B3 - STAFF B3 - RESEARCH B3 - PUBLICATIONS
         
  
  

 

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 VWFs 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 VWFs 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 are interested in VWF as a multimeric, mechanosensitive protein and its shear-induced regulation of its functionality.

In the first funding period we showed 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. 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.

During the next funding period, we will study VWF binding under shear flow, with the focus on the dynamic structure-function relationship of both, VWF wild type and VWF mutants like the C4 polymorphism. In particular, we are interested in the interactions underlying collective network formation, and binding to endothelium cells and collagen. To this end, we will use two quantitative techniques: FCS and Microscale Thermophoresis (MST). A particular technical challenge is the measurement of binding affinities under shear flow, which we will solve by two-color cross-correlation using pulsed lasers (FCCS). 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.  

 

 

 
 
 
  

Figure: (left) VWF in the blood vessel and during coagulation. Different sized VWF multimers persist at low shear rates in an inactivated coiled state. Shear stress elongates VWF and its binding sites are exposed. Specific intermolecular VWF binding is possible. The metalloprotease ADAMTS13 regulates VWF size distribution by shear-dependent proteolysis. VWF binds to endothelial cells, platelets and collagen. VWF and platelets form collective networks during hemostasis. (right) Measurement principle of FCS using recombinant GFP-VWF. FCS allows for measuring the diffusion constant, the detection of binding events, self-assembly and conformational changes in buffer as well as in complex media like blood plasma.

 
  
  
 
     
B3 - STAFF B3 - RESEARCH B3 - PUBLICATIONS