Likewise, no platelet adhesion was detected using other negative control peptides, containing GpVI- or integrin-binding sequences but not terminal cysteine. Lastly, comparison of platelets from wild-type and FcRγ−/− knockout mice, which lack GpVI [6], confirm that platelet adhesion to CRP is mediated by GpVI interacting with (GPO)n and not via some unsuspected interaction with terminal cysteine residues. This forces us to conclude that cysteine is required for tethering the peptide to the plate. Finally, using whole blood perfusion experiments [22], we observed thrombus formation upon GFOGERcys and CRPcys-prepared surfaces, but not upon GFOGER and CRP-prepared surfaces (Fig. 7), where

the latter peptides lack cysteine. This was the case regardless of whether the slide had been pre-washed with acid or alkali, although washing the surface with sodium hydroxide prior to coating led to the deposition of noticeably Quizartinib chemical structure larger thrombi. Inclusion of cysteine in collagenous peptides offers the possibility of cross-linking, an important property exemplified by CRPcys-XL, a potent platelet agonist, whereas monomeric

CRP is an antagonist or at best a partial agonist [18]. Polymerization of the peptide by deliberate, random cross-linking using SPDP introduces higher-order structure into the peptide, which can only then support the clustering of GpVI which leads to activatory signaling in platelets. This parallels find more the behavior of collagen itself, where fibrillar but not monomeric collagen preparations can activate platelets. The active peptide aggregate within CRPcys-XL can be calculated to have an average Stokes Radius of 8.6 nm from its elution volume (Fig. 3). This is similar to the 8.9 nm Stokes Radius of the rod-like 86 kDa glycoprotein, extensin [32], which has the mass and length of ∼8 CRPcys

triple helices. Therefore, the active CRPcys-XL aggregate must contain at least 8, and probably many more, CRPcys triple helices, as the peptide Mirabegron helices are unlikely to align lengthwise [8]. The elution volume from gel filtration of CRPcys also showed that a single CRPcys helix at 10 °C has a Stokes’ Radius of ∼2 nm (its length being 11 nm). As the molecular mass varies with the cube of the Stokes Radius, we can estimate that a CRPcys-XL aggregate contains up to 80 CRPcys triple helices. A second valuable property of terminal cysteine residues in peptides is that they enhanced the adhesion of the peptide to plastics (Fig. 6a), glass (Fig. 7), and gold [26] without further modification. This may be a specific property of cysteine sidechains, or may result from multiple, co-operative, weak interactions that become possible with oxidized polymeric peptides. Using peptides lacking cysteine may mean that binding sites remain undiscovered as both peptide and target protein may be washed off surfaces. It is also likely that less peptide will be needed for surface coating.