Altered properties of fibrin clots have been associated with bleeding and thrombotic disorders, including hemophilia or trauma and heart attack or stroke. Clotting factors, such as thrombin and tissue factor, or blood plasma proteins, such as fibrinogen, play critical roles in fibrin network polymerization. The concentrations and combinations of these proteins affect the structure and stability of clots, which can lead to downstream complications. The present work includes clots made from plasma and purified fibrinogen and shows how varying fibrinogen and activation factor concentrations affect the fibrin properties under both conditions. We used a combination of scanning electron microscopy, confocal microscopy, and turbidimetry to analyze clot/fiber structure and polymerization. We quantified the structural and polymerization features and found similar trends with increasing/decreasing fibrinogen and thrombin concentrations for both purified fibrinogen and plasma clots. Using our compiled results, we were able to generate multiple linear regressions that predict structural and polymerization features using various fibrinogen and clotting agent concentrations. This study provides an analysis of structural and polymerization features of clots made with purified fibrinogen or plasma at various fibrinogen and clotting agent concentrations. Our results could be utilized to aid in interpreting results, designing future experiments, or developing relevant mathematical models.

Turbidimetry is an experimental technique often used to study the structure of filamentous networks. To extract structural properties such as filament diameter from turbidimetric data, simplifications to light scattering theory must be employed. In this work, we evaluate the applicability of three commonly utilized turbidimetric analysis approaches, each using slightly different simplifications. We make a specific application towards analyzing fibrin fibers, which form the structural scaffold of blood clots, but the results are generalizable. Numerical simulations were utilized to assess the applicability of each approach across a range of fiber lengths and diameters. Simulation results indicated that all three turbidimetric approaches commonly underestimate fiber diameter, and that the “Carr-Hermans” approach, utilizing wavelengths in the range of 500–800 nm, provided <10% error for the largest number of diameter/length combinations. These theoretical results were confirmed, under select conditions, via the comparison of fiber diameters extracted from experimental turbidimetric data, with diameters obtained using super-resolution microscopy.

Fibrin forms the structural scaffold of blood clots and has great potential for biomaterial applications. Creating recombinant expression systems of fibrinogen, fibrin’s soluble precursor, would advance the ability to construct mutational libraries that would enable structure–function studies of fibrinogen and expand the utility of fibrin as a biomaterial. Despite these needs, recombinant fibrinogen expression systems, thus far, have relied on the time-consuming creation of stable cell lines. Here we present tests of a transient fibrinogen expression system that can rapidly generate yields of 8–12 mg/L using suspension HEK Expi293^TM cells. We report results from two different plasmid systems encoding the fibrinogen cDNAs and two different transfection reagents. In addition, we describe a novel, affinity-based approach to purifying fibrinogen from complex media such as human plasma. We show that using a high-affinity peptide which mimics fibrin’s knob ‘A’ sequence enables the purification of 50–75% of fibrinogen present in plasma. Having robust expression and purification systems of fibrinogen will enable future studies of basic fibrin(ogen) biology, while paving the way for the ubiquitous use of fibrin as a biomaterial.

The coagulation cascade generates a three-dimensional fibrin gel that seals wounds and prevents the loss of blood. Extensive research has defined the structural and mechanical characteristics of fibrin gels and the various biochemical, physiological, and pathological factors that alter those properties. However, there has been no direct visualization of fibrin polymerization with the spatial and temporal resolution to characterize the fibrin branching process. Thus, a mechanistic explanation for the origins of fibrin structures remains unresolved, with several hypothesized models for how fibrin branching occurs. Using SimView light-sheet microscopy, we captured videos of fibrin fibers forming junctions and gelling. Results: We observed four distinct pathways for fibrin fibers to interact and bind, all predicated on the observation that tumbling and diffusing fibers collide and stick together. Branching occurred due to the binding of mature fibers, not through the splitting of fibers or through the lateral association of small fibrin oligomers (protofibrils), as had been previously hypothesized. Diffusion simulations of rod-like fibers confirmed the branching mechanisms, the relative frequency of the various binding pathways, and the distribution of fiber angles after binding. Scanning Electron Microscopy (SEM) imaging also corroborated the relative frequency of various types of fibrin junctions. Moreover, SEM enabled the visualization of junctions at different timepoints, demonstrating that additional fibrin strands adhere to the initial binding site, strengthening the interaction. These results establish a mechanistic framework for fibrin branching and gel formation and define an innovative experimental approach for future interrogations of fibrin polymerization.