# Minimal Collagen-Binding Epitope of Glycoprotein VI in Human and Mouse Platelets
## 4 Discussion  
The biochemical properties of GPVI that allow binding of CRP were investigated in human and mouse platelets, and results showed that the CRP-binding affinity of human GPVI was significantly higher than mouse GPVI [21]. Similar to other studies, we could confirm that the human and mouse CRP-binding surface of the D1 domain is variable; several amino acids were changed during evolution, thereby changing the electrostatic surface potential, which possibly modifies the ligand-binding affinity of GPVI. In ancient mammalian species, such as _Sorex araneus_, we detected the most variable peptide sequence  
Figure 5: EA-20 antibody does not induce clustering or shedding of GPVI. (**A,B**) Washed human platelets were stimulated with Col-I (2.5 ug/mL) in the presence of control IgG (Ctrl) or EA-20 antibody and/or Losartan (50 uM). Platelet aggregation was measured using light transmission aggregometry and recorded for 10 min. Representative aggregation curves are shown. (**C**) Human PRP were stimulated with Col-I (10 ug/mL), thrombin (1U), a combination of Col-I (10 ug/mL) and thrombin (1U), or GPVI shedding-inducing-reagent NEM (2 mM), in the absence or presence of EA-20 antibody. After 1 h incubation, platelet-free plasma (PFP) was collected and soluble GPVI (sGPVI) levels, indicative of platelet GPVI-shedding, were determined using ELISA assay. Mean \(\pm\) standard deviation (SD), Ctrl (IgG control antibody), and EA-20. Each point represents one individual (**B,C**) healthy human donor. 1-way ANOVA and [PERSON]’s post hoc test, \({}^{*}\)\(p<\) 0.05, \({}^{***}\)\(p<\) 0.0001; ns, not significant. of the D1 domain (\({}^{48}\)CQGPP**DMDVY**RLEKGL**SGATY**RDQ**YU**NISAMILGS**[82]), which is definitively involved in collagen and CRP-binding of GPVI in many species, including human and mouse. Therefore, we speculate that this modification in _Sorex araneus_ may strongly reduce the collagen-binding affinity of GPVI. However, it may not influence the interaction with other GPVI ligands, such as laminin or fibrin, because these amino acid changes do not influence the main structure of GPVI, as we observed in 3D GPVI models of human, bovine, mouse, and sorex species. JAQ-1 is an anti-mouse GPVI monoclonal antibody, which inhibits the major collagen and CRP-binding site in platelets [41]. Interestingly, JAQ-1 showed specific interaction with mouse and human GPVI, but, unexpectedly, it cannot bind platelets isolated from different mammalian species, such as rat, rabbit, guinea pig, dog, and swine, experimentally supporting our finding that the collagen and CRP-binding surface of GPVI diverged during evolution [42]. Although the JAQ-1 antibody recognized an epitope on hGPVI, it also induced an abnormal aggregation response in human platelets on collagen [42]. Unhydroxylated collagen, isolated from transgenic plants, or unhydroxylated CRP (GPP10), is not able to bind or activate platelets [43]. A single amino acid substitution of GPVI at position K61 (\({}^{48}\)CQGPPGVDLY**RLEK**[62]) could increase the binding affinity of GPVI to hydroxylproline-containing motifs, indicating that hydroxylation of collagen fibers is essential for GPVI interaction. Interestingly, in many mammalian species, this amino acid was spontaneously mutated from K to Q, or E or N, during evolution, supporting the idea that this amino acid substitution may increase the collagen-binding affinity of GPVI in these species. Our docking results could confirm that the hydroxyl group of collagen could directly bind several amino acids on GPVI, and possibly stabilize the protein complex. However, we also found that the K61 residue is not involved in the hydroxyl groups or GPO-binding (Supplementary Figure S8), supporting our hypothesis that the observed amino acid substitution (K/**Q**, K/**E**, or K/**N**) may influence collagen-binding to GPVI in different species. However, this mechanism is independent of GPO-binding.