## 2 Results
### GPVI- and Syk-Dependent Parameters of Thromus Formation on Collagen Peptides  
To assess how the five collagen peptides supported whole-blood thrombus formation, we applied these as microspots (M1-5) in a microfluidic device, as described before [9]. The microspots were supplemented with VWF-BP (peptide binding plasma VWF) to allow for GPlb-V-IX-mediated trapping of platelets. Whole-blood perfusion was performed at a wall-shear rate of 1000 s\({}^{-1}\). Through end-stage (3.5 min) multicolor microscopic imaging, it was possible to analyze up to eight thrombus and platelet characteristics: Overall platelet deposition (parameter P1, see Table 1); platelet aggregation (P2); thrombus signature, i.e., morphology, multilayers, and contraction (P3-5); platelet procaagulant activity, measured as PS exposure (P6); platelet activation parameter CD62P expression (P7); and fibrinogen binding to activated integrin \(\alpha_{\text{IB}}\beta_{3}\) (P8). Typically, the collagen peptides containing (GPO)\({}_{n}\) (M1-3) produced large thrombi with aggregated platelets with high levels of activation markers, i.e., PS exposure, CD62P expression, and integrin activation (Figure 2). In contrast, the non-GPVI-stimulating (GPP)\({}_{n}\) peptide GFOGER-GPP (M4) caused the formation of smaller thrombi with residual CD62P expression and integrin activation, but essentially no PS exposure. Quantification of the raw image data confirmed high parameter values for all (GPO)\({}_{n}\) microspots from M1-3, indicating a strong ability to support thrombus formation (Figure S2). Interestingly, when comparing the two GFOGER peptides with or without the GPVI-binding motif (M1 or M4), the latter still induced residual platelet activation, in spite of lower thrombus signature scores (P4-5) and limited PS exposure (P6). Furthermore, M1 (GFOGER), which had a supposedly higher-affinity \(\alpha_{2}\beta_{1}\) binding motif than M3 (GAOGER), was less effective in promoting almost all thrombus parameters (P1,2,3-5,7,8). The differences between microspots were visualized in a univariate scaled heatmap of all parameters (Figure 3A). Together, the data suggested that the earlier distinction made between high- and low-affinity \(\alpha_{2}\beta_{1}\) binding sites (established under static conditions [11; 15]) became confusing in part when immobilized collagen peptides were exposed to platelets in flowing whole blood. On the other hand, the apparent lack of both GPVI and \(\alpha_{2}\beta_{1}\) binding sites, as with M5, resulted in almost no stable platelet adhesion and activation. Parallel flow runs on all microspots from M1-5 were performed with blood samples pretreated with Syk-IN (maximum effective dose of 20 \(\upmu\)M) or DMSO vehicle. This resulted in marked reductions in the majority of thrombus parameters (Figure 3A). A subtraction heatmap pinpointing only relevant changes (\(p<0.05\)) indicated that for M1-4, essentially all parameters except for P1 (platelet deposition) were reduced by Syk inhibition (Figure 3B). Most drastic complete reductions were seen with PS exposure (P6) on the \"active\" (GPO)\({}_{n}\) surfaces of M1-3. Surprisingly, Syk inhibition also affected platelet activation at the supposedly non-GPVI (GPP)\({}_{n}\) surface of M4. The other microspot, M5, was inactive in the absence of Syk-IN. A summative plot was made indicating how individual (scaled) parameters were changed by Syk inhibition across all microspots (Figure 3C). This revealed a complete reduction in P6 (PS exposure),along with strong reductions in P2 (aggregate coverage), P4 (thrombus multilayer), P5 (thrombus contraction), and P8 (fibrinogen binding). Less affected were P3 (thrombus morphology) and P7 (CD62P expression). Figure 2: Thromus formation on immobilized collagen peptides with or without a (GPO)\({}_{\text{n}}\) motif.