# Minimal Collagen-Binding Epitope of Glycoprotein VI in Human and Mouse Platelets
## 4 Discussion
Using site-directed mutagenesis of GPVI, they identified the amino acid residue K79 (\({}^{63}\)SSSRYQDQAVLFIPAMKRSL[82]), which interacted with the inhibitory single-chain antibody fragment (scFv) of the 10B12 antibody [21]. This result suggests that the collagen-binding site of GPVI is located in close proximity to the amino acid of K79. Recently, [PERSON] et al. published the crystal structure of GPVI interacting with collagen or CRP. We recognized that the structure of GPVI in complex with CRP (PDB code: 5 OU8) and the dimeric conformation of GPVI (PDB code: 2 GI7), which we received from the protein structure databank ([[http://www.rcsb.org](http://www.rcsb.org)]([http://www.rcsb.org](http://www.rcsb.org))), were similar to that published in [PERSON] et al., where the authors also identified the ligand-bound structure on the D1 domain of GPVI [30]. Using solid-state assays with mutant variants of GPVI, they could identify several amino acid residues essential for collagen- and CRP-binding. Two amino acid residues (R58 and E60) are located on the minimal collagen-binding epitope detected by the EA-20 antibody (\({}^{48}\)CQGPPGVDLYRLEKI[62]), and mutations of these residues could strongly reduce ligand-binding of GPVI. Interestingly, a similar epitope was also observed in the human leukocyte-associated Ig-like receptor (LAIR-1), and mutation of this site also reduced collagen binding [30]. Furthermore, at close proximity to the minimal epitope detected by the EA-20 antibody, Y67 and D69 mutations (\({}^{63}\)SSSRYQDQAVLFIPAMKRSL[82]) moderately changed the biochemical properties of GPVI. Based on the crystal structure of GPVI, the current model showed that the active form of GPVI does not change conformation after ligand binding, supporting the idea that the collagen-binding surface on GPVI is similar in resting and activated platelets [30]. They could also confirm earlier published results by [PERSON] et al, showing distinct sites between dimerization (D2 domain) and collagen binding (D1 domain) [30]. It is still an open question whether receptor clustering or dimerization is a necessary step for GPVI activation. Our docking results suggest that dimers could effectively bind collagen fibers, including symmetric amino acid residues located on the D1 domain of each monomer. We also agree that dimerization seems to not change the conformation of the collagen-binding surface of GPVI; therefore, repetitive GPO and hydroxyl-groups of collagen can spontaneously interact with GPVI dimers. We speculate that dimers of GPVI form a cluster around the collagen fibers, which is an essential step in stabilizing the protein complex. Monomeric GPVI can form fewer bridges with hydroxyl-groups and GPO motifs, which may be not sufficient to keep collagen fibers on the platelet surface under shear condition, but may effectively bind collagen at static condition. The crystal structure of human GPVI identified by [PERSON] et al. opens a gate for an in silico docking study to identify novel drugs which may selectively inhibit interaction between GPVI and collagen. We could experimentally prove [PERSON] et al. 's model, using our functional blocking EA-20 antibody, which specifically inhibits collagen and CRP interactions with GPVI. As a proof of principle, we evaluated its inhibitory effects on collagen-induced platelet adhesion, activation, and thrombus growth. Experimental studies also highlighted the relevance of targeting of GPVI-binding with collagen or collagen-like domain coding proteins in arterial thrombosis, ischemic stroke, and tumor metastasis [28; 49; 50; 51; 52]. Although recent studies highlighted GPVI as a safe-anti thrombotic target in patients with stroke, site-specific targeting approaches for GPVI are still missing. Future investigation is necessary to evaluate the antithrombotic potential of \({}^{48}\)CQGPPGVDLYRLEKL.62 minimal epitope blockade in vivo using a humanized GPVI mouse model.