Elimination of fibrin γ-chain cross-linking by FXIIIa increases pulmonary embolism arising from murine inferior vena cava thrombi.

The onset of venous thromboembolism, including pulmonary embolism, represents a significant health burden affecting more than 1 million people annually worldwide. Current treatment options are based on anticoagulation, which is suboptimal for preventing further embolic events. In order to develop better treatments for thromboembolism, we sought to understand the structural and mechanical properties of blood clots and how this influences embolism in vivo. We developed a murine model in which fibrin γ-chain cross-linking by activated Factor XIII is eliminated (FGG3X) and applied methods to study thromboembolism at whole-body and organ levels. We show that FGG3X mice have a normal phenotype, with overall coagulation parameters and platelet aggregation and function largely unaffected, except for total inhibition of fibrin γ-chain cross-linking. Elimination of fibrin γ-chain cross-linking resulted in thrombi with reduced strength that were prone to fragmentation. Analysis of embolism in vivo using Xtreme optical imaging and light sheet microscopy demonstrated that the elimination of fibrin γ-chain cross-linking resulted in increased embolization without affecting clot size or lysis. Our findings point to a central previously unrecognized role for fibrin γ-chain cross-linking in clot stability. They also indirectly indicate mechanistic targets for the prevention of thrombosis through selective modulation of fibrin α-chain but not γ-chain cross-linking by activated Factor XIII to reduce thrombus size and burden, while maintaining clot stability and preventing embolism.

Cryptic Extensibility in von Willebrand Factor Revealed by Molecular Nanodissection.

Von Willebrand factor (VWF) is a multimer with a variable number of protomers, each of which is a head-to-head dimer of two multi-domain monomers. VWF responds to shear through the unfolding and extension of distinct domains, thereby mediating platelet adhesion and aggregation to the injured blood vessel wall. VWF's C1-6 segment uncoils and then the A2 domain unfolds and extends in a hierarchical and sequential manner. However, it is unclear whether there is any reservoir of further extensibility. Here, we explored the presence of cryptic extensibility in VWF by nanodissecting individual, pre-stretched multimers with atomic force microscopy (AFM). The AFM cantilever tip was pressed into the surface and moved in a direction perpendicular to the VWF axis. It was possible to pull out protein loops from VWF, which resulted in a mean contour length gain of 217 nm. In some cases, the loop became cleaved, and a gap was present along the contour. Frequently, small nodules appeared in the loops, indicating that parts of the nanodissected VWF segment remained folded. After analyzing the nodal structure, we conclude that the cryptic extensibility lies within the C1-6 and A1-3 regions. Cryptic extensibility may play a role in maintaining VWF's functionality in extreme shear conditions.

Cryoprecipitate transfusion in trauma patients attenuates hyperfibrinolysis and restores normal clot structure and stability: Results from a laboratory sub-study of the FEISTY trial.

BACKGROUND: Fibrinogen is the first coagulation protein to reach critical levels during traumatic haemorrhage. This laboratory study compares paired plasma samples pre- and post-fibrinogen replacement from the Fibrinogen Early In Severe Trauma studY (FEISTY; NCT02745041). FEISTY is the first randomised controlled trial to compare the time to administration of cryoprecipitate (cryo) and fibrinogen concentrate (Fg-C; Riastap) in trauma patients. This study will determine differences in clot strength and fibrinolytic stability within individuals and between treatment arms. METHODS: Clot lysis, plasmin generation, atomic force microscopy and confocal microscopy were utilised to investigate clot strength and structure in FEISTY patient plasma. RESULTS: Fibrinogen concentration was significantly increased post-transfusion in both groups. The rate of plasmin generation was reduced 1.5-fold post-transfusion of cryo but remained unchanged with Fg-C transfusion. Plasminogen activator inhibitor 1 activity and antigen levels and Factor XIII antigen were increased post-treatment with cryo, but not Fg-C. Confocal microscopy analysis of fibrin clots revealed that cryo transfusion restored fibrin structure similar to those observed in control clots. In contrast, clots remained porous with stunted fibres after infusion with Fg-C. Cryo but not Fg-C treatment increased individual fibre toughness and stiffness. CONCLUSIONS: In summary, our data indicate that cryo transfusion restores key fibrinolytic regulators and limits plasmin generation to form stronger clots in an ex vivo laboratory study. This is the first study to investigate differences in clot stability and structure between cryo and Fg-C and demonstrates that the additional factors in cryo allow formation of a stronger and more stable clot.

Plasmin-driven fibrinolysis in a quasi-two-dimensional nanoscale fibrin matrix.

Fibrin plays a fundamentally important role during hemostasis. To withstand the shear forces of blood flow and prevent embolisation, fibrin monomers form a three-dimensional polymer network that serves as an elastic scaffold for the blood clot. The complex spatial hierarchy of the fibrin meshwork, however, severely complicates the exploration of structural features, mechanical properties and molecular changes associated with the individual fibers of the clot. Here we developed a quasi-two-dimensional nanoscale fibrin matrix that enables the investigation of fibrin properties by topographical analysis using atomic force microscopy. The average thickness of the matrix was ∼50 nm, and structural features of component fibers were accessible. The matrix could be lysed with plasmin following rehydration. By following the topology of the matrix during lysis, we were able to uncover the molecular mechanisms of the process. Fibers became flexible but retained axial continuity for an extended time period, indicating that lateral interactions between protofibrils are disrupted first, but the axial interactions remain stable. Nearby fibers often fused into bundles, pointing at the presence of a cohesional force between them. Axial fiber fragmentation rapidly took place in the final step. Conceivably, the persisting axial integrity and cohesion of the fibrils assist to maintain global clot structure, to prevent microembolism, and to generate a high local plasmin concentration for the rapid, final axial fibril fragmentation. The nanoscale fibrin matrix developed and tested here provides a unique insight into the molecular mechanisms behind the structural and mechanical features of fibrin and its proteolytic degradation.

Nano-thrombelastography of fibrin during blood plasma clotting.

Hemostasis is a complex process that relies on the sensitive balance between the formation and breakdown of the thrombus, a three-dimensional polymer network of the fibrous protein fibrin. Neither the details of the fibrinogen-fibrin transition, nor the exact mechanisms of fibrin degradation are fully understood at the molecular level. In the present work we investigated the nanoscale-changes in the viscoelasticity of the 3D-fibrin network during fibrinogenesis and streptokinase (STK)-induced fibrinolysis by using a novel application of force spectroscopy, named nano-thrombelastography. In this method the changes in the bending of an oscillating atomic-force-microscope (AFM) cantilever in human blood-plasma droplet were followed as a function of time. Whereas the global features of the time-dependent change in cantilever deflection corresponded well to a macroscopic thrombelastogram, the underlying force spectra revealed large, sample-dependent oscillations in the range of 3-50nN and allowed the separation of elastic and viscous components of fibrin behavior. Upon STK treatment the nano-thrombelastogram signal decayed gradually. The decay was driven by a decrease in thrombus elasticity, whereas thrombus viscosity decayed with a time delay. In scanning AFM images mature fibrin appeared as 17-nm-high and 12-196-nm-wide filaments. STK-treatment resulted in the decrease of filament height and the appearance of a surface roughness with 23.7nm discrete steps that corresponds well to the length of a fibrinogen monomer. Thus, the initial decay of thrombus elasticity during fibrinolysis may be caused by the axial rupture of fibrin fibers.

Fibrinogen αC-region acts as a functional safety latch: Implications for a fibrin biomechanical behaviour model.

Fibrin has unique biomechanical properties which are essential for its role as a scaffold for blood clots. Fibrin is highly extensible and demonstrates significant strain stiffening behaviour, which is essential for stress-distribution in the network. Yet the exact structures of fibrin at the sub-fibre level that contribute to its unique biomechanical characteristic are unknown. Here we show how truncations of the fibrinogen αC-region impact the biomechanical properties of fibrin fibres. Surprisingly, absence of the complete αC-region did not influence the low strain modulus of fibrin fibres but led to premature fibre rupture and decreased extensibility. Intermediate effects were observed with partial deletion of the αC-region, reflected by intermediate rupture stress and toughness. However, overall strain-stiffening behaviour remained even in absence of the αC-region, indicating that strain stiffening is not due to stress being transferred from the αC-region to the protofibril backbone. Upon stress-relaxation, decay constants and their relative contribution to the total relaxation remained similar at all strains, showing that a distinct relaxation process is present until fibre rupture. However, relative contribution of fast relaxation was maximal only in crosslinked fibres if the flexible αC-connector was present. These data show that the αC-region is not the main load-bearing structure within fibrin fibres and point to a critical role for the protofibril backbone instead. We present a revised structural model based on protofibril branching that fully explains the unique biomechanical behaviour of fibrin fibres, while the αC-region primarily acts as a safety latch at the highest of strains. STATEMENT OF SIGNIFICANCE: The findings presented in this paper reveal critically important details about how the molecular structure of fibrin contributes to its unique mechanical properties which are essential to fulfil its function as the scaffold of blood clots. In this work we used engineered proteins with alterations in an important but highly disordered area of the molecule called αC-region and we provide direct evidence for the first time for how the absence of either the globular αC-domain, or the complete αC-region impacts the mechanical behaviour of individual fibrin fibres. Using these results we developed a new structural model of protofibril organisation within fibrin fibres that fully explains their strain stiffening, relatively low modulus and their high, largely variable, extensibility.

A novel method to quantify fibrin-fibrin and fibrin-α2-antiplasmin cross-links in thrombi formed from human trauma patient plasma.

BACKGROUND: The widespread use of the antifibrinolytic 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 (F)XIIIa catalyzes the formation of fibrin-fibrin and fibrin-α2-antiplasmin (α2AP) cross-links, which increases clot mechanical strength and resistance to fibrinolysis. OBJECTIVES: Here, we developed 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. METHODS: Fibrinogen alpha (FGA) chain (FGA-FGA), fibrinogen gamma (FGG) chain (FGG-FGG), and FGA-α2AP cross-links were quantified using liquid chromatography-mass spectrometry (LC-MS) and parallel reaction monitoring in paired plasma samples from trauma patients prefibrinogen and postfibrinogen replacement. Differences in the abundance of cross-links in trauma patients receiving cryoprecipitate (cryo) or fibrinogen concentrate (Fg-C) were analyzed. RESULTS: The abundance of cross-links was significantly increased in trauma patients postcryo, but not Fg-C transfusion (P < .0001). The abundance of cross-links was positively correlated with the toughness of individual fibrin fibers, the peak thrombin concentration, and FXIII antigen (P < .05). CONCLUSION: 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 with Fg-C, highlighting the importance of FXIII in clot formation and stability in trauma patients.

Structural hierarchy of mechanical extensibility in human von Willebrand factor multimers.

The von Willebrand factor (VWF) is a multimeric glycoprotein composed of 80- to 120-nm-long protomeric units and plays a fundamental role in mediating platelet function at high shear. The exact nature of the shear-induced structural transitions have remained elusive; uncovering them requires the high-resolution quantitative analysis of gradually extended VWF. Here, we stretched human blood-plasma-derived VWF with molecular combing and analyzed the axial structure of the elongated multimers with atomic force microscopy. Protomers extended through structural intermediates that could be grouped into seven distinct topographical classes. Protomer extension thus progresses through the uncoiling of the C1-6 domain segment, rearrangements among the N-terminal VWF domains, and unfolding and elastic extension of the A2 domain. The least and most extended protomer conformations were localized at the ends and the middle of the multimer, respectively, revealing an apparent necking phenomenon characteristic of plastic-material behavior. The structural hierarchy uncovered here is likely to provide a spatial control mechanism to the complex functions of VWF.

Building block aspect ratio controls assembly, architecture, and mechanics of synthetic and natural protein networks.

Fibrous networks constructed from high aspect ratio protein building blocks are ubiquitous in nature. Despite this ubiquity, the functional advantage of such building blocks over globular proteins is not understood. To answer this question, we engineered hydrogel network building blocks with varying numbers of protein L domains to control the aspect ratio. The mechanical and structural properties of photochemically crosslinked protein L networks were then characterised using shear rheology and small angle neutron scattering. We show that aspect ratio is a crucial property that defines network architecture and mechanics, by shifting the formation from translationally diffusion dominated to rotationally diffusion dominated. Additionally, we demonstrate that a similar transition is observed in the model living system: fibrin blood clot networks. The functional advantages of this transition are increased mechanical strength and the rapid assembly of homogenous networks above a critical protein concentration, crucial for in vivo biological processes such as blood clotting. In addition, manipulating aspect ratio also provides a parameter in the design of future bio-mimetic and bio-inspired materials.

Why fibrin biomechanical properties matter for hemostasis and thrombosis.

Polymeric fibrin displays unique structural and biomechanical properties that contribute to its essential role of generating blood clots that stem bleeds. The aim of this review is to discuss how the fibrin clot is formed, how protofibrils make up individual fibrin fibers, what the relationship is between the molecular structure and fibrin biomechanical properties, and how fibrin biomechanical properties relate to the risk of thromboembolic disease. Fibrin polymerization is driven by different types of bonds, including knob-hole interactions displaying catch-slip characteristics, and covalent crosslinking of fibrin polypeptides by activated factor XIII. Key biophysical properties of fibrin polymer are its visco-elasticity, extensibility and resistance to rupture. The internal packing of protofibrils within fibers changes fibrin biomechanical behavior. There are several methods to analyze fibrin biomechanical properties at different scales, including AFM force spectroscopy, magnetic or optical tweezers and rheometry, amongst others. Clinically, fibrin biomechanical characteristics are key for the prevention of thromboembolic disorders such as pulmonary embolism. Future studies are needed to address unanswered questions regarding internal molecular structure of the fibrin polymer, the structural and molecular basis of its remarkable mechanical properties and the relationship of fibrin biomechanical characteristics with thromboembolism in patients with deep vein thrombosis and ischemic stroke.

Citrullinated fibrinogen forms densely packed clots with decreased permeability.

BACKGROUND: Fibrin, the main scaffold of thrombi, is susceptible to citrullination by PAD (peptidyl arginine deiminase) 4, secreted from neutrophils during the formation of neutrophil extracellular traps. Citrullinated fibrinogen (citFg) has been detected in human plasma as well as in murine venous thrombi, and it decreases the lysability and mechanical resistance of fibrin clots. OBJECTIVE: To investigate the effect of fibrinogen citrullination on the structure of fibrin clots. METHODS: Fibrinogen was citrullinated with PAD4 and clotted with thrombin. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to measure fiber thickness, fiber height/width ratio, and fiber persistence length in clots containing citFg. Fiber density was measured with laser scanning microscopy (LSM) and permeability measurements were carried out to estimate the porosity of the clots. The intra-fiber structure of fibrin was analyzed with small-angle X-ray scattering (SAXS). RESULTS: SEM images revealed a decrease in the median fiber diameter that correlated with the fraction of citFg in the clot, while the fiber width/length ratio remained unchanged according to AFM. With SAXS we observed that citrullination resulted in the formation of denser clots in line with increased fiber density shown by LSM. The permeability constant of citrullinated fibrin decreased more than 3-fold indicating significantly decreased porosity. SAXS also showed largely preserved periodicity in the longitudinal assembly of fibrin monomers. CONCLUSION: The current observations of thin fibers combined with dense packing and low porosity in the presence of citFg can provide a structural framework for the mechanical fragility and lytic resistance of citrullinated fibrin.