Internal tension in a collapsed polymer under shear flow and the connection to enzymatic cleavage of von Willebrand factor

By means of Brownian hydrodynamics simulations we show that the tension distribution along the contour of a single collapsed polymer in shear flow is inhomogeneous and above a threshold shear rate exhibits a double-peak structure when hydrodynamic interactions are taken into account. We argue that the tension maxima close to the termini of the polymer chain reflect the presence of polymeric protrusions. We establish the connection to shear- induced globule unfolding and determine the scaling behavior of the maximal tensile forces and the average protrusion length as a function of shear rate, globule size, and cohesive strength. A quasi-equilibrium theory is employed in order to describe the simulation results. Our results are used to explain experimental data for the shear-sensitive enzymatic degradation of von Willebrand factor

Exponential Size Distribution of von Willebrand Factor

AbstractVon Willebrand Factor (VWF) is a multimeric protein crucial for hemostasis. Under shear flow, it acts as a mechanosensor responding with a size-dependent globule-stretch transition to increasing shear rates. Here, we quantify for the first time, to our knowledge, the size distribution of recombinant VWF and VWF-eGFP using a multilateral approach that involves quantitative gel analysis, fluorescence correlation spectroscopy, and total internal reflection fluorescence microscopy. We find an exponentially decaying size distribution of multimers for recombinant VWF as well as for VWF derived from blood samples in accordance with the notion of a step-growth polymerization process during VWF biosynthesis. The distribution is solely described by the extent of polymerization, which was found to be reduced in the case of the pathologically relevant mutant VWF-IIC. The VWF-specific protease ADAMTS13 systematically shifts the VWF size distribution toward smaller sizes. This dynamic evolution is monitored using fluorescence correlation spectroscopy and compared to a computer simulation of a random cleavage process relating ADAMTS13 concentration to the degree of VWF breakdown. Quantitative assessment of VWF size distribution in terms of an exponential might prove to be useful both as a valuable biophysical characterization and as a possible disease indicator for clinical applications

Direct Detection of Antibody Concentration and Affinity in Human Serum Using Microscale Thermophoresis

The direct quantification of both the binding affinity and absolute concentration of disease-related biomarkers in biological fluids is particularly beneficial for differential diagnosis and therapy monitoring. Here, we extend microscale thermophoresis to target immunological questions. Optically generated thermal gradients were used to deplete fluorescently marked antigens in 2- and 10-fold-diluted human serum. We devised and validated an autocompetitive strategy to independently fit the concentration and dissociation constant of autoimmune antibodies against the cardiac β1-adrenergic receptor related to dilated cardiomyopathy. As an artificial antigen, the peptide COR1 was designed to mimic the second extracellular receptor loop. Thermophoresis resolved antibody concentrations from 2 to 200 nM and measured the dissociation constant as 75 nM. The approach quantifies antibody binding in its native serum environment within microliter volumes and without any surface attachments. The simplicity of the mix and probe protocol minimizes systematic errors, making thermophoresis a promising detection method for personalized medicine