# Minimal Collagen-Binding Epitope of Glycoprotein VI in Human and Mouse Platelets
## 3 Results
### Collagen-Binding with GPVI Dimer in Humans  
Experimental evidence suggests that only GPVI dimers strongly bind collagen, whereas monomeric GPVI only weakly binds this ligand [15]. Using a competitive platelet aggregation assay, it has been shown that soluble dimeric GPVI-Fc fusion protein inhibits collagen-mediated platelet aggregation, while soluble monomeric GPVI-Fc fusion protein has only a minor, or no effect, on collagen-binding [15]. To prove this concept, we performed a molecular docking study using the recently published crystal structure of the human GPVI dimer and human collagen to validate the binding surface of GPVI (Figure 2A). Collagen structures were docked between the dimer of GPVI, which is consistent with the experimental structure (PDB code: 5 OU8). Several amino acid residues were depicted in the D1 domain of the human GPVI dimer (green line: R58, E60, L62, Y67, R87, S89, Q91, W96) which possibly interact with collagen residues (orange line: Hyp3, Hyp6, P5), thereby stabilizing the protein complex (Figure 2A,B). The predicted collagen-binding site of the human GPVI dimer overlapped with the mouse GPVI dimer. Of note, the amino acid residues of GPVI, which are within the distance of 3 A with those of collagen, are considered as interacting residues. The list of interacting amino acid residues, as well as the corresponding distances, is provided in Table 1. \begin{table}
\begin{tabular}{l l l l} \hline \hline
**Residue** & **Closest Residues** & **Distance, Å** & **Specific Interactions** \\ \hline  & collagen-1 : 4:Gly & 2.7 & 1x hb to collagen-1 : 4:Gly \\  & collagen-1 : 3:Hyp & 3.0 & 1x hb to collagen-1 : 3:Hyp \\
**GPVI:58:Arg \({}^{*}\)** & collagen-1 : 6:Hyp & 3.2 & \\  & collagen-2 : 5:Pro & 3.3 & \\  & collagen-2 : 4:Gly & 3.9 & \\
**GPVI:60:Glu \({}^{*}\)** & collagen-1 : 6:Hyp & 2.8 & 2x clash to collagen-1 : 6:Hyp \\
**GPVI:62:Leu \({}^{*}\)** & collagen-2 : 5:Pro & 3.4 & \\ \hline
**GPVI:62:Leu \({}^{*}\)** & collagen-2 : 8:Pro & 4.0 & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Binding mode of collagen with GPVI. * Symbol depicts residues which are in the peptide (CQGPGVDLYRLEKL). The closest distances are presented. Figure 2: Binding mode of collagen with GPVI. (**A**) The docking simulation of collagen and GPVI dimer. The interaction between collagen and the blocking peptide residues in GPVI. The complex structure (PDB code: 5 OU8) of GPVI and collagen-peptide was retrieved from the protein structure databank. GPVI is shown in green; peptide residues in GPVI and collagen are shown in magenta and orange, respectively. (**B**) The zoomed-in interaction between collagen and peptide residues in GPVI. The interaction residues are depicted as sticks. Red dash lines indicate hydrogen bonds between residues. EA-20 Antibody Inhibits GPVI-Mediated Platelet Activation and Thrombus Formation on Collagen, but Not on Fibrin  
To confirm our bioinformatic results experimentally, we designed a rabbit polyclonal peptide antibody (clone name: EA-20) which recognized a linear epitope on the surface of GPVI (\({}^{48}\)CQGPPVDLYRLEKL\({}^{62}\)) and potentially inhibits the interaction between GPVI and collagen (Figure 2A,B, Table 1). Since the peptide sequence is conserved between human and mouse GPVI (only \({}^{53}\)G changed to D in mouse), we synthetized peptides with wobble amino acids of D/G in the same batch, linking KLH and immunized rabbits to obtain a rabbit IgG polyclonal antibody (EA-20) which was later affinity purified. We studied the effects of the EA-20 antibody on both mouse and human platelets responding to collagen and fibrin, using static and flow adhesion assays. First, washed mouse and human platelets were allowed to adhere to the collagen and fibrin-coated surfaces under static conditions (Figure 3A,B). We found that the EA-20 antibody significantly reduced the adhesion of mouse and human platelets to collagen (Figure 3A,B,E).