Mechanical and biophysical properties of VWF in single molecule experiments

Von Willebrand Factor (VWF) is a long, filamentous protein that contains various important domains responsible for unfolding, cleavage and adhesion. The length and the adhesion of themechano-sensitive VWF are tuned by the shear rate in the blood stream making VWF to a molecular shear force sensor. The knowledge of the (shear) force induced molecular mechanics und function of VWF domains is of key importance for a detailed physiological understanding of hemostasis and blood clotting. Atomic force microscopy (AFM) based single molecule force experiments are particularly suited to scrutinize binding forces and mechanical properties along the molecular backbone with a sensitivity of a few pN. Molecular adhesion and recognition forces as well as VWF domain folding/unfolding forces and forces influencing enzymatic activity will be measured by dynamic force spectroscopy. Furthermore the interaction strengths of VWF with cells (platelets, endothelial cells) and the controversially discussed release of factor VIII naturally bound to VWF will be investigated. This project is embedded in a strong interdisciplinary environment. In particular defined VWF oligomers and concatemers of VWF domains and well-directed point mutations and pathologic type 2 mutations will be delivered. Force measurements on those samples will gain a detailed structural and functional picture with respect to alterations in mechanical and in adhesion properties of VWF. In addition by force mapping and recognition imaging (TREC) on living cells, domains for receptors binding VWF will be localized on the surface of platelets and endothelial cells at the nanometer scale. The strong background in AFM technology and experience with cell adhesion force measurements are pooled complementary in this project: High-resolution topography, recognition imaging and single molecular recognition force measurements on the one hand; cell to cell adhesion force spectroscopy and protein unfolding force measurements on the other.

The von Willebrand factor (VWF) is a protein that plays an initial key role in blood clotting. It helps blood platelets, which stop bleeding by clumping, to bind to exposed collagen in areas of injury and thus cause wound closure. Sites in VWF for binding collagen are located in domains A1 and A3 of VWF. Collagen type III is believed to interact with the A3-domain, and collagen type VI with the VWF domain A1. The forces and the dynamic behavior of these interactions were investigated with single molecule force spectroscopy (SMFS), a method which can be used to study the binding between single molecules.In this project, we elucidated the key initial steps in blood clotting by studying interactions between VWF and collagen. Thereby we explained the clinical relevance of three mutations (S1731T, Q1734H and H1786R), which are located in VWF domain A3 each, in respect to collagen binding. Interactions between collagen type III or VI and the S1731T mutant showed no significant difference in stability when compared to the construct without a mutation. These results are in good agreement with the observation that patients carrying this mutation exhibit no or only moderate bleeding symptoms. The A3-domain mutations Q1734H and H1786R formed a slightly more stable complex with collagen III, probably due to additional hydrogen bonds. The same mutants on collagen VI resulted in a drastic increase in bond stability. Since VWF domain A1 is known as the main binding partner for collagen type VI, we conclude that the VWF A1 domain might compensate for mutations in domain A3 which could explain the inconspicuous bleeding tendency found in patients carrying these VWF A3 domain mutations. VWF-collagen interactions are important for the defective interaction between one VWF domain and one type of collagen can be compensated by alternative binding events.VWF domain A1 is also responsible for mediating blood platelet adhesion under flow in the blood through the platelet receptor GPIb?. The adjacent domain A2 is unfolded under shear upon which it exposes a site that can be cleaved by a protease to prevent unwanted thrombosis. In a state with no injury, VWF is incapable of binding platelets. This behavior has been associated with a shielding of the GPIb? binding site in domain A1 by domain A2. Nevertheless, the exact shielding mechanism has not been clarified yet.We thus probed the interaction strength between the A1 and A2 domains and the unfolding behavior of A2 using SMFS. Domain A2 remained largely folded during the dissociation process from A1, keeping the VWF protected against cleavage and degradation. In this manner VWF can fulfill it biological task, before it is remove by the protease.This work was done within the scope of the project SHENC (Shear flow regulation of HEmostasis - bridging the gap between Nanomechanics and Clinical presentation). Our subproject focused on interaction studies between single molecules.