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  • We aim to apply the same technology to target other

    2020-01-11

    We aim to apply the same technology to target other collagen-binding proteins using motifs from collagens that are specific for other receptors or matrix components. Previously, we synthesized THP libraries, called Toolkits, composed of an active guest sequence flanked by five GPP host triplets that form a triple helix [18]. Screening of collagen binding proteins against Toolkits II and III (encompassing the sequences of collagen II and III respectively), led to the identification of a THP ligand for both Discoidin Domain Receptors (DDRs) [19,20] and von Willebrand Factor (VWF) [18,[21], [22], [23]]. The shared binding motif GPRGQOGVMGFO, referred to as VWFIII in the literature, is derived from the active sequence of Toolkit III-23, GPOGPSGPRGQOGVMGFOGPKGNDGAO. A similar motif, GARGQOGVMGFO, is present in collagen II, and an equivalent sequence, GARGQAGVMGFO, occurs in the α1 chain of collagen I (bold text represents differences in sequence). Discoidin Domain Receptors, like the collagen-binding integrins, constitute a major collagen-binding receptor family; DDR1 and DDR2 are widely distributed in mammalian tissues and play important roles in development and wound repair [20,24,25]. They are tyrosine kinase receptors and their auto-phosphorylation is induced by binding to various types of collagen, including collagens I, II and III [19,22,26,27]. Finally, DDR2 is suspected of being present in cardiac mesenchymal stem Amoxapine involved in tissue repair following myocardial infarction [28] as well as epicardium-derived cardiac fibroblasts [29]. In this regard, DDR2 may play an important role in cardiovascular regenerative medicine. VWF is also of major interest in tissue engineering, being a key component of platelet thrombus formation. Following vascular damage, multimers of VWF are recruited to the exposed subendothelial collagen and interact with the platelet glycoprotein GpIb/V/IX complex [30]. This initial association of platelets with the vessel wall allows subsequent binding of collagen receptors, integrin α2β1 and glycoprotein VI (GPVI), leading to strong platelet adhesion and activation [31]. VWF-mediated platelet recruitment might be useful in certain engineered devices, such as aortic stents, where thrombus deposition or tissue sealant activity is an important objective. The synthetic sequence GPRGQOGVNleGFO, with norleucine replacing methionine in VWFIII to avoid oxidation, has a similar affinity for DDR1 [32] and the A3 domain of VWF, and a higher affinity for DDR2 [33]. We therefore produced synthetic peptides containing this binding motif, flanked with 5 GPP triplets and with a 6-aminohexanoic acid linker on the N-terminus. This peptide is referred to as VWFIIINle in this study. We then end-stapled three VWFIIINle peptide strands to stabilise their subsequent triple-helical conformation. Many attempts to covalently link THP strands have been reported, through cysteine knots [34] or peptide elongation from di-lysine [35] but in our hands, these strategies gave poor results. Instead, we used a method described by Khew et al., where three peptide strands on solid support are covalently linked at their N-terminus using a short diglutamate-containing peptide (a tri-acid), generating a THP with an increased thermal stability and with a one-residue stagger between chains, as well as a single free N-terminus for derivatisation [36,37]. Next, end-stapled THPs were covalently linked to collagen matrices. Although THPs can be passively adsorbed by collagen fibers [21,38], we wished to prevent possible ligand elution from the collagen structure, from the perspective of long-term use in regenerative medicine. Our strategy uses a photoreactive group, diazirine, grafted on the N-terminus of the end-stapled THP, which can be readily activated by long wavelength UV light [[39], [40], [41]], producing a THP bearing a single reactive group. Thus, we have developed a controlled, efficient and rapid derivatisation of collagen matrices with pre-assembled and functional THPs.