# Minimal Collagen-Binding Epitope of Glycoprotein VI in Human and Mouse Platelets
## 4 Discussion
Studies with the inhibitory monoclonal antibody JAQ-1, which induces irreversible internalization and shedding of the GPVI receptor [11; 41], indicated the existence of two collagen-binding sites on GPVI, and the primary site overlaps with the CRP site [44]. Using a combinatorial library of linear pentadecamer peptides by phage display technology, [PERSON] et al. screened monoclonal antibodies (mABs) to identify the binding epitopes of collagen on GPVI. Interestingly, 9O12 mAB binds several phage peptide sequences (1:SCGLGVVCGAALVA, 2:GQELLACGLFSVCLS, 3:GQRSSVGGCGLLHVC, and 4:KNGVFLCGLLGLVCD) which correlated with a consensus motif of CGLxxVC. This conserved motif was not found in human GPVI. However, they proposed a weak homology region on human GPVI (\({}^{41}\)EKPVTLRCQGPPGVDLY**RLE**[60]), suggesting that this amino acid sequence may be a collagen- and/or CRP-binding epitope of GPVI [23]. Furthermore, they showed that 9O12 mAB only recognized a functional epitope, since the 9O12 mAB only recognizes GPVI under non-reducing condition in western blot experiments [23; 45]. Using site-directed mutagenesis and a heterologous system to overexpression several mutant variants of GPVI, they could identify that mutation of G50, V54, and L56 to alanine in human GPVI could inhibit collagen- or CRP-binding, to different degrees, in certain experimental conditions [23]. Interestingly, the 9O12 mAB could bind all these mutant forms of GPVI, but the affinity was highly variable. Therefore, they concluded that (i) unknown residues, other than V54 and L56, seem to be part of the GPVI epitope for 9O12 mAB, and (ii) V54 and L56 are critically involved in GPVI interaction with collagen and CRP [23; 46]. Interestingly, these indicated amino acids exist in our peptide (\({}^{48}\)CQGPPGVDLY**RLEK**[62]), used for the generation of the EA-20 antibody. We further analyzed the possible contribution of residues 48-55 (\({}^{48}\)CQGPPGVD**[55]) to the GPVI-collagen binding. We found that these residues have no direct contact with collagen, (Supplementary Figure S9), while 56-62 residues (\({}^{56}\)LYRLEK**[62]) are directly involved in collagen binding. Nevertheless, intramolecular interactions of amino acid residues, such as hydrogen bond and hydrophobic interactions, could maintain the secondary structure and proper folding of this peptide se quence. Amino acid residues of \({}^{48}\)CQGPPGVD[55] show larger contact area, which probably supports the interaction between the EA-20 antibody and GPVI. Recently, the humanized monoclonal antibody glenzocimab (ACT017), which is a derivative of the 9O12 IgG antibody, was positively evaluated in clinical trials (Phase1b/2a studies), showing reduced intracerebral hemorrhage and mortality in patients with stroke [28; 47]. In contrast to the previous study with the 9O12 antibody, crystallization studies mapped the glenzocimab-binding site in the D2 domain of GPVI, which is far away from the collagen-binding site [48]. Furthermore, glenzocimab inhibited GPVI function by preventing GPVI dimerization and inducing steric hindrance that prohibited binding to adjacent GPVI-binding sites on collagen, CRP, and fibrin [48]. [PERSON] et al. showed a putative CRP-binding site using two different computational algorithms (PatchDock and FTDock) [12]. Both docking programs positioned the CRP-binding site of GPVI within the D1 domain involving several amino acid residues (\({}^{63}\)SSSRYQDQAVLFIPAMKRSL[82]), located at close proximity to our peptide sequence that we used for the generation of the EA-20 antibody, although it is not overlapped. Nevertheless, they also reported that residues of V54 and L56 might form a secondary binding site for CRP and collagen. Another group used a three-dimensional model of the human D1/D2 domain of GPVI. This approach was based on the crystal structures of the killer Ig-like receptors, which showed a homologues structure to GPVI [21].