# Minimal Collagen-Binding Epitope of Glycoprotein VI in Human and Mouse Platelets
## 3 Results
### Collagen-Binding with GPVI Dimer in Humans
In sharp contrast, no differences were observed on the fibrin-coated surface (Figure 3A,B,E), indicating that the EA-20 antibody can selectively block collagen interaction with GPVI in both species. Next, we analyzed the blocking effect of the EA-20 antibody under thrombus formation on the collagen-coated surface, at shear rate of 1000 s\({}^{-1}\), using human or mouse whole blood in the flow chamber. Mouse blood treated with EA-20 antibody exhibited strongly reduced platelet adhesion and impaired thrombus formation (Figure 3C,F). Similar results were obtained using human blood, although the inhibition of thrombus formation was less severe than that observed with mouse platelets (Figure 3D,F). It is important to note that thrombus formation was not completely abolished, indicating a partially defective thrombus formation, possibly maintained by GPVI and fibrin interaction. Using mouse and human washed platelets, aggregation responses to collagen were also severely impaired (Figure 4A,E; Supplementary Figure S4A,E). In the humanized mouse model of GPVI, expressing only the human GPVI isoform on the mouse platelet surface, the EA-20 antibody was tested and aggregation response to collagen was also reduced (Figure 4C; Supplementary Figure S4C). Interestingly, CRP-induced aggregation response was strongly impaired in the presence of the EA-20 antibody in humanized GPVI or wild-type mouse platelets (Figure 4B,D; Supplementary Figure S4B,D), but, in sharp contrast, abnormal hyper-aggregation was observed in human platelets when a low dose of CRP was applied (Figure 4F; Supplementary Figure S4F). However, such an aggregation effect was not observed in response to a low dose of collagen (Supplementary Figure S5). Human immunoglobulin G (IgG) Fc receptor, gamma Ila (FcryRIIa) receptor, is expressed in human platelets and fully activated by IgG. However, the FcryRIIa receptor is not expressed in mouse platelets. Therefore, we assumed that the abnormal response to CRP was triggered by rabbit IgG of the EA-20 antibody, activating FcryRIIa receptor signaling, a mechanism \begin{table}
\begin{tabular}{l l l l} \hline \hline
**Residue** & **Closest Residues** & **Distance, Å** & **Specific Interactions** \\ \hline  & collagen-1 : 6:Hyp & 3.3 & \\
**GPVI:67:Tyr** & collagen-1 : 3:Hyp & 3.7 & \\  & collagen-1 : 5:Pro & 3.7 & \\  & collagen-1 : 4:Cly & 4.0 & \\  & collagen-2 : 6:Hyp & 3.1 & 2x hb to collagen-2 : 6:Hyp \\
**GPVI:87:Arg** & collagen-2 : 7:Gly & 3.2 & \\  & collagen-2 : 8:Pro & 3.2 & \\
**GPVI:89:Ser** & collagen-2 : 5:Pro & 3.7 & \\  & collagen-1 : 3:Hyp & 2.7 & 1x hb to collagen-1 : 3:Hyp \\
**GPVI:91:Gln** & collagen-2 : 2:Pro & 3.9 & \\  & collagen-2 : 3:Hyp & 3.2 & 1x hb to collagen-2 : 3:Hyp \\  & collagen-2 : 5:Pro & 3.8 & \\
**GPVI:96:Trp** & collagen-2 : 4:Gly & 3.8 & \\  & collagen-1 : 3:Hyp & 3.8 & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Cont. that is absent in mouse platelets. To prove this concept experimentally, human seroblock was used to block IgG-mediated clustering and activation of the FcyRIIIa receptor. After CRP activation, treatment with seroblock could revert EA-20-induced hyper-aggregation of human platelets (Figure 4F; Supplementary Figure S4F). Of note, the EA-20 antibody did not influence the platelet aggregation response to non-GPVI agonists, such as thrombin, TRAP-6, and ADP (Supplementary Figures S6 and S7). Altogether, these results indicate that the EA-20 antibody selectively inhibits the interaction between GPVI and collagen, but does not influence other platelet receptor functions or fibrin binding to GPVI. Figure 3: EA-20 antibody selectively impairs platelet adhesion and thrombus formation to Col-I-coated surfaces under static and flow conditions. Representative immunofluorescence and phase contrast microscopy images of mouse and human platelets adhering to Col-I and fibrin under (**A**,**B**) static and (**C**,**D**) flow conditions, respectively.