α−α Cross-Links Increase Fibrin Fiber Elasticity and Stiffness

Fibrin fibers, which are ∼100 nm in diameter, are the major structural component of a blood clot. The mechanical properties of single fibrin fibers determine the behavior of a blood clot and, thus, have a critical influence on heart attacks, strokes, and embolisms. Cross-linking is thought to fortify blood clots; though, the role of α–α cross-links in fibrin fiber assembly and their effect on the mechanical properties of single fibrin fibers are poorly understood. To address this knowledge gap, we used a combined fluorescence and atomic force microscope technique to determine the stiffness (modulus), extensibility, and elasticity of individual, uncross-linked, exclusively α–α cross-linked (γQ398N/Q399N/K406R fibrinogen variant), and completely cross-linked fibrin fibers. Exclusive α–α cross-linking results in 2.5× stiffer and 1.5× more elastic fibers, whereas full cross-linking results in 3.75× stiffer, 1.2× more elastic, but 1.2× less extensible fibers, as compared to uncross-linked fibers. On the basis of these results and data from the literature, we propose a model in which the α-C region plays a significant role in inter- and intralinking of fibrin molecules and protofibrils, endowing fibrin fibers with increased stiffness and elasticity

A novel method to quantify fibrin-fibrin and fibrin-α2AP cross-links in thrombi formed from human trauma patient plasma.

The widespread use of the anti-fibrinolytic agent, tranexamic acid (TXA), interferes with the quantification of fibrinolysis by dynamic laboratory assays such as clot lysis, making it difficult to measure fibrinolysis in many trauma patients. At the final stage of coagulation, Factor XIIIa (FXIIIa) catalyses the formation of fibrin-fibrin and fibrin-α2-antiplasmin (α2AP) cross-links which increases clot mechanical strength and resistance to fibrinolysis. Here, we develop a method to quantify fibrin-fibrin and fibrin-α2AP cross-links that avoids the challenges posed by TXA in determining fibrinolytic resistance in conventional assays. Fibrinogen alpha chain (FGA-FGA), fibrinogen gamma chain (FGG-FGG) and FGA-α2AP cross-links were quantified using liquid-chromatography-mass spectrometry (LC-MS) and parallel reaction monitoring (PRM) in paired plasma samples from trauma patients pre- and post-fibrinogen replacement. Differences in the abundance of cross-links in trauma patients receiving cryoprecipitate (cryo) or fibrinogen concentrate (Fg-C) were analysed. The study found that the abundance of cross-links was significantly increased in trauma patients post-cryo, but not Fg-C, transfusion (p < 0.0001). The abundance of cross-links was positively correlated with the toughness of individual fibrin fibres, the peak thrombin concentration and FXIII antigen (p < 0.05). We have developed a novel method that allows us to quantify fibrin cross-links in trauma patients who have received TXA, providing an indirect measure of fibrinolytic resistance. Using this novel approach we have avoided the effect of TXA and shown that cryo increases fibrin-fibrin and fibrin-α2AP cross-linking when compared to Fg-C, highlighting the importance of FXIII in clot formation and stability in trauma patients

Fibrinogen splice variation and cross-linking: Effects on fibrin structure/function and role of fibrinogen γ’ as thrombomobulin II

Fibrin is an important matrix protein that provides the backbone to the blood clot, promoting tissue repair and wound healing. Its precursor fibrinogen is one of the most heterogenous proteins, with an estimated 1 million different forms due to alterations in glycosylation, oxidation, single nucleotide polymorphisms, splice variation and other variations. Furthermore, ligation by transglutamimase factor XIII (cross-linking) adds to the complexity of the fibrin network. The structure and function of the fibrin network is in part determined by this natural variation in the fibrinogen molecule, with major effects from slice variation and cross-linking. This mini-review will discuss the direct effects of fibrinogen αEC and fibrinogen γ’ splice variation on clot structure and function and also discuss the additional role of fibrinogen γ’ as thrombomodulin II. Furthermore, the effects of cross-linking on clot function will be described. Splice variation and cross-linking are major determinants of the structure and function of fibrin and may therefore impact on diseases affecting bleeding, thrombosis and tissue repair

Plasma from patients with pulmonary embolism show aggregates that reduce after anticoagulation

BACKGROUND: Microclots, a term also used for amyloid fibrin(ogen) particles and henceforth named aggregates, have recently been reported in the plasma of patients with COVID-19 and long COVID. These aggregates have been implicated in the thrombotic complications of these diseases. METHODS: Plasma samples from 35 patients with acute pulmonary embolism were collected and analysed by laser scanning confocal microscopy and scanning electron microscopy before and after clotting. RESULTS: Here we confirm the presence of aggregates and show that they also occur in the plasma of patients with pulmonary embolism, both before and after clotting. Aggregates vary in size and consist of fibrin and platelets. We show that treatment with low-molecular weight heparin reduces aggregates in the samples of patients with pulmonary embolism. Double centrifugation of plasma does not eliminate the aggregates. CONCLUSIONS: These data corroborate the existence of microclots or aggregates in diseases associated with venous thromboembolism. Important questions are raised regarding their pathophysiological relevance and further studies are warranted to investigate whether they represent cause or consequence of clinical thrombosis

Automated fiber diameter and porosity measurements of plasma clots in scanning electron microscopy images

Scanning Electron Microscopy (SEM) is a powerful, high-resolution imaging technique widely used to analyze the structure of fibrin networks. Currently, structural features, such as fiber diameter, length, density, and porosity, are mostly analyzed manually, which is tedious and may introduce user bias. A reliable, automated structural image analysis method would mitigate these drawbacks. We evaluated the performance of DiameterJ (an ImageJ plug-in) for analyzing fibrin fiber diameter by comparing automated DiameterJ outputs with manual diameter measurements in four SEM data sets with different imaging parameters. We also investigated correlations between biophysical fibrin clot properties and diameter, and between clot permeability and DiameterJ-determined clot porosity. Several of the 24 DiameterJ algorithms returned diameter values that highly correlated with and closely matched the values of the manual measurements. However, optimal performance was dependent on the pixel size of the images—best results were obtained for images with a pixel size of 8–10 nm (13–16 pixels/fiber). Larger or smaller pixels resulted in an over- or underestimation of diameter values, respectively. The correlation between clot permeability and DiameterJ-determined clot porosity was modest, likely because it is difficult to establish the correct image depth of field in this analysis. In conclusion, several DiameterJ algorithms (M6, M5, T3) perform well for diameter determination from SEM images, given the appropriate imaging conditions (13–16 pixels/fiber). Determining fibrin clot porosity via DiameterJ is challenging