Binding and conformational changes of VWF under shear

• Applicant: Professor Dr. Joachim Rädler
• Subject Area: Biophysics
• Term: from 2011 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 172540668

The multimeric von Willebrand factor (VWF) plays a pivot role as mechanosensitive protein in hemostasis. In succession of injury, VWF promotes adhesion of platelets to collagen as well as platelet aggregation forming filamentous networks, which cover the injured epithelial tissue. Most of VWF´s functions are known to be shear-dependent, since the multimer possesses structural elements that unfold under flow-induced tension. Hereby, the multimer size and the domain structure are important parameters for VWF´s shear-dependent functionality. Yet, many polymorphisms of VWF exist with hitherto unknown consequences for human health. It is believed that quantifying the molecular affinities of VWF wildtype and mutants, especially under shear and blood plasma conditions, will reveal undiscovered disparities. We showed in the first funding period that Fluorescence Correlation Spectroscopy (FCS) is well suited to investigate VWF under blood plasma conditions and proved to be sensitive to characterize disease-related VWF mutants. In a first set of experiments, recombinant plasmatic eGFP-VWF was found to exhibit an exponential size distribution, which we attributed to a step-growth polymerization process during biosynthesis. Furthermore, we developed a novel approach to quantify the kinetics of VWF cleavage by ADAMTS13 as a function of shear flow, combining the FCS setup with a home-build shear cell. We measured the binding constants of protein disulfide isomerase (PDI) to full-length VWF wild type and mutants using FCS and Microscale Thermophoresis (MST). Our measurements suggest that PDI is an enzyme facilitating VWF dimerization. For the next funding round, we propose to study VWF binding under shear flow, with the focus on the dynamic structure-function relationship of both, VWF wild type and VWF mutants. In particular, we are interested in the interactions underlying collective network formation, i.e. VWF crosslinking and binding to GPIIb/IIIa with respect to C4 domain polymorphism. Furthermore, full binding isotherms of VWF binding to vesicles mimicking endothelial cell as well as to collagen type III and VI as a function of A1/A3 domain mutations will be measured. To this end, we will use two quantitative techniques: FCS and MST. A particular technical challenge is the measurement of binding affinities under shear flow, which we will solve by two-color cross-correlation using a pulsed interleaved excitation (PIE)-FCS set-up. Here, we will address the role of the C4 polymorphism on the stem unfolding and binding of VWF with and without flow. Structural studies will be complemented by Small Angle X-ray Scattering (SAXS) and Transmission Electron Microscopy (TEM). Finally, we will expand our studies on ADAMTS13 enzymatic cleavage to pathophysiological conditions such as e.g. found in the inflammatory milieu.

• DFG Programme: Research Units
• Subproject of: FOR 1543: Shear Flow Regulation of Hemostasis - Bridging the Gap between Nanomechanics and Clinical Presentation
• Major Instrumentation: Microscopy setup for TCSPC
• Instrumentation Group: 5080 Optisches Mikroskopzubehör