Advancing multimer analysis of von Willebrand factor by single-molecule AFM imaging

The formation of hemostatic plugs at sites of vascular injury crucially involves the multimeric glycoprotein von Willebrand factor (VWF). VWF multimers are linear chains of N-terminally linked dimers. The latter are formed from monomers via formation of the C-terminal disulfide bonds Cys2771-Cys2773', Cys2773-Cys2771', and Cys2811-Cys2811'. Mutations in VWF that impair multimerization can lead to subtype 2A of the bleeding disorder von Willebrand Disease (VWD). Commonly, the multimer size distribution of VWF is assessed by electrophoretic multimer analysis. Here, we present atomic force microscopy (AFM) imaging as a method to determine the size distribution of VWF variants by direct visualization at the single-molecule level. We first validated our approach by investigating recombinant wildtype VWF and a previously studied mutant (p. Cys1099Tyr) that impairs N-terminal multimerization. We obtained excellent quantitative agreement with results from earlier studies and with electrophoretic multimer analysis. We then imaged specific mutants that are known to exhibit disturbed C-terminal dimerization. For the mutants p. Cys2771Arg and p. Cys2773Arg, we found the majority of monomers (87 +/- 5% and 73 +/- 4%, respectively) not to be C-terminally dimerized. While these results confirm that Cys2771 and Cys2773 are crucial for dimerization, they additionally provide quantitative information on the mutants' different abilities to form alternative C-terminal disulfides for residual dimerization. We further mutated Cys2811 to Ala and found that only 23 +/- 3% of monomers are not C-terminally dimerized, indicating that Cys2811 is structurally less important for dimerization. Furthermore, for mutants p. Cys2771Arg, p. Cys2773Arg, and p. Cys2811Ala we found 'even-numbered' non-native multimers, i.e. multimers with monomers attached on both termini;a multimer species that cannot be distinguished from native multimers by conventional multimer analysis. Summarizing, we demonstrate that AFM imaging can provide unique insights into VWF processing defects at the single-molecule level that cannot be gained from established methods of multimer analysis

Mutual A domain interactions in the force sensing protein von Willebrand factor

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets

DNA binds to a specific site of the adhesive blood-protein von Willebrand factor guided by electrostatic interactions.

Neutrophils release their intracellular content, DNA included, into the bloodstream to form neutrophil extracellular traps (NETs) that confine and kill circulating pathogens. The mechanosensitive adhesive blood protein, von Willebrand Factor (vWF), interacts with the extracellular DNA of NETs to potentially immobilize them during inflammatory and coagulatory conditions. Here, we elucidate the previously unknown molecular mechanism governing the DNA-vWF interaction by integrating atomistic, coarse-grained, and Brownian dynamics simulations, with thermophoresis, gel electrophoresis, fluorescence correlation spectroscopy (FCS), and microfluidic experiments. We demonstrate that, independently of its nucleotide sequence, double-stranded DNA binds to a specific helix of the vWF A1 domain, via three arginines. This interaction is attenuated by increasing the ionic strength. Our FCS and microfluidic measurements also highlight the key role shear-stress has in enabling this interaction. Our simulations attribute the previously-observed platelet-recruitment reduction and heparin-size modulation, upon establishment of DNA-vWF interactions, to indirect steric hindrance and partial overlap of the binding sites, respectively. Overall, we suggest electrostatics-guiding DNA to a specific protein binding site-as the main driving force defining DNA-vWF recognition. The molecular picture of a key shear-mediated DNA-protein interaction is provided here and it constitutes the basis for understanding NETs-mediated immune and hemostatic responses

Lignite planning, structural change and coal phase-out in Germany

Lignite planning in the Rhineland, central German and Lusatian coalfields is a core spatial development planning task at the federal state and regional levels. The Lignite Planning Information Group and Initiative (Informations- und Initiativkreis Braunkohlenplanung) was founded in 1994 at the ARL - Academy for Territorial Development in the Leibniz Association to provide a platform for expert discussion. Starting from experiences with the Rhineland and the structural upheavals in the new federal states in the early 1990s, it has since continuously addressed new technical and legal requirements involving resettlement, water balance issues, environmental assessments, the energy transition and the common good. Against the backdrop of rapid change and geopolitical events, the combination of structural change and the politically initiated phase-out of lignite-based power generation in a time frame between 'ideally 2030' and no later than the end of 2038 constitutes a challenge that will have to be met by the active players from the perspective of both federal state and regional planning and of regional development. This position paper takes stock of the situation across federal states and across coalfields and describes the required actions for lignite planning as a basis for reaching conclusions about a process with far-reaching national consequences. The various aspects of this process are subject to constant change and call for proactive strategies to exploit opportunities, tap potential, and effectively identify and avoid negative developments

The nuclear GYF protein CD2BP2/U5–52K is required for T cell homeostasis

The question whether interference with the ubiquitous splicing machinery can lead to cell-type specific perturbation of cellular function is addressed here by T cell specific ablation of the general U5 snRNP assembly factor CD2BP2/U5–52K. This protein defines the family of nuclear GYF domain containing proteins that are ubiquitously expressed in eukaryotes with essential functions ascribed to early embryogenesis and organ function. Abrogating CD2BP2/U5–52K in T cells, allows us to delineate the consequences of splicing machinery interferences for T cell development and function. Increased T cell lymphopenia and T cell death are observed upon depletion of CD2BP2/U5–52K. A substantial increase in exon skipping coincides with the observed defect in the proliferation/differentiation balance in the absence of CD2BP2/U5–52K. Prominently, skipping of exon 7 in Mdm4 is observed, coinciding with upregulation of pro-apoptotic gene expression profiles upon CD2BP2/U5–52K depletion. Furthermore, we observe enhanced sensitivity of naïve T cells compared to memory T cells to changes in CD2BP2/U5–52K levels, indicating that depletion of this general splicing factor leads to modulation of T cell homeostasis. Given the recent structural characterization of the U5 snRNP and the crosslinking mass spectrometry data given here, design of inhibitors of the U5 snRNP conceivably offers new ways to manipulate T cell function in settings of disease

Gain-of-Function Variant pPro2555Arg of von Willebrand Factor Increases Aggregate Size through Altering Stem Dynamics

The multimeric plasma glycoprotein (GP) von Willebrand factor (VWF) is best known for recruiting platelets to sites of injury during primary hemostasis. Generally, mutations in the VWF gene lead to loss of hemostatic activity and thus the bleeding disorder von Willebrand disease. By employing cone and platelet aggregometry and microfluidic assays, we uncovered a platelet GPIIb/IIIa-dependent prothrombotic gain of function (GOF) for variant p.Pro2555Arg, located in the C4 domain, leading to an increase in platelet aggregate size. We performed complementary biophysical and structural investigations using circular dichroism spectra, small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, molecular dynamics simulations on the single C4 domain, and dimeric wild-type and p.Pro2555Arg constructs. C4-p.Pro2555Arg retained the overall structural conformation with minor populations of alternative conformations exhibiting increased hinge flexibility and slow conformational exchange. The dimeric protein becomes disordered and more flexible. Our data suggest that the GOF does not affect the binding affinity of the C4 domain for GPIIb/IIIa. Instead, the increased VWF dimer flexibility enhances temporal accessibility of platelet-binding sites. Using an interdisciplinary approach, we revealed that p.Pro2555Arg is the first VWF variant, which increases platelet aggregate size and shows a shear-dependent function of the VWF stem region, which can become hyperactive through mutations. Prothrombotic GOF variants of VWF are a novel concept of a VWF-associated pathomechanism of thromboembolic events, which is of general interest to vascular health but not yet considered in diagnostics. Thus, awareness should be raised for the risk they pose. Furthermore, our data implicate the C4 domain as a novel antithrombotic drug target

Training of attention functions in children with attention deficit hyperactivity disorder

Pharmacological treatment of children with ADHD has been shown to be successful; however, medication may not normalize attention functions. The present study was based on a neuropsychological model of attention and assessed the effect of an attention training program on attentional functioning of children with ADHD. Thirty-two children with ADHD and 16 healthy children participated in the study. Children with ADHD were randomly assigned to one of the two conditions, i.e., an attention training program which trained aspects of vigilance, selective attention and divided attention, or a visual perception training which trained perceptual skills, such as perception of figure and ground, form constancy and position in space. The training programs were applied in individual sessions, twice a week, for a period of four consecutive weeks. Healthy children did not receive any training. Alertness, vigilance, selective attention, divided attention, and flexibility were examined prior to and following the interventions. Children with ADHD were assessed and trained while on ADHD medications. Data analysis revealed that the attention training used in the present study led to significant improvements of various aspects of attention, including vigilance, divided attention, and flexibility, while the visual perception training had no specific effects. The findings indicate that attention training programs have the potential to facilitate attentional functioning in children with ADHD treated with ADHD drugs

Business model validity in early-stage technology ventures’ business plans

Corporate entrepreneurship (CE) supports the strategic renewal of established companies. Corporate venturing represents one key concept of CE that supports companies to strengthen their innovation capabilities. For the successful implementation of corporate ventures dual structures are recommended. The question, how the interface should be designed, plays a crucial role. Although it seems to be an important factor, this aspect requires further attention. One relevant element of the interface design are the different roles of the individuals that are interacting within the interface. This study is based on nine interviews that are representing six internal corporate ventures within one large German corporate from the ICT sector. The results that were mirrored with short case studies of 25 additional companies of the data sample, contribute to a better understanding of the interface design by adding insights about roles in corporate entrepreneurship. This deeper understanding about roles allows to draw conclusions on the interface design from a structural point of view