## 2 Results
### GPVI-Induced and Syk-Dependent Platelet Activation by Different Collagens  
Subendothelial collagen types I and III are considered to be the major platelet-activating collagens in the vessel wall, acting via GPVI and \(\alpha_{2}\beta_{1}\)[30]. Equine standard collagen (collagen-H), likely a modified type I collagen, is the most commonly used collagen in studies of GPVI-induced platelet activation. This prompted us to compare four collagen preparations for their ability to support  
Figure 3: Effect of Syk inhibition on parameters of thrombus formation on immobilized collagen peptides. Blood samples pre-incubated with vehicle (Ctrl) or Syk-IN (20 uM) flowed over microspots M1–5, and the thrombi formed were imaged to obtain parameters P1–8, as in Figure 2. The effects of Syk-IN were assessed per blood sample, surface, and parameter. Mean values from individual blood samples (\(n\) = 5–7) were univariate-scaled to 0–10 per parameter across all surfaces M1–9. (**A**) Heatmap of scaled parameters, demonstrating the mean effects of Syk-IN. The rainbow color code indicates scaled values between 0 (blue) and 10 (red). (**B**) Subtraction heatmap representing the scaled effects of Syk-IN, filtered for relevant changes (\(p<0.05\), paired Student’s \(t\)-test per surface and parameter). The color code represents decreases (green) or increases (red) in comparison to control runs. (**C**) Cumulative inhibitory effect per parameter over all microspots, indicating relevant changes. the GPVI-PLCy2-Ca\({}^{2+}\) activation pathway: The fibrous collagen-H (M6), human fibrillar collagen-I (M7), a degraded collagen-I (M8), and human fibrillar collagen-III (M9). Realizing that the very high molecular mass of collagens results in a heterogeneous interaction with platelets in suspension, we evaluated the [Ca\({}^{2+}\)]i rises induced by these collagens. Markedly, the four collagens (M6-9) evoked a biphasic rise in [Ca\({}^{2+}\)]i, with an initial plateau level and a later second phase that was highest for M7 and M9 (Figure 4A,B). In absolute levels, the rises in [Ca\({}^{2+}\)]i obtained with M6, 7, and 9 at a late time point of 600 s were 2-3-fold lower than those seen with the (GPO)\({}_{n}\)-containing collagen peptides (Figure 4 vs. Figure 1). This difference was likely due to the high molecular mass of the fibrillar-type collagens, which slowed down the rate and extent of diffusion-limited interactions with platelets, but it was also likely due to the higher density of the activation motif within the peptides. In addition, it appeared that Syk inhibition completely suppressed the [Ca\({}^{2+}\)]i transients induced by the standard collagen-H (M6), but it did not alter the transients of other collagens (Figure 4). In the presence of indomethacin (10 uM, a thromboxane A\({}_{2}\) pathway inhibitor), AR-C69931 MX (10 uM, a P2Y\({}_{12}\) receptor inhibitor), or MRS2179 (100 uM, a P2Y\({}_{1}\) receptor inhibitor), the rises in [Ca\({}^{2+}\)]i with collagens I-III were suppressed by 15-28%, 31-32%, or 17-31%, respectively, in a nonredundant way (data not shown). Taken together, this suggested the presence of a Syk-independent pathway for Ca\({}^{2+}\) mobilization of suspended natural collagens, which in part came from autocrine activation mechanisms. Figure 4: Syk inhibition differently affecting platelet Ca\({}^{2+}\) rises by collagens. Fura-2-loaded platelets in 96-well plates were pre-incubated with Syk-IN (5 uM) or were left untreated before stimulation with different collagens (M6–9, 10 ug/mL). Changes in [Ca\({}^{2+}\)]i were continuously monitored per well-plate row by ratio fluorometry using a FlexStation 3. Collagens were injected at 60 s (arrow), and they reached platelets in a diffusion-limited way. (**A**) Calibrated [Ca\({}^{2+}\)]i traces recorded for 600 s in the absence (black, control) or presence (gray) of a Syk inhibitor. Traces are representative of three experiments. (**B**) Quantification of [Ca\({}^{2+}\)]i rises after 300 s (top graph) and 600 s (bottom graph) for M1–5. Means \(\pm\) SEM (\(n=3\)).