Structural characterization of a novel GPVI-nanobody complex reveals a biologically active domain-swapped GPVI dimer

Abstract

GPVI is the major signalling receptor for collagen on platelets. We have raised 54 nanobodies (Nb), grouped into 33 structural classes based on their complementary determining region 3 (CDR3) loops, against recombinant GPVI-Fc (dimeric GPVI) and have characterised their ability to bind recombinant GPVI, resting and activated platelets, and to inhibit platelet activation by collagen. Nanobodies from six different binding classes showed the strongest binding to recombinant GPVI-Fc suggesting that there was not a single dominant class. The most potent three, Nb2, 21 and 35, inhibited collagen-induced platelet aggregation with nanomolar IC50 values and inhibited platelet aggregation under flow. The binding KD of the most potent Nb, Nb2, against recombinant monomeric and dimeric GPVI was 0.6 and 0.7 nM, respectively. The crystal structure of monomeric GPVI in complex with Nb2 revealed a binding epitope adjacent to the CRP binding groove within the D1 domain. In addition, a novel conformation of GPVI involving a domain swap between the D2 domains was observed. The domain swap is facilitated by the outward extension of the C-C' loop which forms the domain swap hinge. The functional significance of this conformation was tested by truncating the hinge region so that the domain swap cannot occur. Nb2 was still able to displace collagen and CRP binding to the mutant, but signalling was abolished in a cell-based NFAT-reporter assay. This demonstrates that the C-C' loop region is important for GPVI signalling but not ligand binding and suggests the domain-swapped structure may represent an active GPVI conformation.

Role of Tyrosine Kinase Syk in Thrombus Stabilisation at High Shear

“…The complete list of reagents is described in Supplementary Materials . The anti-GPVI nanobody 21 (Nb21) was generated as described [ 11 ]. The human plaque homogenate was obtained from 10 patients with symptomatic carotid artery stenosis undergoing carotid endarterectomy, then homogenised and pooled as described [ 12 ].…”

“…Furthermore, we have compared the effect of Syk inhibition to that of the feedback agonists, adenosine diphosphate (ADP) and thromboxane A 2 (TxA 2 ), and of the tyrosine kinases Src and Btk. We have also investigated the effect of a recently described nanobody, Nb21, to GPVI that blocks platelet activation by collagen [ 11 ]. The results show that inhibition of Syk promotes thrombus breakdown on collagen and plaque homogenate thereby demonstrating a critical role for the kinase in the stability of this region.…”

Evidence that GPVI is Expressed as a Mixture of Monomers and Dimers, and that the D2 Domain is not Essential for GPVI Activation

“…Thus, the greater binding observed by Miura et al 5 to recombinant GPVI-Fc is most likely due to the increase in avidity. However, interestingly, a recent study 40 has found that a small loop in the D2 domain is critical for collagen and CRP signalling. The explanation for this contrasting result is unknown.…”

Platelet activation by charged ligands and nanoparticles: platelet glycoprotein receptors as pattern recognition receptors

“…GPVI has several areas of charge in its two Ig domains as shown in Figure 4A, and contains a highly negatively charged stalk that is highly O-glycosylated and charged due to sialylation. 80,81 Site directed mutagenesis studies 82 along with co-crystallization of GPVI with CRP and a blocking nanobody show critical charged residues (predominantly positively charged) at positions E21, R38, E40, R46, K59, R60, R67 and R166 in the D1 domain. [82][83][84] The crystallization of GPVI with a nanoparticle or dendrimer could reveal whether these residues or other residues provide a surface on GPVI to mediate binding of charged ligands.…”

“…80,81 Site directed mutagenesis studies 82 along with co-crystallization of GPVI with CRP and a blocking nanobody show critical charged residues (predominantly positively charged) at positions E21, R38, E40, R46, K59, R60, R67 and R166 in the D1 domain. [82][83][84] The crystallization of GPVI with a nanoparticle or dendrimer could reveal whether these residues or other residues provide a surface on GPVI to mediate binding of charged ligands. This will also help determine whether nanoparticles and other charged ligands bind directly to specific epitopes or in ligand-binding regions on GPVI, or if GPVI activation is mainly driven by the general charge of the nanoparticles/ligands.…”