Methods to attach polypeptides to lipid bilayers are often indirect, ineffective and can represent a substantial bottleneck in the formation of functionalized lipid-based materials. Although the polyhistidine tag (his-tag) has been transformative in its simplicity and efficacy in binding to immobilized metals, the successful application of this approach has been challenging in physiological settings. Here we show that lipid bilayers containing porphyin-phospholipid that is chelated with cobalt, but not other metals, can effectively capture his-tagged proteins and peptides. The binding follows a Co(II) to Co(III) transition and occurs within the sheltered hydrophobic bilayer, resulting in essentially irreversible attachment in serum or in million-fold excess of competing imidazole. Using this approach we anchored homing peptides into the bilayer of preformed and cargo-loaded liposomes to enable tumour-targeting without disrupting the bilayer integrity. As a further demonstration, a synthetic HIV-derived protein fragment was bound to immunogenic liposomes for potent antibody generation for an otherwise non-antigenic peptide.

Von Willebrand factor (VWF) is the largest glycoprotein in blood. It plays a crucial role in primary hemostasis via its binding interaction with platelet and endothelial cell surface receptors, other blood proteins and extra-cellular matrix components. This protein is found as a series of repeat units that are disulfide bonded to form multimeric structures. Once in blood, the protein multimer distribution is dynamically regulated by fluid shear stress which has two opposing effects: it promotes the aggregation or self-association of multiple VWF units, and it simultaneously reduces multimer size by facilitating the force-dependent cleavage of the protein by various proteases, most notably ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type repeats, motif 1 type 13). In addition to these effects, fluid shear also controls the solution and substrate-immobilized structure of VWF, the nature of contact between blood platelets and substrates, and the biomechanics of the GpIbα–VWF bond. These features together regulate different physiological and pathological processes including normal hemostasis, arterial and venous thrombosis, von Willebrand disease, thrombotic thrombocytopenic purpura and acquired von Willebrand syndrome. This article discusses current knowledge of VWF structure–function relationships with emphasis on the effects of hydrodynamic shear, including rapid methods to estimate the nature and magnitude of these forces in selected conditions. It shows that observations made by many investigators using solution and substrate-based shearing devices can be reconciled upon considering the physical size of VWF and the applied mechanical force in these different geometries.

The cleavage of the A2-domain of Von Willebrand Factor (VWF) by the metalloprotease ADAMTS13 regulates VWF size and platelet thrombosis rates. Reduction or inhibition of this enzyme activity leads to thrombotic thrombocytopenic purpura (TTP). We generated a set of novel molecules called VWF-A2 FRET proteins’, where variants of YFP (Venus) and CFP (Cerulean) flank either the entire VWF-A2 domain (175 amino acids) or truncated fragments (141, 113, 77 amino acids) of this domain. These proteins were expressed in E. coli in soluble form, and they exhibited Fluorescence/Förster Resonance Energy Transfer (FRET) properties. Results show that introduction of Venus/Cerulean itself did not alter the ability of VWF-A2 to undergo ADAMTS13 mediated cleavage. The smallest FRET protein, XS-VWF, detected plasma ADAMTS13 activity down to 10% of normal levels. Tests of acquired and inherited TTP could be completed within 30 min. VWF-A2 conformation changed progressively, and not abruptly, upon increasing urea concentration. While proteins with 77 and 113 VWF-A2 residues were cleaved in the absence of denaturant, 4M urea was required for the efficient cleavage of larger constructs. Overall, VWF-A2 FRET proteins can be applied both for the rapid diagnosis of plasma ADAMTS13 activity, and as a tool to study VWF-A2 conformation dynamics.

Key Points• VWF A2-domain intracellular proteolysis within ECs is enhanced upon disrupting calcium binding (eg, in VWD type 2A mutants).• VWF string cleavage on ECs is calcium independent and is strongly dependent on platelet binding.

Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594–1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly ignored in immobilized peptide-substrate assays.