Biology subjects

Hudson, N. E.

Publications and source records attributed to Hudson, N. E..

3 recordsLinked to original sources

bioRxiv2026

Indium Tin Oxide (ITO) Substrates Enable Coating-Free SEM Imaging and Simplified Preparation of Purified Fibrinogen Clots

BackgroundScanning electron microscopy (SEM) is widely used to determine fibrin fiber structural properties such as fiber diameter and fiber length. However, conventional SEM preparation protocols are time-consuming and typically require conductive sputter coating. The coating process introduces an additional layer onto the sample surface and may influence measurements of nanoscale fiber structure. Furthermore, preparation of purified fibrinogen clots often follows protocols originally developed for plasma clots, resulting in unnecessary processing steps. ObjectiveTo evaluate indium tin oxide (ITO) as a flat, conductive substrate for SEM imaging of fibrin fibers, investigate the effects of sputter coating on measured fiber diameter, and develop a simplified SEM preparation protocol for purified fibrinogen clots. MethodsPlatelet-poor plasma clots and purified fibrinogen clots were formed on ITO substrates and imaged by SEM following 0 s, 45 s, or 90 s sputter coating. Fibrin fiber diameters were quantified and compared across coating conditions. For purified fibrinogen clots, an ITO-based simplified preparation protocol, in which clots were formed and imaged directly on the conductive ITO surface, was compared with a previously developed, standardized SEM protocol, in which clots were formed in microtube lids and subsequently transferred onto carbon tape for imaging. ResultsFiber diameter measurements were affected by sputter coating duration, with increasing coating time resulting in larger apparent fiber diameters. Plasma and purified fibrinogen clots exhibited distinct fiber diameter distributions and coating responses. For purified fibrinogen clots, the simplified ITO-based protocol produced fiber diameter measurements that were not significantly different from those obtained using the standardized lid-to-carbon-tape workflow when identical coating times were applied. ConclusionsITO provides a practical conductive substrate for SEM imaging of fibrin fibers and enables substantial simplification of purified fibrinogen clot preparation. When coating conditions are matched, the simplified ITO-based protocol yields fiber diameter measurements comparable to those obtained using the previously standardized lid-to-carbon-tape workflow. These findings support the use of ITO as an alternative conductive imaging substrate and provide a simplified workflow for SEM analysis of purified fibrinogen clots. By reducing washing and transfer steps, this workflow may also provide a useful platform for future controlled studies of fibrin interactions with added proteins or other associated components.

biophysics↗

bioRxiv2026

Nonlinear Relationships of Fibrin Network Structure as a Function of Fibrinogen and Thrombin Concentrations for Purified Fibrinogen and Plasma Clots

BackgroundFibrinogen levels are associated with bleeding disorders and thrombotic disease. Thrombin converts fibrinogen to fibrin, producing the load-bearing fibrin scaffold that governs clot mechanics and transport. ObjectiveQuantitatively map how initial fibrinogen and thrombin concentrations, [Fgn]0 and [T[h]r]0, determine fibrin architecture in human plasma and a purified fibrinogen system. MethodsScanning electron microscopy was used to quantify single-fiber morphology--fiber diameter and branch-to-branch segment length from a standardized sample-preparation protocol. Confocal microscopy was used to quantify network architecture--projected fiber density and pore/bubble size. Results and ConclusionsAcross plasma and purified systems, fibrin structural parameters were quantitatively captured by compact multiplicative scaling laws of the form Y = k[Fgn]0[T[h]r]0{beta}. Unlike prior studies, which examined narrower condition ranges without establishing predictive equations across a systematic fibrinogen-thrombin concentration matrix, this framework defines distinct, quantitative roles for fibrinogen and thrombin in fibrin assembly. The magnitudes and signs of the exponents and {beta} indicate that thrombin primarily controls individual fiber growth kinetics, strongly shortening branch-to-branch segment length and modestly thinning fibers, whereas fibrinogen primarily controls space filling, strongly increasing fiber density, reducing pore/bubble size, and thickening fibers. For matched [Fgn]0 and [T[h]r]0, compared to plasma clots, purified fibrinogen formed denser networks with thinner and shorter fibers, suggesting that the plasma biochemical environment partially inhibits thrombin activity. Fiber length analysis further suggests that each thrombin molecule nucleates one fiber segment. Together, these parameterized scaling relations provide a predictive quantitative framework linking clot composition to fibrin microstructure in plasma and purified fibrinogen clots.

biophysics↗

bioRxiv2022

Chorography and conformational dynamism of the Soluble Human Fibrinogen in solution

Fibrinogen is a soluble, multi-subunit and multi-domain dimeric protein, which, upon its proteolytic cleavage by thrombin, is converted to insoluble fibrin initiating polymerization that substantially contributes to clot growth. The consentaneous structural view of the soluble form of fibrinogen is relatively straight and rigid-like. However, fibrinogen contains numerous, transiently-accessible "cryptic" epitopes for hemostatic and immunologic proteins, suggesting that fibrinogen exhibits conformational flexibility, which may play functional roles in its temporal and spatial interactions. Hitherto, there has been limited information on the solution structure and internal flexibility of soluble fibrinogen. Here, utilizing an integrative, biophysical approach involving temperature-dependent hydrogen-deuterium exchange mass spectrometry, small angle X-ray scattering, and negative stain electron microscopy, we present a holistic, conformationally dynamic model of human fibrinogen in solution. Our data reveal a high degree of internal flexibility, accommodated by four major and distinct flexible sites along the central axis of the protein. We propose that the fibrinogen structure in solution consists of a complex, conformational landscape with multiple local minima, rather than a single, rigid topology. This is further supported by the location of numerous point mutations that are linked to dysfibrinogenemia, and post-translational modifications, residing near fibrinogen flexions. This work provides a molecular basis for the structural "dynamism" of fibrinogen that is expected to influence the broad swath of functionally diverse macromolecular interactions and fine-tune the structural and mechanical properties of blood clots.

biochemistry↗