## 2 Results  
### GPVI-Dependent and Syk-Dependent Platelet Activation by Collagen Peptides  
As a first estimation of the potency of distinct collagen peptides to act as ligands for platelet GPVI, we examined their ability to stimulate PLCy2-mediated rises in cytosolic Ca2+ using Fura-2-loaded platelets. For a selective inhibitor of the GPVI pathway, we used the compound PRT-060318 (Syk-IN), which was earlier used to identify Syk-dependent activation processes in platelets mediated by GPVI [21; 25] or CLEC2 [27]. The Syk-IN compound was found to phenocopy the effects of Syk depletion on platelet responses in \(S\psi k^{-/-}\) bone marrow chimeric mice [28]. Moreover, in human platelets, Syk-IN selectively blocked the GPVI/Syk-dependent tyrosine phosphorylation and aggregation responses induced by fibrin [29]. To confirm the selectivity of Syk-IN as an inhibitor of GPVI-induced responses of human platelets, we monitored its effect (using 5 uM throughout) on the aggregation of platelets induced by CRP-XL, thrombin, or stable ADP. This inhibitor caused the complete inhibition of aggregation only with the GPVI agonist CRP-XL, whereas with thrombin or ADP it was ineffective (Figure S1A). This was in agreement with earlier studies performed with mouse platelets [21; 25]. Additional control experiments with Fura-2-loaded human platelets indicated that Syk-IN did not suppress thrombin- or ADP-induced Ca2+ rises (Figure S1B). \begin{table}
\begin{tabular}{c c c c} \hline \hline  & **Microspot** & \multicolumn{2}{c}{Platelet Receptors} \\ \cline{3-4}  & _GPVI_ & \(a_{2}\beta_{1}\) & _GPIb_ \\ \hline M1 & GFOGER-GPO + VWF-BP & ++ & ++ & ++ \\ M2 & CRP-XL + VWF-BP & ++ & o & ++ \\ M3 & GAOGER-GPO + VWF-BP & ++ & + & ++ \\ M4 & GFOGER-GPP + VWF-BP & (o)* & ++ & ++ \\ M5 & VWF-BP & o & ++ \\ M6 & collagen-H (Horm type) & ++ & ++ & ++ \\ M7 & collagen-I (human) & n.a. & n.a. & ++ \\ M8 & monomeric collagen-I (human) & n.a. & n.a. & ++ \\ M9 & collagen-III (human) & n.a. & n.a. & ++ \\ \hline  & **Parameter** & range & scaling \\ \hline  & _Bright-Field Images_ & & & \\ \hline P1 & **platelet deposition (\% SAC)** & 0–51.52 & 0–10 \\ P2 & platelet aggregate coverage (\% SAC) & 0–21.09 & 0–10 \\ P3 & thrombus morphological score & 0–4.10 & 0–10 \\ P4 & thrombus multilayer score & 0–2.60 & 0–10 \\ P5 & thrombus contraction score & 0–2.94 & 0–10 \\ \hline  & _Fluorescence Images_ & & \\ \hline P6 & PS exposure (\% SAC) & 0–13.91 & 0–10 \\ P7 & CD62P expression (\% SAC) & 0–46.71 & 0–10 \\ P8 & fibrinogen binding (\% SAC) & 0–28.33 & 0–10 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Overview of composition of microspots (M1–9), platelet receptors implicated in thrombus formation. Also indicated are the analyzed thrombus parameters (P1–8) from bright-field and fluorescence microscopic images. Measured ranges and scaling for heatmap analysis were as indicated. GP: Glycoprotein; PS: Phosphatidylserine; VWF-BP: von Willebrand factor binding peptide, SAC: Surface area coverage, n.a., not assessed. Figure 1: Syk inhibition affecting platelet Ca\({}^{2+}\) rises by collagen peptides with (GPO)\({}_{n}\) motif. Fura-2-loaded platelets in 96-well plates were pre-incubated with Syk-IN (5 \(\upmu\)M) or left untreated before stimulation with collagen peptide (M1-5, 10 \(\upmu\)g/mL). Changes in [Ca\({}^{2+}\)]\({}_{i}\) were recorded over time per well-plate row by ratio fluorometry using a FlexStation 3. Peptides were injected into wells at 60 s (arrow) and reached platelets in a diffusion-limited way. (**A**) Calibrated [Ca\({}^{2+}\)] traces recorded over 600 s in the absence (black, control) or presence (gray) of a Syk inhibitor. Traces are representative of three experiments. (**B**) Quantification for M1-5 of increased [Ca\({}^{2+}\)]\({}_{i}\) at 300 s (top graph) or 600 s (bottom graph). Means \(\pm\) SEM (\(n\) = 3). Paired Student’s \(t\)-tests; \({}^{*}p<0.05\), \({}^{**}p<0.01\).