Background: Fibrinogen is a plasma protein forming the fibrin scaffold of blood clots. Its mechanical properties therefore affect the risk of bleeding as well as thrombosis. There has been much recent interest in the biophysical mechanisms controlling fibrin mechanics; however, the role of molecular heterogeneity of the circulating fibrinogen in determining clot mechanical function remains poorly characterized. Objectives: By comparing 2 fibrinogen variants where the only difference is the Aα-chain length, with one variant having a globular domain at its C-terminus, this study aimed to reveal how the molecular structure impacts the structure and mechanics of fibrin networks. Methods: We characterized the mechanical response to large shear for networks formed from 2 recombinant fibrinogen variants: the most prevalent variant in circulation with a molecular weight of 340 kDa (recombinant human fibrinogen [rFib] 340) and a minor variant with a molecular weight of 420 kDa (rFib420). Results: We show that the elastic properties of the 2 variants are identical when fibrin is cross-linked with factor XIIIa but differ strongly in its absence. Uncross-linked rFib420 networks are softer and up to 3-fold more extensible than rFib340 networks. Electron microscopy imaging showed that the 2 variants formed networks with a comparable structure, except at 4 mg/mL, where rFib420 formed denser networks. Conclusion: We propose that the α_EC domains of rFib420 increase the extensibility of uncross-linked fibrin networks by promoting protofibril sliding, which is blocked by FXIIIa cross-linking. Our findings can help explain the functional role of different circulating fibrinogen variants in blood clot mechanics and tissue repair.

Giant unilamellar vesicles (GUVs) are cell-sized aqueous compartments enclosed by a phospholipid bilayer. Due to their cell-mimicking properties, GUVs have become a widespread experimental tool in synthetic biology to study membrane properties and cellular processes. In stark contrast to the experimental progress, quantitative analysis of GUV microscopy images has received much less attention. Currently, most analysis is performed either manually or with custom-made scripts, which makes analysis time-consuming and results difficult to compare across studies. To make quantitative GUV analysis accessible and fast, we present DisGUVery, an open-source, versatile software that encapsulates multiple algorithms for automated detection and analysis of GUVs in microscopy images. With a performance analysis, we demonstrate that DisGUVery's three vesicle detection modules successfully identify GUVs in images obtained with a wide range of imaging sources, in various typical GUV experiments. Multiple predefined analysis modules allow the user to extract properties such as membrane fluorescence, vesicle shape, and internal fluorescence from large populations. A new membrane segmentation algorithm facilitates spatial fluorescence analysis of nonspherical vesicles. Altogether, DisGUVery provides an accessible tool to enable high-throughput automated analysis of GUVs, and thereby to promote quantitative data analysis in synthetic cell research.

Collagen forms the structural scaffold of connective tissues in all mammals. Tissues are remarkably resistant against mechanical deformations because collagen molecules hierarchically self-assemble in fibrous networks that stiffen with increasing strain. Nevertheless, collagen networks do fracture when tissues are overloaded or subject to pathological conditions such as aneurysms. Prior studies of the role of collagen in tissue fracture have mainly focused on tendons, which contain highly aligned bundles of collagen. By contrast, little is known about fracture of the orientationally more disordered collagen networks present in many other tissues such as skin and cartilage. Here, we combine shear rheology of reconstituted collagen networks with computer simulations to investigate the primary determinants of fracture in disordered collagen networks. We show that the fracture strain is controlled by the coordination number of the network junctions, with less connected networks fracturing at larger strains. The hierarchical structure of collagen fine-tunes the fracture strain by providing structural plasticity at the network and fiber level. Our findings imply that low connectivity and plasticity provide protective mechanisms against network fracture that can optimize the strength of biological tissues.

Subjects with diabetes mellitus (DM) have an increased risk of arterial thrombosis, to which changes in clot structure and mechanics may contribute. Another contributing factor might be an increased formation of neutrophil extracellular traps (NETs) in DM. NETs are mainly formed during the acute phase of disease and form a network within the fibrin matrix, thereby influencing clot properties. Previous research has shown separate effects of NETs and DM on clot properties, therefore our aim was to study how DM affects clot properties in a model resembling an acute phase of disease with NETs formation. Clots were prepared from citrated plasma from subjects with and without DM with the addition of NETs, induced in neutrophils by S. aureus bacteria or phorbol myristate acetate (PMA). Structural parameters were measured using scanning electron microscopy, mechanical properties using rheology, and sensitivity to lysis using a fluorescence-based fibrinolysis assay. Plasma clots from subjects with DM had significantly thicker fibers and fewer pores and branch points than clots from subjects without DM. In addition, fibrinolysis was significantly slower, while mechanical properties were similar between both groups. In conclusion, in a model of acute NETs formation, DM plasma shows prothrombotic effects on fibrin clots.

Fibrin is an elastomeric protein forming highly extensible fiber networks that provide the scaffold of blood clots. Here we reveal the molecular mechanisms that explain the large extensibility of fibrin networks by performing in situ small angle X-ray scattering measurements while applying a shear deformation. We simultaneously measure shear-induced alignment of the fibers and changes in their axially ordered molecular packing structure. We show that fibrin networks exhibit distinct structural responses that set in consecutively as the shear strain is increased. They exhibit an entropic response at small strains (<5%), followed by progressive fiber alignment (>25% strain) and finally changes in the fiber packing structure at high strain (>100%). Stretching reduces the fiber packing order and slightly increases the axial periodicity, indicative of molecular unfolding. However, the axial periodicity changes only by 0.7%, much less than the 80% length increase of the fibers, suggesting that fiber elongation mainly stems from uncoiling of the natively disordered αC-peptide linkers that laterally bond the molecules. Upon removal of the load, the network structure returns to the original isotropic state, but the fiber structure becomes more ordered and adopts a smaller packing periodicity compared to the original state. We conclude that the hierarchical packing structure of fibrin fibers, with built-in disorder, makes the fibers extensible and allows for mechanical annealing. Our results provide a basis for interpreting the molecular basis of haemostatic and thrombotic disorders associated with clotting and provide inspiration to design resilient bio-mimicking materials. Statement of Significance: Fibrin provides structural integrity to blood clots and is also widely used as a scaffold for tissue engineering. To fulfill their biological functions, fibrin networks have to be simultaneously compliant like skin and resilient against rupture. Here, we unravel the structural origin underlying this remarkable mechanical behaviour. To this end, we performed in situ measurements of fibrin structure across multiple length scales by combining X-ray scattering with shear rheology. Our findings show that fibrin sustains large strains by undergoing a sequence of structural changes on different scales with increasing strain levels. This demonstrates new mechanistic aspects of an important biomaterial's structure and its mechanical function, and serves as an example in the design of biomimicking materials.

Filamentous proteins are responsible for the superior mechanical strength of our cells and tissues. The remarkable mechanical properties of protein filaments are tied to their complex molecular packing structure. However, since these filaments have widths of several to tens of nanometers, it has remained challenging to quantitatively probe their molecular mass density and three-dimensional packing order. Scanning transmission electron microscopy (STEM) is a powerful tool to perform simultaneous mass and morphology measurements on filamentous proteins at high resolution, but its applicability has been greatly limited by the lack of automated image processing methods. Here, we demonstrate a semi-automated tracking algorithm that is capable of analyzing the molecular packing density of intra- and extracellular protein filaments over a broad mass range from STEM images. We prove the wide applicability of the technique by analyzing the mass densities of two cytoskeletal proteins (actin and microtubules) and of the main protein in the extracellular matrix, collagen. The high-throughput and spatial resolution of our approach allow us to quantify the internal packing of these filaments and their polymorphism by correlating mass and morphology information. Moreover, we are able to identify periodic mass variations in collagen fibrils that reveal details of their axially ordered longitudinal self-assembly. STEM-based mass mapping coupled with our tracking algorithm is therefore a powerful technique in the characterization of a wide range of biological and synthetic filaments