## 3 Discussion
### Collagen and GPVI-Dependent Platelet Activation  
Fibrillar type I and type III collagens are among the vessel wall components that most strongly activate platelets [7; 30]. Due to the structural complexity of multiple adjacent triple helices in these collagens, little is known about how platelet receptors bind to the fibrils, although there is evidence that the copresence of multiple binding sites in a collagen fiber enforces platelet adhesion and activation [31; 32]. Recent high-resolution microscopy has indicated that multiple copies of GPVI dimerize and cluster along the fibers of such collagens, a process that is considered to enforce GPVI-dependent platelet activation [33; 34]. Previous sequence analysis has shown that both type I and III collagens are made for up to 10% of GPO triplets, with \(\alpha_{2}\beta_{1}\) binding sequences present in both cases, e.g., GFOGER in collagen-I and GAOGER in collagen-III [35].  
Here, we compared the effects of preparations of human fibrillar collagen-I and collagen-III to the standard collagen-H, i.e., a commercial equine type I-enriched preparation with fewer defined supramolecular characteristics [36]. Markedly, added to suspended platelets, collagen-H (M6) was the only collagen that induced Syk-dependent [Ca\({}^{2+}\)]\({}_{\text{i}}\) rises, whereas the other collagens (M7,9) induced low [Ca\({}^{2+}\)]\({}_{\text{i}}\) rises that were insensitive to Syk inhibition. When microspotted, collagen-H triggered the formation of large-size thrombi, with high parameters of platelet aggregation and activation, i.e., responses that are known to be strongly GPVI-dependent [9] and that in the present setting were consistently affected by Syk inhibition.  
In addition, we tested a protease-treated, monomeric collagen-I preparation (M8), which appeared to be inactive in supporting thrombus formation with no appreciable effect of Syk inhibition. This findingsupports the notion that the fibrillar structure of immobilized collagens helps to expose receptor (GPVI) binding sites upon stretching under flow conditions.  
In comparison to collagen-H, the immobilized type I (M7) and type III (M9) collagens triggered the formation of smaller thrombi with lower platelet activation parameters. Yet, for the fibrillar collagens, the summed suppressive effects of Syk inhibition were remarkably similar to those seen for collagen-H and the (GPO)\({}_{\text{n}}\)-containing collagen peptides. Given the similar abundance of GPO triplets in both collagen-I and -III [15], these findings point to a limited role of GPVI-induced activation under flow conditions. In agreement with such a role for GPVI, others have shown that immobilized collagens can induce GPVI dimer clustering in adhered and spreading platelets [34]. In this setting, immobilized collagen-III was found to be more effective in cluster formation than collagen-H or CRP-XL. Furthermore, the inhibition of Syk did not abrogate the GPVI clustering. These findings suggest that there is not a direct link between GPVI cluster formation and the strength of the GPVI-Syk-PLCy2 signal. However, under flow conditions, the additional involvement of VWF/GPIb-V-IX and integrin \(\alpha_{2}\beta_{1}\) interactions [13; 16] might enforce the GPVI clustering pattern, but this still needs to be demonstrated.