Focus of our research is the von Willebrand factor (VWF). With a size of up to 1 GDa, multimeric VWF is the largest soluble glycoprotein in mammals. It plays an essential role in platelet-dependent primary hemostasis and as a carrier protein that protects factor VIII from degradation. Many of the multiple functions of VWF have been related to the multidomain structure of VWF (Figure 1) that contains binding sites, e.g. for platelet membrane glycoprotein Ib (GPIb), collagen, factor VIII, sites for dimerization and multimerization and cleavage sites for furin and ADAMTS13 (1,2).
Figure 1: Multi-domain structure of VWF. The various functional sites for dimerisation, protein interactions, proteolytic cleavage and others, many of which are shear-sensitive, are indicated.
Apart from its function in hemostasis VWF further appears also to be a key player in angiogenesis, tumor metastasis and inflammation (3-7). The biosynthesis of VWF high molecular weight multimers (HMWM) starts with VWF pre-pro-monomers that are dimerized in the ER and subsequently further posttranslationally modified in the Golgi apparatus before they are stored in Weibel-Palade bodies (WPB’s) (8,9). Upon agonist stimulation endothelial cells secrete VWF-HMWM from WPB’s. These multimers can interact to form even larger biopolymers or strings on the cells’ surface exposing binding sites, for example the A1-domain that includes multiple binding sites (e.g. for the α-chain of GPIbα on the plasma membrane of platelets (10)). The potentially prothrombotic VWF strings are size-regulated by ADAMTS13 that cleaves VWF within the A2-domain only in the presence of hydrodynamic stress (11,12). Therefore, VWF activity as well as its susceptibility to proteolytic cleavage by ADAMTS13 are shear force-regulated and perturbation of VWF’s response to shear flow predicts devastating consequences. Mutations in VWF, causing the potentially life threatening von Willebrand disease (VWD) (13) - a bleeding disorder that arises from qualitative and quantitative deficiencies of VWF (1) - may be directly related to such perturbations. The multifaceted role of shear stress in the regulation of VWF function and mutation-related dysfunction are major challenges in our project. To address these challenges, an interdisciplinary approach is required ranging from molecular biology, theoretical and experimental biophysics to cell physiology and clinical expertise to develop a holistic picture of VWF as a shear-regulated protein on the one hand and the globular FVIII transporter on the other. To tackle all challenges, SHENC is arranged in three areas (Figure 2).
Figure 2. SHENC is composed of an interdisciplinary consortium that provided the expertise to investigate VWF with respect to different levels of complexity. Area A’s expertise provides critical clinical data and facilitates the investigation of VWF with respect to clinical representation by in vitro shear flow assays and mouse models. Area B‘s expertise allows to unravel the biophysical function of VWF by studying collective networks from mesoscopic (B1, B2, B4) to microscopic scales. The single-to-view molecule studies (B3) finally exchange with the expertise of area C, where Molecular Dynamics simulations, X-ray studies and Atomic Force Microscopy experiments explore the structure-function relationship of VWF.
By combining the expertise of all SHENC areas VWF can be investigated with decreasing complexity and increasing detail resolution from area A to C. Expertise of area A: Clinical, Functional and Genetic Aspects: The groups of R. Schneppenheim and U. Budde (A1) represent international reference institutions for the phenotypic and molecular diagnosis of VWD. “Our expertise in analyzing phenotype/genotype correlations in VWD with the help of recombinant protein technologies contributed significantly to the identification of structural and functional defects of VWF in bleeding disorders and thromboembolism (14,15). Further our expertise encompasses fluidic shear assays, cone and plate analysis and imaging (16)”.
The group of S.W. Schneider (A2) has outstanding expertise in the field of endothelial cell and VWF physiology upon tumor spreading, inflammation and hemostasis (5,12,17,18). “We established a so-called “artifical” microvessel by using microfluidic devices (such as Bioflux system, ibidi microchambers and surface acoustic waves provided by B1) together with RICM (reflectance interference contrast microscopy). We further establish culture of human endothelial cells in those microchannels where they can be perfused with whole human blood”. Expertise of area B: Polymer and Cellular Hydrodynamics: The group of A. Wixforth (B1) and our external collaborator M.F. Schneider (Boston University, SHENC guest professor) are pioneers of surface acoustic wave (SAW) driven microfluidics in biological physics (19). “Employing this technology we - together with S.W. Schneider (A2) and the group of R.R. Netz (B4) - discovered the phenomenon of shear driven uncoiling of individual VWF fibers and its adhesion to surfaces (12,20,21).
This finding is one of the cornerstones of the founding of SHENC and demonstrates the fruitful cooperation between cell biologists and biophysicists”. The group of G. Gompper and D.A. Fedosov (B2) has an extensive expertise in computer simulations of bioflows and flows of soft objects in complex geometries. “Our simulations are based on mesoscale hydrodynamics approaches such as multi-particle-collision dynamics (22) and dissipative particle dynamics. These particle-based simulation techniques are very flexible and can be used to investigate the flow behavior of polymers and proteins such as VWF, colloidal suspensions, and membrane systems (e.g. blood cells)”. The soft matter group of J.O. Rädler (B3) has a long standing expertise in the field of polymer and membrane science using single molecule microscopy, fluorescence correlation spectroscopy (FCS) and small angle X-ray scattering. “In particular, the combination of FCS and microfluidics has been used for in situ measurement of shear profiles (23), binding affinities (24-26) and macromolecular conformations (27) (28). Our biophysical techniques facilitate the quantitative investigation of VWF binding and ADAMTS13’s enzymatic activity under shear flow”. The group of R.R. Netz (B4) “has extensive expertise in coarse-grained and atomistic modeling of the adsorption of polymers in shear (29), the internal friction of polymeric globules (30) and protein aggregates (31), and statics and kinetics aspects of the binding of peptides to surfaces (32,33)”.
Expertise of area C: Molecular Mechanics and Biophysics: C. Baldauf and F. Gräter (C1) work in the fields of computational chemistry and biophysics with a focus on protein mechanics. “Force distribution analysis (FDA), a technique developed in F. Gräter’s group (34), is a perfect tool to study the molecular basis of VWF capabilities to sense flow conditions, as shown in their previous studies on the mechanism of VWF A2 unfolding and activation for ADAMTS13 cleavage in collaboration with A1 (35). C. Baldauf has a distinct track record in the field of peptide structure formation, also towards non-natural peptides for a potential development of new diagnostic and therapeutic agents”. The groups of P. Hinterdorfer and M. Benoit (C2) “are experts in atomic force microscopy (AFM) of biological molecules and cells (36,37). They have established AFM in their laboratories to detect protein-protein interactions and in particular the adhesive forces of single molecules binding to receptors on living cells”. C3. “The core expertise of M. Wilmanns laboratory at EMBL is in high-resolution experimental structural biology, using intensive synchrotron radiation”.
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