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

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

Role of Fibrin Structure in Thrombosis and Vascular Disease

Fibrin clot formation is a key event in the development of thrombotic disease and is the final step in a multifactor coagulation cascade. Fibrinogen is a large glycoprotein that forms the basis of a fibrin clot. Each fibrinogen molecule is comprised of two sets of Aα, Bβ, and γ polypeptide chains that form a protein containing two distal D regions connected to a central E region by a coiled-coil segment. Fibrin is produced upon cleavage of the fibrinopeptides by thrombin, which can then form double-stranded half staggered oligomers that lengthen into protofibrils. The protofibrils then aggregate and branch, yielding a three-dimensional clot network. Factor XIII, a transglutaminase, cross-links the fibrin stabilizing the clot protecting it from mechanical stress and proteolytic attack. The mechanical properties of the fibrin clot are essential for its function as it must prevent bleeding but still allow the penetration of cells. This viscoelastic property is generated at the level of each individual fiber up to the complete clot. Fibrinolysis is the mechanism of clot removal, and involves a cascade of interacting zymogens and enzymes that act in concert with clot formation to maintain blood flow. Clots vary significantly in structure between individuals due to both genetic and environmental factors and this has an effect on clot stability and susceptibility to lysis. There is increasing evidence that clot structure is a determinant for the development of disease and this review will discuss the determinants for clot structure and the association with thrombosis and vascular disease

A fibrin biofilm covers blood clots and protects from microbial invasion

Hemostasis requires conversion of fibrinogen to fibrin fibers that generate a characteristic network, interact with blood cells, and initiate tissue repair. The fibrin network is porous and highly permeable, but the spatial arrangement of the external clot face is unknown. Here we show that fibrin transitioned to the blood-air interface through Langmuir film formation, producing a protective film confining clots in human and mouse models. We demonstrated that only fibrin is required for formation of the film, and that it occurred in vitro and in vivo. The fibrin film connected to the underlying clot network through tethering fibers. It was digested by plasmin, and formation of the film was prevented with surfactants. Functionally, the film retained blood cells and protected against penetration by bacterial pathogens in a murine model of dermal infection. Our data show a remarkable aspect of blood clotting in which fibrin forms a protective film covering the external surface of the clot, defending the organism against microbial invasion

Assessment and determinants of whole blood and plasma fibrinolysis in patients with mild bleeding symptoms

Enhanced clot lysis is associated with bleeding, but assessment of lysis capacity remains difficult. The plasma turbidity lysis and whole blood tissue Plasminogen Activator-Rotational Thromboelastometry (tPA-ROTEM) assays estimate fibrinolysis under more physiological conditions than clinically used assays. We hypothesized that these assays could find signs of enhanced lysis capacity in patients who report bleeding symptoms, but are not diagnosed with bleeding disorders. We also aimed to gain insight in determinants of the results of these lysis assays. Data from 240 patients with and 95 patients without self-reported bleeding symptoms were obtained, who were included in a study that primarily aimed to assess prevalence of haemostaticabnormalities in preoperative patients. ROTEM and turbidity assays were performed with rtPA. Blood counts, fibrinolysis and coagulation factor activities were determined. Data were analysed using multivariable linear regression models. Remarkably, patients reporting bleeding symptoms showed signs of significantly impaired lysis capacity in the tPA-ROTEM, but not in the turbidity lysis assay. In these patients, the tPA-ROTEM results depended on FII, FXII, plasminogen, α2-antiplasmin, PAI-1 and TAFI levels. The turbidity lysis results were significantly influenced by fibrinogen, α2-antiplasmin, PAI-1 and TAFI. In conclusion, the tPA-ROTEM and the turbidity lysis assay could not detect enhanced fibrinolytic capacity in patients with bleeding symptoms. This suggests that these symptoms are not caused by enhanced fibrinolytic activity. As both assays were sensitive to important determinants of fibrinolysis they may be able to detect a fibrinolytic imbalance, but this needs to be validated in patients with known hypo- or hyperfibrinolytic disorders

Nanoscale Probing Reveals that Reduced Stiffness of Clots from Fibrinogen Lacking 42 N-Terminal Bβ-Chain Residues Is Due to the Formation of Abnormal Oligomers

Removal of Bβl-42 from fibrinogen by Crotalus atrox venom results in a molecule lacking fibrinopeptide B and part of a thrombin binding site. We investigated the mechanism of polymerization of desBβ1-42 fibrin. Fibrinogen trinodular structure was clearly observed using high resolution noncontact atomic force microscopy. E-regions were smaller in desBβ1-42 than normal fibrinogen (1.2 nm ± 0.3 vs. 1.5 nm ± 0.2), whereas there were no differences between the D-regions (1.7 nm ± 0.4 vs. 1.7 nm ± 0.3). Polymerization rate for desBβ1-42 was slower than normal, resulting in clots with thinner fibers. Differences in oligomers were found, with predominantly lateral associations for desBβ1-42 and longitudinal associations for normal fibrin. Clot elasticity as measured by magnetic tweezers showed a G′ of ∼1 Pa for desBβ1-42 compared with ∼8 Pa for normal fibrin. Spring constants of early stage desBβ1-42 single fibers determined by atomic force microscopy were ∼3 times less than normal fibers of comparable dimensions and development. We conclude that Bβ1-42 plays an important role in fibrin oligomer formation. Absence of Bβ1-42 influences oligomer structure, affects the structure and properties of the final clot, and markedly reduces stiffness of the whole clot as well as individual fibrin fibers