Glycation of the high affinity NGF-receptor and RAGE leads to reduced ligand affinity.

AGEs are posttranslational modifications generated by irreversible non-enzymatic crosslinking reactions between sugars and proteins - a reaction referred to as glycation. Glycation, a feature of ageing, can lead to non-degradable and less functional proteins and enzymes and can additionally induce inflammation and further pathophysiological processes such as neurodegeneration. In this study we investigated the influence of glycation on the high affinity NGF-receptor TrkA and the AGE-receptor RAGE. We quantified the binding affinity of the TrkA-receptor and RAGE to their ligands by surface plasmon resonance (SPR) and compared these to the binding affinity after glycation. At the same time, we established a glycation procedure using SPR. We found that glycation of TrkA reduced the affinity to NGF by a factor of three, which could be shown to lead to a reduction of NGF-dependent neurite outgrowth in PC12 cells. Glycation of RAGE reduced binding affinity of AGEs by 10-fold.

ADAMTS13 content and VWF multimer and triplet structure in commercially available VWF/FVIII concentrates.

ADAMTS13 is a metalloproteinase that cleaves von Willebrand factor (VWF) into smaller multimers in vivo. This cleavage creates both the typical multimeric size distribution and the characteristic triplet band distribution of VWF. Here we analysed ADAMTS13 content, VWF multimeric size distribution and VWF triplet structure in five commercial VWF/factor VIII (FVIII) concentrates. The relative distribution of ADAMTS13 activity values corresponded well to the ADAMTS13 antigen values for all examined concentrates except Haemate HS®, which had markedly higher ADAMTS13 antigen/activity ratio, with Fanhdi® and Haemate HS® displaying the most intense ADAMTS13 signal. Interestingly, ADAMTS13 levels did not correlate with the high molecular weight multimer content of the concentrates, but did correlate with VWF triplet distribution. Densitometric quantification showed that Wilate®, Immunate® and Willfact® displayed human plasma-like VWF triplet distribution, whereas Fanhdi® and Haemate HS® showed enhanced content of the faster migrating triplet band, which corresponded well to their higher ADAMTS13 content. In summary, Immunate®, Willfact® and Wilate® had lower levels of ADAMTS13 antigen and activity and exhibited a plasma-like VWF triplet structure. Fanhdi® and Haemate HS® had higher ADAMTS13 content and an altered triplet structure. The possible impact of these observations on function and clinical efficacy of VWF/FVIII concentrates is discussed.

Flow-based measurements of von Willebrand factor (VWF) function: binding to collagen and platelet adhesion under physiological shear rate.

INTRODUCTION: VWF circulates in plasma as a series of heterogeneous multimers, mediating platelet tethering, translocation and finally adhesion to areas of injured endothelium under physiological high arterial blood flow. VWF-platelet binding requires conformational changes in VWF, which are induced by immobilization and shear. Because of unavailability of a simple flow-based measurement system, VWF activity assays are generally performed under static conditions. We describe an easily reproducible in vitro flow-chamber model using commercially available flow devices to examine VWF-collagen binding and VWF-mediated platelet adhesion under physiological flow conditions. METHODS: The collagen surface of the flow-chamber was analyzed by atomic force microscopy. Collagen-bound VWF was characterized by multimer analysis and multi labelling immunofluorescence detection of exposed GPIb binding domains. Platelet adhesion was captured by time-lapse microscopy. RESULTS: The described flow-chamber system facilitates multimer analysis of collagen-bound VWF, whereas all VWF multimers bound to collagen under physiological low to high shear rates. Multi labelling immunofluorescence detection exhibited exposed GPIb binding domains co-localized with VWF molecules. VWF-dependent platelet adhesion using time-lapse microscopy showed values comparable to experiments done with whole blood, and platelet adhesion was dependent on the VWF concentration. CONCLUSIONS: The established flow-chamber model represents an easy-to-set-up and customized tool for the characterization of VWF-binding to collagen as well as the determination of VWF-dependent platelet adhesion under defined flow conditions in real-time.