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Unraveling the Mechano-Regulation of Von Willebrand Factor

Subject Area Biophysics
Biochemistry
Term from 2017 to 2023
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 386143268
 
Final Report Year 2024

Final Report Abstract

In the DFG-project “Unraveling the Mechano-Regulation of Von Willebrand Factor”, we have investigated the blood glycoprotein von Willebrand Factor (VWF). In this project, we used a combination of single-molecule biophysical techniques and high-resolution structure determination to better understand the force response and structural basis for mechanical activation of VWF. We employed two highly complementary force spectroscopy techniques: magnetic tweezers, which in particular allow us to apply and resolve very small forces (< 1 pN), and an atomic force microscope, which can readily exert and monitor very high forces at the molecular scale (> 1 nN). For both approaches, we developed new attachment chemistries that enables more stable and longer-lived attachment of individual VWF dimers for single-molecule manipulation than what is possible with previously used coupling strategies. Using magnetic tweezers, we were able to show that the VWF stem, consisting of the 6 C-domains “zipping up” across the dimer, opens under relatively low forces, with a midpoint force of 1 pN at the physiological pH of blood (pH 7.4). In preliminary work, we have started to address the role of pH and mutations in the VWF stem. In addition, we were able to use magnetic tweezers to show how Ca2+ stabilizes the folding of the VWF A2 domain, which is known to unfold under mechanical load, and revealed the dynamics and pH-dependencies of an interface in the D’D3 domain, which is believed to play an important role in the biosynthesis of VWF. Using AFM force spectroscopy, we obtained preliminary results, probing the VWF under much higher forces than previously possible, up to > 2 nN. The results suggest that some disulfide bridges in the protein might open under high mechanical load. In addition, a construct with VWF dimers in an inverted geometry shows that the strong D4 interaction across the dimer can occur even if the dimer is not linked via the CK domains in the stem. Furthermore we could resolve the inverted dimers and VWF with multimerization defects leading to subtype 2A of the bleeding disorder von Willebrand Disease by AFM imaging. We also used various biophysical methods to decipher the VWF structure. Specifically, we determined the structures of the C4 and C6 domains, allowing to deduce general principles of its structural and functional properties. Three publications resulted from this work. We also structurally characterized a VWF construct covering the entire segment from the D4 to the C-terminal CK domain, using negative stain electron microscopy. The inherent flexibility between these domains has hampered highresolution structural analysis, so far. In summary, our combined structural and single-molecule force spectroscopy approach has resulted in several key methodological developments, in particular the development of novel, ultra-stable attachment strategies in both magnetic tweezers and the AFM. Using these approaches, we were able to provide new insights about the interaction and mechano-regulation of several VWF domains. The work lays the foundation for understanding activation of VWF, both in its regular function, but also in the context of gain-of-function mutations relevant in human disease. Our project was severely affected by the COVID pandemic, with lab shut down and disruption of workflows and collaboration efforts. As such, the most surprising and least anticipated impact on our project was clearly external and not based on unforeseen scientific roadblocks or discoveries. Nonetheless, we were able to obtain a string of exciting results, published in leading scientific journals, as well as several lines of promising preliminary data that forms a basis for future work.

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