STED lithography in microfluidics for 3D thrombocyte aggregation testing.

Three-dimensional photopolymerization techniques such as multiphoton polymerization lithography (MPL) and stimulated emission depletion (STED) lithography are powerful tools for fabricating structures in the sub-µm range. Combining these techniques with microfluidics enables us to broaden the range of their applications. In this study, we show a microfluidic device enhanced with MPL structures carrying STED-lithographically written nanoanchors that promote binding of the von Willebrand factor (vWF). The density of vWF is adjusted by varying the number of the nanoanchors on the 3D structures. This allows us to study the impact of the density of vWF on the activation of thrombocytes. The activation of the thrombocytes seems to decrease with the density of vWF on the 3D scaffolds inside the microfluidic channels.

Interaction of von Willebrand factor domains with collagen investigated by single molecule force spectroscopy.

von Willebrand factor (VWF) is a huge multimeric protein that plays a key role in primary hemostasis. Sites for collagen binding, an initial event of hemostasis, are located in the VWF-domains A1 and A3. In this study, we investigated single molecule interactions between collagen surfaces and wild type VWF A1A2A3 domain constructs, as well as clinically relevant VWF A3 domain point mutations, such as p.Ser1731Thr, p.Gln1734His, and p.His1786Arg. For this, we utilized atomic force microscopy based single molecular force spectroscopy. The p.Ser1731Thr mutant had no impact on the VWF-collagen type III and VI interactions, while the p.Gln1734His and p.His1786Arg mutants showed a slight increase in bond stability to collagen type III. This effect probably arises from additional hydrogen bonds that come along with the introduction of these mutations. Using the same mutants, but collagen type VI as a binding partner, resulted in a significant increase in bond stability. VWF domain A1 was reported to be essential for the interaction with collagen type VI and thus our findings strengthen the hypothesis that the VWF A1 domain can compensate for mutations in the VWF A3 domain. Additionally, our data suggest that the mutations could even stabilize the interaction between VWF and collagen without shear. VWF-collagen interactions seem to be an important system in which defective interactions between one VWF domain and one type of collagen can be compensated by alternative binding events.

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.

Polyphosphazene Based Star-Branched and Dendritic Molecular Brushes.

A new synthetic procedure is described for the preparation of poly(organo)phosphazenes with star-branched and star dendritic molecular brush type structures, thus describing the first time it has been possible to prepare controlled, highly branched architectures for this type of polymer. Furthermore, as a result of the extremely high-arm density generated by the phosphazene repeat unit, the second-generation structures represent quite unique architectures for any type of polymer. Using two relativity straight forward iterative syntheses it is possible to prepare globular highly branched polymers with up to 30 000 functional end groups, while keeping relatively narrow polydispersities (1.2-1.6). Phosphine mediated polymerization of chlorophosphoranimine is first used to prepare three-arm star polymers. Subsequent substitution with diphenylphosphine moieties gives poly(organo)phosphazenes to function as multifunctional macroinitiators for the growth of a second generation of polyphosphazene arms. Macrosubstitution with Jeffamine oligomers gives a series of large, water soluble branched macromolecules with high-arm density and hydrodynamic diameters between 10 and 70 nm.

Force-sensitive autoinhibition of the von Willebrand factor is mediated by interdomain interactions.

Von Willebrand factor (VWF) plays a central role in hemostasis. Triggered by shear-stress, it adheres to platelets at sites of vascular injury. Inactivation of VWF has been associated to the shielding of its adhesion sites and proteolytic cleavage. However, the molecular nature of this shielding and its coupling to cleavage under shear-forces in flowing blood remain unknown. In this study, we describe, to our knowledge, a new force-sensory mechanism for VWF-platelet binding, which addresses these questions, based on a combination of molecular dynamics (MD) simulations, atomic force microscopy (AFM), and microfluidic experiments. Our MD simulations demonstrate that the VWF A2 domain targets a specific region at the VWF A1 domain, corresponding to the binding site of the platelet glycoprotein Ibα (GPIbα) receptor, thereby causing its blockage. This implies autoinhibition of the VWF for the binding of platelets mediated by the A1-A2 protein-protein interaction. During force-probe MD simulations, a stretching force dissociated the A1A2 complex, thereby unblocking the GPIbα binding site. Dissociation was found to be coupled to the unfolding of the A2 domain, with dissociation predominantly occurring before exposure of the cleavage site in A2, an observation that is supported by our AFM experiments. This suggests that the A2 domain prevents platelet binding in a force-dependent manner, ensuring that VWF initiates hemostasis before inactivation by proteolytic cleavage. Microfluidic experiments with an A2-deletion VWF mutant resulted in increased platelet binding, corroborating the key autoinhibitory role of the A2 domain within VWF multimers. Overall, autoinhibition of VWF mediated by force-dependent interdomain interactions offers the molecular basis for the shear-sensitive growth of VWF-platelet aggregates, and might be similarly involved in shear-induced VWF self-aggregation and other force-sensing functions in hemostasis.

Activation induced morphological changes and integrin αIIbß3 activity of living platelets.

Platelets are essential in hemostasis. Upon activation they undergo a shape-change accompanied with receptor presentation. Atomic force microscopy (AFM) imaging and single molecule force spectroscopy (SMFS) were used as powerful tools for exploring morphological changes as well as receptor activities of platelets. Imaging time series was accomplished with and without fixation steps at the single platelet level. Hereby the response of mechanical stimulation of the platelet by the AFM cantilever tip was directly observed. We demonstrate that living and fixed platelets develop filopodia after a short activation time followed by their disappearance including cellular bleb formation. Thereafter a second filopodia formation (filopodia extrusion) was observed; those filopodia subsequently disappeared again, and finally platelets detached from the support due to cell death. We determined the influence of mechanical stress on the chronology of morphological changes of platelets and demonstrated shear force induced filopodia formation. Through recordings over several hours, topographical AFM images over the full platelet lifetime - from early activation up to apoptosis - are presented. SMFS measurements on living platelets allowed determining the activation state of the most prominent membrane receptor integrin αIIbß3 at all different phases of activation. αIIbß3 was fully activated, independent of the morphological state.

YidC from Escherichia coli Forms an Ion-Conducting Pore upon Activation by Ribosomes.

The universally conserved protein YidC aids in the insertion and folding of transmembrane polypeptides. Supposedly, a charged arginine faces its hydrophobic lipid core, facilitating polypeptide sliding along YidC's surface. How the membrane barrier to other molecules may be maintained is unclear. Here, we show that the purified and reconstituted E. coli YidC forms an ion-conducting transmembrane pore upon ribosome or ribosome-nascent chain complex (RNC) binding. In contrast to monomeric YidC structures, an AlphaFold parallel YidC dimer model harbors a pore. Experimental evidence for a dimeric assembly comes from our BN-PAGE analysis of native vesicles, fluorescence correlation spectroscopy studies, single-molecule fluorescence photobleaching observations, and crosslinking experiments. In the dimeric model, the conserved arginine and other residues interacting with nascent chains point into the putative pore. This result suggests the possibility of a YidC-assisted insertion mode alternative to the insertase mechanism.

Single molecule force spectroscopy data and BD- and MD simulations on the blood protein von Willebrand factor.

We here give information for a deeper understanding of single molecule force spectroscopy (SMFS) data through the example of the blood protein von Willebrand factor (VWF). It is also shown, how fitting of rupture forces versus loading rate profiles in the molecular dynamics (MD) loading-rate range can be used to demonstrate the qualitative agreement between SMFS and MD simulations. The recently developed model by Bullerjahn, Sturm, and Kroy (BSK) was used for this demonstration. Further, Brownian dynamics (BD) simulations, which can be utilized to estimate the lifetimes of intramolecular VWF interactions under physiological shear, are described. For interpretation and discussion of the methods and data presented here, we would like to directly point the reader to the related research paper, "Mutual A domain interactions in the force sensing protein von Willebrand Factor" (Posch et al., 2016) [1].