## 2 Results
### GPVI- and Syk-Dependent Platelet Responses in Thrombus Formation on Collagens  
The same collagen preparations (M6-9) were also applied as microspots to test their ability to support thrombus formation under flow. As indicated in Figure 5, collagen-H (M6) was the most potent in provoking the formation of large aggregates of platelets with high PS exposure, granule secretion, and fibrinogen binding, in agreement with the known high GPVI- and \(\alpha_{2}\beta_{1}\)-activating potency of this collagen when immobilized [9; 11; 12]. In comparison, the fibrillar type I (M7) and III (M9) collagens formed only small aggregates of platelets with remaining secretion and fibrinogen binding, with M9 causing residual PS exposure (Figure 5). The degraded collagen-I (M8) caused mostly single-platelet adhesion with incidental small-sized aggregates. The same information was obtained from the raw mean values of individual parameters with these surfaces (Figure S3). Heatmapping of the eight scaled parameter values confirmed that overall microspot thrombogenicity decreased in the order of M6 \(>\) M7,9 \(>\) M8 (Figure 6A). Treatment of the blood with Syk-IN left platelet deposition (P1) unchanged, but it decreased the thrombus signature and the platelet activation parameters (P2-5; P7,8) for several collagens. A subtraction heatmap was built with a filter for relevant changes (\(p<0.05\)). For collagen-H (M6) as well as for fibrillar collagen-I and -III (M7,9), it showed a reduction in almost all parameters except for P1 in the presence of Syk-IN (Figure 6B). The parameters of platelet aggregation and contraction (P2,4,5) and platelet activation (P6 for M6, and P7,8, for M7,9) were the most reduced. Figure 5: Thrombus formation on immobilized collagens. Whole blood was perfused over microspots M6 (collagen-H), M7 (fibrillar collagen-I), M8 (degraded collagen-I), and M9 (collagen-III). The wall-shear rate was 1000 s\({}^{-1}\), and the perfusion time was 3.5 min. Representative bright-field microscopic images at the end stage are shown for an analysis of platelet deposition (parameter P1) and thrombus characteristics (P2–5). In addition, end-stage three-color fluorescence images for an analysis of PS exposure (AF568 annexin A5, P6), CD62P expression (AF647 \(\alpha\)-CD62P, P7), and fibrinogen binding (FITC, P8) are shown. Scale bars represent 50 \(\upmu\)m.  
To obtain an overall insight into the effect of Syk inhibition, a summative plot was again constructed for each scaled parameter across all collagen microspots (Figure 6C). Importantly, this revealed a highly similar effect of Syk inhibition, as previously seen for the collagen peptides. Summing up the values for M6-9, we noticed a near-complete reduction in P6 (M6, PS exposure), along with strong reductions in P2 (platelet aggregate coverage), P4 (thrombus multilayer), P5 (thrombus contraction), P7 (CD62P expression), and P8 (fibrinogen binding) compared to vehicle-treated blood. Less affected by Syk inhibition was P3 (thrombus morphology), while platelet adhesion (P1) was unchanged. Figure 6: Effect of Syk inhibition on parameters of thrombus formation on immobilized collagen. Whole blood pre-incubated with vehicle (Ctrl) or Syk-IN (20 \(\upmu\)M) was perfused over microspots M6–9, and thrombus formation was imaged to obtain parameters P1–8, as in Figure 5. The effects of Syk-IN were calculated per blood sample, surface, and parameter. Mean values for all blood samples (\(n\) = 5–7) were univariate-scaled to 0–10 per parameter across all surfaces of M1–9. (**A**) A heatmap of the scaled parameters showing the mean effects of Syk-IN. The rainbow color code gives scaled values between 0 (blue) and 10 (red). (**B**) A subtraction heatmap representing the scaled effects of Syk-IN, filtered for relevant changes (\(p\) < 0.05, paired Student’s \(t\)-tests per surface and parameter). The color code represents decreases (green) or increases (red) in comparison to control runs.