38, SER44, ARG46 and TYR47 on GPVI) and six paratope residues (GLU1, ASP98, GLU102, ASP107, ASP108 and ASP111 on 10B12), were newly found and also might be important for the 10B12–GPVI binding. The seven predicted dispensable residues on GPVI were had been illustrated in previous mutation experiments.ConclusionsThe present computer strategy combining homology modeling, rigid body docking and MD simulation was illustrated to be effective in mapping paratope on antithrombotic antibody 10B12 to epitope on GPVI, and have large potential in drug discovery and antibody research.

GPVI Interaction with Polymerized Fibrin Boosts Thrombin Generation and Thrombus Growth

Background: Fibrin, the end product of the coagulation cascade, consolidates the platelet plug at site of thrombosis: polymerized fibrin supports platelet adhesion under low and high shear rate conditions (Hantgan RR et al., Thromb Haemost 1992) and triggers platelet procoagulant activity (Beguin S et al., Blood 1999). These responses are largely independent of the integrin αIIβ3 and are carried by a yet ill-defined receptor. Platelet glycoprotein VI (GPVI) has a well-established key role in the initiation of thrombosis since it supports collagen-mediated platelet activation but it has recently been recognized to interact with other macromolecules such as fibronectin, vitronectin and laminins. We hypothesized GPVI could be the “missing” platelet receptor of fibrin.Aim of the study: to challenge the hypothesis that glycoprotein VI (GPVI) could be a functional fibrin receptorMethods: Thrombin generation was measured using calibrated automated thrombogram (CAT) in PRP from healthy volunteers, four GPVI-deficient patients and one patient with a fibrinogen deficiency. CAT was also performed on washed platelets mixed with prothrombin complex (FII, FVII, FIX, FX), antithrombin and fibrinogen. GPVI was blocked using the Fab of the monoclonal antibody 9O12. Fibrin polymerization was blocked using the GPRP peptide. GPVI binding to fibrin was measured in vitro using recombinant soluble GPVI (GPVI-Fc). Flow based adhesion assays were performed in capillary chambers coated with polymerized fibrin at variable shear rates and platelet morphological changes analyzed by scanning electron microscopy. The formation of fibrin-platelet thrombi was visualized by perfusing recalcified blood containing A647 fibrinogen in flow chambers (Vena8 Fluoro+ Cellix) coated with collagen and tissue factor. In a second step, the perfusion of hirudinated blood in which platelets were stained by A488-RAM1 allowed to visualize platelet recruitment by fibrin rich clots.Results: Thrombin generation triggered by tissue factor was impaired in the PRP of patients with a GPVI deficiency or in the presence of the Fab 9O12 as indicated by a respective decrease in the peak height of 45 and 25% as compared to controls. This effect was observed regardless the trigger of thrombin generation and required platelet activation. Measuring thrombin generation in a purified system showed that fibrinogen dose-dependently increased the thrombin peak by up to 150% at 3 mg/mL but the Fab 9O12 blunted this effect. Moreover, the Fab 9O12 had no effect on thrombin generation in the PRP of a fibrinogen-deficient patient confirming a GPVI/fibrin(ogen)interplay. Blocking fibrin polymerization by GPRP reduced the thrombin peak in normal PRP, in fibrinogen-supplemented PRP of the fibrinogen-deficient patient and in purified conditions. In contrast GPRP had no effect on the thrombin peak in normal PRP containing the Fab 9O12 and in the PRP of GPVI-deficient patients. The proof that GPVI specifically interacts with fibrin was obtained in a binding assay showing a dose-dependent binding of GPVI-Fc to fibrin polymers that was reversed by the Fab 9O12. Platelets adhered to polymerized fibrin resulting in platelet shape change and exposure of phosphatidylserine. Platelet adhesion on a fibrin network was observed at low (300 s-1) and high (1500 s-1) shear rates with the formation of small contractile thrombi. Adhesion was decreased by 62% for 9O12-treated platelets and by 43% with the blood of GPVI-deficient mice as compared to controls. Importantly, lack of GPVI or its blockade decreased stationary adhesion indicating that GPVI is required to stabilize the interactions between platelets and fibrin. Finally when hirudinated blood was perfused at a shear rate of 1500 s-1 onto preformed fibrin-rich clots, the Fab 9O12 decreased the recruitment of platelets by up to 93%.Conclusions: Here we show for the first time that GPVI acts as a receptor for polymerized fibrin with two major functions: GPVI interaction with polymerized fibrin triggers (i) a new loop amplifying thrombin generation and (ii) platelet recruitment at the clot surface. These, so far, unrecognized properties of GPVI confer it a key role in the maturation of the thrombus by facilitating its growth and stabilization in addition to its well-known effect in the initiation of thrombus formation. DisclosuresJandrot-Perrus:Acticor Biotech: Other. Gachet:Acticor Biotech: Other.

Constitutive Dimerization of Glycoprotein VI (GPVI) in Resting Platelets Is Essential for Binding to Collagen and Activation in Flowing Blood

The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (K(d)) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triple-helical (GPO)(10)-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ~29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ~39 and ~44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation.

Platelet Glycoprotein VI Dimerization, an Active Process Inducing Receptor Competence, Is an Indicator of Platelet Reactivity

The immune receptor homologue glycoprotein VI (GPVI)/FcR receptor γ chain complex is primarily responsible for platelet activation by collagen. There is growing evidence that optimal binding of GPVI to collagen depends on the assembly of GPVI dimers. The valence of GPVI on resting platelets needs to be clearly established because platelet avidity for collagen would be greater if GPVI is constitutively expressed as a dimer than as a monomer. Using a monoclonal antibody (9E18) that preferentially binds to GPVI dimers, we found that GPVI was maintained in a monomeric form on human resting platelets under the control of intraplatelet cAMP concentration. Activation by soluble agonists or von Willebrand factor induced a shift toward GPVI dimerization related to increased platelet adhesion to collagen. A correlation between platelet binding of 9E18 and P-selectin exposure was observed in patients experiencing coronary artery disease, and antagonists of the ADP receptor P2Y12 limited ADP-induced GPVI dimerization. The rapid assembly of highly competent dimers of GPVI at sites of vascular lesion represents an important step in the sequence of events leading to platelet activation by collagen. GPVI dimers could represent a new marker to analyze platelet reactivity.

Convulxin Forms a Dimer in Solution and Can Bind Eight Copies of Glycoprotein VI: Implications for Platelet Activation

Convulxin (CVX) is a C-type lectin-like protein from the venom of the South American rattlesnake that functions as a potent agonist of the platelet collagen receptor glycoprotein VI (GPVI). Although CVX is widely used as a platelet agonist, the molecular basis for its extremely high potency is not clear. In order to delineate possible mechanisms for CVX-induced GPVI activation, we used analytical ultracentrifugation to determine the assembly state of CVX in solution and surface plasmon resonance in order to understand the affinity, kinetics, and stoichiometry of GPVI binding to CVX. We show here that CVX exists in solution as a dimer of alpha4beta4 rings, yielding eight potential binding sites for GPVI. Binding studies confirm that all eight sites are able to bind GPVI tightly, each with high picomolar or low nanomolar affinity. Reanalysis of previously determined crystal structures of CVX revealed the dimer in both structures. The dimeric nature of CVX and its ability to bind eight GPVI molecules suggest that it might be capable of binding to GPVI expressed on two opposing surfaces. Agglutination assays using GPVI-coated beads confirm that CVX is able to bridge distinct GPVI-coated surfaces and indicate that CVX agglutination of platelets is dependent on GPVI binding. Thus, in addition to clustering up to eight GPVI receptors, CVX may facilitate platelet activation by bridging platelets directly.

Novel synthetic collagen fibers, poly(PHG), stimulate platelet aggregation through glycoprotein VI

Novel synthetic collagen fibers, poly(PHG) made by polycondensation of Pro-Hyp-Gly, spontaneously assume polymeric structure with molecular weights greater than 105. Its application for biomaterials has been explored, but that for a platelet agonist has not been investigated. Poly(PHG)-induced platelet aggregation independently of thromboxane A2 and integrin α2β1. Poly(PHG)-induced tyrosine phosphorylation of glycoprotein VI (GPVI)-related molecules and failed to activate GPVI/FcRγ-deficient platelets. Binding of GPVI to poly(PHG) was confirmed by a surface plasmon resonance spectroscopy, suggesting that poly(PHG) activates platelets through GPVI. Poly(PHG) is an useful research tool to investigate GPVI-mediated signals and a substitute for collagen in platelet functional assays.

Identification and Characterization of a Human Platelet Glycoprotein VI Peptidomimetic Permitting Molecular Imaging of Fibrosis.

Glycoprotein VI (GPVI), the main receptor for platelet activation by collagen, has been shown to play an important role in thrombosis, vascular remodelling and atherothrombosis. GPVI which belongs to the immunoglobulin receptor family, binds to fibrillar type I and type III collagens of vascular as well as non-vascular origin. 9O12.2, a high affinity monoclonal antibody directed to the GPVI extracellular domain, blocks GPVI binding to collagen and possess antithrombotic properties (Ohlmann et al J.Thromb. Haemost. 2008,6:1013). We have hypothesized that the 9O12.2 epitope overlaps, at least in part, with the collagen-binding site on GPVI and (ii) that molecules mimicking the 9O12.2 epitope can be expected to be antithrombotic by competing with platelet GPVI for binding to collagen and/or to act as tracers for collagen in vivo. A bacterial random 12 mer cyclic peptide library was screened against the 9O12.2 IgG. Twenty clones were selected. Sequencing the inserts revealed 9 peptidic motifs with 7 identical residues. One sequence was selected to synthesize a biotin-coupled constrained peptide. (designated collagelin). Surface plasmon resonance (SPR) analysis showed that 9O12.2 IgG bound to immobilized collagelin (KD 10−6M) and that binding was inhibited in the presence of soluble recombinant (sr)GPVI or after disulfide bridge reduction as expected for a molecule mimicking the 9O12.2 epitope known to be conformational (Lecut et al. J.Biol.Chem. 2004, 279:52293). Using SPR and solid phase assays, we observed that collagelin bound to immobilized fibrillar collagen (KD10−7M) and that binding was inhibited by 9O12.2 IgG and by rsGPVI, indicating that collagelin mimics at least in part the collagen-binding site of GPVI. Collagelin did not inhibit collagen-induced platelet aggregation in vitro. However, histochemical analysis demonstrated that it bound to collagen on sections of rat aortas and of rat tail tendon. We then hypothesized that collagelin could be retained in vivo at sites of collagen accumulation, thus allowing isotopic imaging of fibrosis. Collagelin and a control peptide (same size and cyclic,) were labeled either indirectly using 99mTc-streptavidin or directly with 99mTc and iv injected into rats presenting fibrotic scars of myocardial infarction. Radiolabeled collagelin uptake in fibrosis areas was demonstrated in vivo by planar and tomographic scintigraphy. Mean heart-to-lung ratios were of 2.76±0.36 and 2.08±0.17 for 99m Tc-streptavidin-coupled collagelin and 99m Tc-collagelin respectively. Ex vivo, autoradiography on frozen heart sections showed a clear uptake of labeled-collagelin in the infarct collagen-rich scars with mean scar to remote myocardium activity ratios of 2.52±0.2 and 2.92±.053 for 99mTc-streptavidin-coupled collagelin and 99mTc-collagelin respectively as compared with 1.82±0.32 and 1.61±0.23 for the control peptides (p<0.006 and 0.01). In conclusion, we have produced a peptide which partly mimics the collagen binding site of GPVI, specifically binds to collagen and appears to be a specific tool for direct targeting of collagen in vitro and in vivo. Collagelin or derived molecules thus potentially have a large field of applications, as a tracer of fibrotic lesions, in non-invasive vascular as vascular pathologies.

Inhibition of collagen-induced platelet aggregation by anopheline antiplatelet protein, a saliva protein from a malaria vector mosquito

During blood feeding, mosquitoes inject saliva containing a mixture of molecules that inactivate or inhibit various components of the hemostatic response to the bite injury as well as the inflammatory reactions produced by the bite, to facilitate the ingestion of blood. However, the molecular functions of the individual saliva components remain largely unknown. Here, we describe anopheline antiplatelet protein (AAPP) isolated from the saliva of Anopheles stephensi, a human malaria vector mosquito. AAPP exhibited a strong and specific inhibitory activity toward collagen-induced platelet aggregation. The inhibitory mechanism involves direct binding of AAPP to collagen, which blocks platelet adhesion to collagen and inhibits the subsequent increase in intracellular Ca(2+) concentration ([Ca(2+)]i). The binding of AAPP to collagen effectively blocked platelet adhesion via glycoprotein VI (GPVI) and integrin alpha(2)beta(1). Cell adhesion assay showed that AAPP inhibited the binding of GPVI to collagen type I and III without direct effect on GPVI. Moreover, intravenously administered recombinant AAPP strongly inhibited collagen-induced platelet aggregation ex vivo in rats. In summary, AAPP is a malaria vector mosquito-derived specific antagonist of receptors that mediate the adhesion of platelets to collagen. Our study may provide important insights for elucidating the effects of mosquito blood feeding against host hemostasis.

Studies of the Affinity and Stoichiometry of Convulxin Binding to Glycoprotein VI: Clues into Its Potent Agonism.

The primary cell-surface receptor that mediates platelet activation upon exposure to collagen is glycoprotein VI (GPVI). GPVI can be activated by fibrous collagen, soluble collagen mimics, or several snake venom proteins. Upon binding of these agonists, GPVI signaling activates key integrins and triggers secretion of platelet granule contents, leading in turn to activation of neighboring circulating platelets. One of the most potent GPVI agonists from snake venom is convulxin (CVX), an α4β4 C-type lectin-like protein from the South American rattlesnake, Crotalus durissus terrificus. Using analytical ultracentrifugation, we have discovered that CVX exists in solution as a stable dimer of α4β4 rings, creating eight potential binding sites for GPVI. Using a combination of kinetic and equilibrium surface plasmon resonance binding experiments, we show that CVX binds GPVI with high picomolar to low nanomolar affinity at each site, and that all eight sites are likely to be available for binding. Analysis of the previously published crystal structures of CVX suggests that its potency could be due to both high-affinity binding and clustering of GPVI on a single platelet as well as potential bridging of adjacent platelets via the dimeric CVX architecture.

Selective Blockade of Glycoprotein VI Clustering on Collagen Helices

Platelet activation by collagen relies on the interaction of the receptor glycoprotein VI (GPVI) with collagen helices. We have previously generated two recombinant single chain human antibodies (scFvs) to human GPVI. The first, 10B12, binds to the collagen-binding site on the apical surface between the two immunoglobulin-like domains (D1D2) of the receptor and so directly inhibits GPVI function. The second, 1C3, binds D1D2 independently of 10B12 and has been shown to have a more subtle effect on platelet responses to collagen. Here we have shown that 1C3 potentiates the effect of 10B12 on platelet aggregation induced by collagen and cross-linked collagen-related peptide (CRP-XL). We investigated this by measuring the effect of both scFvs on the binding of D1D2 to immobilized collagen and CRP. As expected, 10B12 completely inhibited binding of GPVI to each ligand in a dose-dependent manner. However, 1C3 inhibited only a proportion of GPVI binding to its ligands, implying that it interferes with another aspect of ligand recognition by GPVI. To further understand the mode of inhibition, we used a unique set of CRPs in which the content of critical glycine-proline-hydroxyproline (GPO) triplets was varied in relation to an "inert" scaffold sequence of GPP motifs. We observed that a stepwise increase in D1D2 binding with (GPO)(2) content was blocked by 1C3. Together these results indicate that 1C3 inhibits clustering of the immunoglobulin-like domains of GPVI on collagen/CRPs, a conclusion that is supported by mapping the 1C3 epitope to the region including isoleucine 148 in D2.

Gain- and loss-of-function mutants confirm the importance of apical residues to the primary interaction of human glycoprotein VI with collagen.

By site-directed mutagenesis of recombinant receptor fragments, we have previously identified residue lysine59 of the platelet collagen receptor glycoprotein VI (GPVI) as being critical for its interaction with the synthetic ligand collagen-related peptide (CRP) and the inhibitory phage antibody 10B12. Lysine59 is proposed to lie on the apical surface of the receptor near the linker joining the two immunoglobulin (Ig)-like extracellular domains. Recently, others have postulated the involvement of a portion of the first domain distant from the interdomain hinge as being involved in an extended collagen-binding site. To extend our knowledge of the primary collagen-binding site of GPVI, a number of neighboring residues on the apical surface of recombinant soluble GPVI were mutated to alanine and binding of these mutants, as well as the lysine59 mutant, to fibrillar collagen was measured. Binding of recombinant GPVI to collagen, like CRP, was dramatically reduced by the mutation of residue lysine59 to glutamate. Remarkably, the mutation of residues arginine60 in domain one and arginine166 in domain two, individually to alanine, which had no significant affect on CRP binding, reduced binding of recombinant GPVI to collagen. Mutation of the residue lysine41 to alanine dramatically increased binding to both CRP and collagen. This mutation abolished 10B12 binding, confirming its position in the epitope of our inhibitory phage antibody. Residues lysine59, arginine60, and arginine166, from both Ig-like domains of GPVI, are critical for collagen binding by the receptor. This provides additional evidence for a basic patch on the apical surface of the receptor as the primary collagen-binding site of GPVI.

Identification of Residues within Human Glycoprotein VI Involved in the Binding to Collagen: EVIDENCE FOR THE EXISTENCE OF DISTINCT BINDING SITES

Glycoprotein VI (GPVI) has a crucial role in platelet responses to collagen. Still, little is known about its interaction with its ligands. In binding assays using soluble or cell-expressed human GPVI, we observed that (i) collagen, and the GPVI-specific ligands collagen-related peptides (CRP) and convulxin, competed with one another for the binding to GPVI and (ii) monoclonal antibodies directed against the extracellular part of the human receptor displayed selective inhibitory properties on GPVI interaction with its ligands. Monoclonal antibody 9E18 strongly reduced the binding of GPVI to collagen/CRP, 3F8 inhibited its interaction with convulxin, whereas 9O12 prevented all three interactions. These observations suggest that ligand-binding sites are distinct, exhibiting specific features but at the same time also sharing some common residues participating in the recognition of these ligands. The epitope of 9O12 was mapped by phage display, along with molecular modeling of human GPVI, which allowed the identification of residues within GPVI potentially involved in ligand recognition. Site-directed mutagenesis revealed that valine 34 and leucine 36 are critical for GPVI interaction with collagen and CRP. The loop might thus be part of a collagen/CRP-binding site.

Human Platelet Glycoprotein VI: Identification of Residues Involved in the Binding to Collagen.

Glycoprotein VI (GPVI), an essential platelet collagen receptor, belongs to the immunoglobulin (Ig) superfamily and presents two Ig-like loops in its extracellular domain (EC1, EC2). Little is known about the structures involved in GPVI binding to collagen and to its specific ligands, convulxin and collagen related peptides (CRP). Our aim was to characterize these sites using different approaches: competition assays, monoclonal antibodies (MoAb) directed against the GPVI extracellular par, phage display, molecular modelling and site-directed mutagenesis. In binding assays using soluble or cell-expressed human GPVI, we observed that: (i) collagen, CRP and convulxin, competed with one another for the binding to GPVI; (ii) GPVI interaction with either ligand is inhibited by at least two MoAb directed to different independent epitopes, and (iii) MoAb displayed selective inhibitory properties on GPVI interaction with each ligand. These observations indicate that the binding sites on GPVI for the diverse ligands seem to be distinct, exhibiting specific features but at the same time sharing common elements. MoAb 9O12 is a potent inhibitor of GPVI binding to collagen. Its epitope was mapped by screening a 15 mer linear peptide library. This allowed the identification of a motif of 7 residues of which 5 are conserved and that presented a relative homology with the GPVI 30–40 sequence. In a computer model, these residues appeared in close vicinity on a surface loop exposed at the surface of the molecule and accessible to ligands. Site-directed mutagenesis revealed that Val34 and Leu36 are critical for GPVI interaction with collagen and CRP. This study allowed the identification of residues within a loop on GPVI EC1 that might be part of a collagen/CRP-binding site.

Platelet receptor glycoprotein VI-mediated adhesion to type I collagen under hydrodynamic flow.

Glycoprotein VI (GPVI) is a platelet receptor that directly binds collagen. It has been shown by expressing GPVI in rat basophilic leukemia (RBL-2H3) cells that GPVI mediates adhesion to type I collagen under static conditions. However, the ability of GPVI to secure adhesion to collagen type I under flow has not been measured. We studied the interaction of GPVI and type I collagen under hydrodynamic flow using RBL-2H3 cells transfected with the GPVI receptor. We found that GPVI-expressing RBL-2H3 cells adhere to collagen under flow, significantly more so than non-GPVI-expressing RBL-2H3 cells. Inhibition of GPVI by the 11A12 anti-GPVI antibody significantly blocks adhesion to collagen, indicating that GPVI specifically interacts with collagen. Probing the role of signaling in GPVI binding to collagen, we used mutants of GPVI and observed that signal transduction did not inhibit adhesion. To test the correlation between receptor expression and adhesion, we tested three GPVI-expressing RBL-2H3 cell lines (A, B, and C) with different levels of receptor expression. At a single shear rate, the level of adhesion increases monotonically with surface expression. The results, using this model cell line, indicate that GPVI is capable of mediating adhesion to collagen under shear, in a density-dependent fashion that is independent of GPVI signaling.

Analysis of the Interaction of Platelet Collagen Receptor Glycoprotein VI (GPVI) with Collagen: A DIMERIC FORM OF GPVI, BUT NOT THE MONOMERIC FORM, SHOWS AFFINITY TO FIBROUS COLLAGEN

Glycoprotein VI (GPVI) is a platelet-specific glycoprotein that has been indicated to react with collagen and activate platelets. Its structure was recently identified by cDNA cloning (Clemetson, J. M., Polgar, J., Magnenat, E., Wells, T. N., and Clemetson, K. J. (1999) J. Biol. Chem. 274, 29019-29024). However, the mechanism of the interaction between collagen and GPVI has not been analyzed in detail because both collagen and GPVI are insoluble molecules. In this study, we expressed the extracellular domain of GPVI as soluble forms as follows: the monomeric form (GPVIex) and the dimeric form of GPVI fused with the human immunoglobulin Fc domain (GPVI-Fc(2)). Purified GPVIex strongly inhibited convulxin (Cvx)-induced platelet aggregation but only weakly inhibited that induced by collagen-related peptide. However, only GPVI-Fc(2), and not GPVIex, inhibited collagen-induced platelet aggregation. The dimeric form of GPVI exhibits high affinity for collagen, as concluded from measurements of GPVI binding to immobilized collagen by both the enzyme-linked immunosorbent assay and surface plasmon resonance methods. GPVI-Fc(2) bound to the surface of immobilized collagen with a dissociation constant (K(D)) of 5.76 x 10(-7) m, but the binding of GPVIex was too weak to allow estimation of this parameter. Cvx did not inhibit the binding of dimeric GPVI to collagen, indicating that the binding site of GPVI to collagen was different from that to Cvx. Taken together, our data indicate that the high affinity binding site for collagen is composed from two chains of GPVI. Furthermore, they suggest that the binding sites for Cvx are different from the collagen-binding sites and do not need to be formed by two GPVI molecules. Because dimeric GPVI is the only form that shows high affinity to fibrous collagen, our results indicate that GPVI would be present as a dimeric form on the platelet. Moreover, surface plasmon resonance indicated that there is no detectable interaction between soluble collagen and GPVI, supporting our previous observation that GPVI only reacts with fibrous collagen.