Structural origins of fibrin clot rheology

The origins of clot rheological behavior associated with network morphology and factor XIIIa-induced cross-linking were studied in fibrin clots. Network morphology was manipulated by varying the concentrations of fibrinogen, thrombin, and calcium ion, and cross-linking was controlled by a synthetic, active-center inhibitor of FXIIIa. Quantitative measurements of network features (fiber lengths, fiber diameters, and fiber and branching densities) were made by analyzing computerized three-dimensional models constructed from stereo pairs of scanning electron micrographs. Large fiber diameters and lengths were established only when branching was minimal, and increases in fiber length were generally associated with increases in fiber diameter. Junctions at which three fibers joined were the dominant branchpoint type. Viscoelastic properties of the clots were measured with a rheometer and were correlated with structural features of the networks. At constant fibrinogen but varying thrombin and calcium concentrations, maximal rigidities were established in samples (both cross-linked and noncross-linked) which displayed a balance between large fiber sizes and great branching. Clot rigidity was also enhanced by increasing fiber and branchpoint densities at greater fibrinogen concentrations. Network morphology is only minimally altered by the FXIIIa-catalyzed cross-linking reaction, which seems to augment clot rigidity most likely by the stiffening of existing fibers.     

The molecular origins of the mechanical properties of fibrin

When normal blood circulation is compromised by damage to vessel walls, clots are formed at the site of injury. These clots prevent bleeding and support wound healing. To sustain such physiological functions, clots are remarkably extensible and elastic. Fibrin fibers provide the supporting framework of blood clots, and the properties of these fibers underlie the mechanical properties of clots. Recent studies, which examined individual fibrin fibers or cylindrical fibrin clots, have shown that the mechanical properties of fibrin depend on the mechanical properties of the individual fibrin monomers. Within the fibrin monomer, three structures could contribute to these properties: the coiled-coil connectors the folded globular nodules and the relatively unstructured αC regions. Experimental data suggest that each of these structures contributes. Here we review the recent work with a focus on the molecular origins of the remarkable biomechanical properties of fibrin clots.     

Evidence that αC region is origin of low modulus, high extensibility, and strain stiffening in fibrin fibers

Fibrin fibers form the structural scaffold of blood clots and perform the mechanical task of stemming blood flow. Several decades of investigation of fibrin fiber networks using macroscopic techniques have revealed remarkable mechanical properties. More recently, the microscopic origins of fibrin's mechanics have been probed through direct measurements on single fibrin fibers and individual fibrinogen molecules. Using a nanomanipulation system, we investigated the mechanical properties of individual fibrin fibers. The fibers were stretched with the atomic force microscope, and stress-versus-strain data was collected for fibers formed with and without ligation by the activated transglutaminase factor XIII (FXIIIa). We observed that ligation with FXIIIa nearly doubled the stiffness of the fibers. The stress-versus-strain behavior indicates that fibrin fibers exhibit properties similar to other elastomeric biopolymers. We propose a mechanical model that fits our observed force extension data, is consistent with the results of the ligation data, and suggests that the large observed extensibility in fibrin fibers is mediated by the natively unfolded regions of the molecule. Although some models attribute fibrin's force-versus-extension behavior to unfolding of structured regions within the monomer, our analysis argues that these models are inconsistent with the measured extensibility and elastic modulus.     

Polydispersion in the diameter of fibers in fibrin networks: Consequences on the measurement of mass–length ratio by permeability and turbidity

The diameters of the fibrin fibers in a clot are not uniform. Morphometric analysis of transmission electron micrographs show a bimodal distribution. The effect of polydispersity of fiber diameter on mass–length ratio calculated from the turbidity and permeability of a clot has been investigated with the aid of a two network model, the networks being called Major and Minor. The fibers in the Major network are many times thicker than those in the Minor network. In a model in which Major network fibers are 10 times thicker than fibers of the Minor network, the fibers in the Minor network make a negligible contribution to the turbidity of the clot. However, they may have a marked effect on its permeability. Experiments with clots made from human fibrinogen show that the Minor network is stabilized by α-polymer and γ-γ linkages and that without such linkages it is washed away during permeation. It remains relatively intact in crosslinked clots. In agreement with the theoretical model, when mass–length ratios calculated from the turbidity are compared with those calculated from the permeability, the latter were reduced in crosslinked clots with an intact Minor network.     

Clots of beta-fibrin. Viscoelastic properties, temperature dependence of elasticity, and interaction with fibrinogen-binding tetrapeptides.

Clots of human beta-fibrin, in which only (or predominantly) the B fibrinopeptide is released, were formed at 14 degrees C by copperhead venom procoagulant enzyme (CVE or venzyme), at pH 8.5, ionic strength 0.45. The shear modulus of elasticity increased slowly and after several days attained a constant value, which was lower than those of alpha-fibrin or alpha beta-fibrin under the same conditions. Before studying the temperature dependence of elasticity, the CVE was then inhibited by introducing phenyl methyl sulfonyl chloride (PMSF) by diffusion. With increasing temperature, the modulus decreased progressively from 5 degrees C to nearly zero at 35 degrees and was essentially reversible with temperature change; recovery of elasticity after change from 34.5 degrees to 14 degrees required approximately 2 d but was considerably faster than the initial buildup of elasticity by CVE at 14 degrees. Creep and creep recovery measurements on unligated clots showed creep rates and irrecoverable deformation that were similar in magnitude to those of alpha-fibrin clots formed with batroxobin and much larger than those of alpha beta-fibrin clots formed with thrombin, under the same conditions. During creep and creep recovery, the differential modulus or compliance remained constant, showing that there was no permanent structural damage, and if network strands are severed in slow flow, they must rejoin in new configurations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced Plasminogen Binding and Delayed Activation Render γ′-Fibrin More Resistant to Lysis than γA-Fibrin

Fibrin (Fn) clots formed from γ′-fibrinogen (γ′-Fg), a variant with an elongated γ-chain, are resistant to lysis when compared with clots formed from the predominant γ_A-Fg, a finding previously attributed to differences in clot structure due to delayed thrombin-mediated fibrinopeptide (FP) B release or impaired cross-linking by factor XIIIa. We investigated whether slower lysis of γ′-Fn reflects delayed plasminogen (Pg) binding and/or activation by tissue plasminogen activator (tPA), reduced plasmin-mediated proteolysis of γ′-Fn, and/or altered cross-linking. Clots formed from γ′-Fg lysed more slowly than those formed from γ_A-Fg when lysis was initiated with tPA/Pg when FPA and FPB were both released, but not when lysis was initiated with plasmin, or when only FPA was released. Pg bound to γ′-Fn with an association rate constant 22% lower than that to γ_A-Fn, and the lag time for initiation of Pg activation by tPA was longer with γ′-Fn than with γ_A-Fn. Once initiated, however, Pg activation kinetics were similar. Factor XIIIa had similar effects on clots formed from both Fg isoforms. Therefore, slower lysis of γ′-Fn clots reflects delayed FPB release, which results in delayed binding and activation of Pg. When clots were formed from Fg mixtures containing more than 20% γ′-Fg, the upper limit of the normal level, the delay in lysis was magnified. These data suggest that circulating levels of γ′-Fg modulate the susceptibility of clots to lysis by slowing Pg activation by tPA and provide another example of the intimate connections between coagulation and fibrinolysis.     

Peroxynitrite may affect fibrinolysis via the reduction of platelet-related fibrinolysis resistance and alteration of clot structure

We tested the hypothesis that in vitro peroxynitrite (ONOO⁻, a product of activated inflammatory cells) may affect fibrinolysis in human blood through the reduction of platelet-related fibrinolysis resistance. It was found that ONOO⁻ (25–300 µM) accelerated lysis of platelet-fibrin clots (in PRP) dose-dependently, whereas fibrinolysis of platelet-free clots was slightly inhibited by ≥1000 µM stressor. Concentrations of ONOO⁻ affecting the lysis of platelet-rich clots, inhibited clot retraction (CR) in a dose-dependent manner. Thromboelastometry (ROTEM) measurements performed in PRP showed that treatment with ONOO⁻ (threshold conc. 100 µM) prolongs clotting time, and reduces alpha angle, and clot formation velocity parameters indicating for reduced thrombin formation rate. In PRP, ONOO⁻ (threshold conc. 100 µM) reduced the collagen-evoked exposure of phosphatidylserine (PS) on platelets’ plasma membrane, the shedding of platelet-derived microparticles (PMP), and inhibited platelet-dependent thrombin generation (measured in artificial system), dose-dependently. As judged by confocal microscopy, similar ONOO⁻ concentrations altered the architecture of clots formed in collagen-treated PRP. Clots formed in the presence of ONOO⁻ were less dense and were composed of thicker fibers, which make them more susceptible to lysis. In platelet-depleted plasma, ONOO⁻ (up to milimolar concentration) did not alter clot structure. Blockage of PS exposed on platelets resulted in an alteration of clot architecture toward more prone to lysis. ONOO⁻, at lysis-affecting concentrations, inhibited the collagen-evoked secretion of fibrinolytic inhibitors from platelets. We conclude that physiologically relevant ONOO⁻ concentrations may accelerate the lysis of platelet-fibrin clots predominantly via downregulation of platelet-related mechanisms including: platelet secretion, clot retraction, platelet procoagulant response, and the alteration in clot architecture associated with it.     

Pro-thrombotic state induced by post-translational modification of fibrinogen by reactive nitrogen species

Formation of nitric oxide-derived oxidants has been linked to development of atherosclerosis and associated thrombotic complications. Although systemic levels of protein nitrotyrosine predict risk for coronary artery disease, neither specific proteins targeted for modification nor functional consequences that might contribute to disease pathogenesis have been defined. Here we report a selective increase in circulating levels of nitrated fibrinogen in patients with coronary artery disease. Exposure of fibrinogen to nitrating oxidants, including those produced by the myeloperoxidase-hydrogen peroxide-nitrite system, significantly accelerates clot formation and factor XIII cross-linking, whereas exposure of fibrinogen to non-nitrating oxidants decelerates clot formation. Clots formed with fibrinogen exposed to nitrating oxidants are composed of large bundles made from twisted thin fibrin fibers with increased permeation and a decrease in storage modulus G' value, suggesting that these clots could be easily deformed by mechanical stresses. In contrast, clots formed with fibrinogen exposed to non-nitrating oxidants showed decreased permeation with normal architecture. Fibrinogen modified by exposure to physiologic nitration systems demonstrated no difference in the rate of plasmin-induced clot lysis, platelet aggregation, or binding. Thus, increased levels of fibrinogen nitration may lead to a pro-thrombotic state via acceleration in formation of fibrin clots. The present results may account, in part, for the association between nitrative stress and risk for coronary artery disease.     

A reconstituted dilute blood clot lysis assay for the medium throughput screening of thrombolytic compounds.

Antithrombotic and clotting factors have long been targets for drug discovery, necessitating the development of blood assays to determine the efficacy of lead compounds prior to animal testing. We have developed a reconstituted blood clot lysis assay which eliminates the need for on-site donors. The assay uses whole blood stored at 4 degrees C obtained from a local blood bank, diluted 1:10 in phosphate buffer. This blood was supplemented with 125I-labeled fibrinogen and the release of radioactive fibrinopeptides from formed clots was measured. The whole blood used in this assay, which had been stored at 4 degrees C for several days, no longer formed solid or retracting clots. Thus, platelets 5-7 days ex vivo (165 x 10(6) platelets) were added to the whole blood in the presence of thrombin (0.80 IU/ml) to form clots. Solid clots formed within 2 min of thrombin addition and began retracting shortly thereafter. In the absence of any thrombolytic agent, clots fully retracted within 2.5 h and remained stable. Thrombin-stimulated clot formation was completely inhibited by heparin. Clots could be lysed in a dose-dependent fashion in the presence of tissue-type plasminogen activator. Clot lysis could be completely inhibited in a dose-dependent fashion with plasminogen activator inhibitor type 1. To demonstrate the utility of this assay as a screen for thrombolytic agents, a 14-amino-acid PAI-1-inhibitory peptide relieved the PAI-1 effect on tPA in a dose-dependent fashion. These data describe an assay for the screening of potential pro-fibrinolytic agents that target PAI-1 inhibition in a human plasma-based system that is versatile, cost-effective, and physiologically relevant and does not rely on the availability of on-site blood donors.     

Conversion of fibrinogen to fibrin. The correlation between clotting kinetics and morphology of fibrin polymerized in different solvent media

The morphology of fibrin strongly depends on solvent medium, as shown by clotting experiments carried out in the presence of different salts. The clots were characterized by electron microscopy and spectrophotometric methods; the kinetics of gelation were determined. In the presence of electrolytes which strongly delay clotting, the strands are thin and few branching points are observed; opposite morphological changes are induced by salts which act as accelerating agents. On the basis of this correlation, and of previous data on the structure of fibrin, a kinetic model of the self-assembly process is outlined. It accounts well for the observed solvent effects on the morphology of the network. An important result emerging from our experiments is that the fibers undergo branching prior to gelation. Branching points arise from the defective growth of the fibers; a simple explanation of the occurrence of branching may be obtained by our self-assembly model.     

Effects of thrombospondin on fibrin polymerization and structure

Thrombospondin (TSP) is a trace protein in plasma but is released in high concentrations from alpha-granules of activated platelets during hemostasis. It binds to the platelet membrane and becomes incorporated into fibrin clots. A variety of approaches were taken to learn the effects of TSP on fibrin polymerization and structure. 125I-TSP and 125I-fibrinogen were used to study the effect of TSP concentration on the extent of TSP and fibrin incorporation. Turbidity at 600 nm was used to monitor the time course of polymerization. Wavelength dependence of the turbidity was used to calculate the mass to length ratio, fiber diameter, and fiber density of fibrin formed in the presence and absence of TSP. Morphologies of control and TSP-containing clots were examined by electron microscopy following critical point drying. The initial TSP concentration influenced the amount of TSP incorporated but did not alter the extent of fibrin polymerization. TSP, in a concentration-dependent manner, reduced the lag time to turbidity rise and caused formation of more numerous but thinner fibers. Except for their diameter, these fibers were identical to fibers of control fibrin in terms of density and morphology. It is proposed that TSP interacts with fibrin intermediates to accelerate fiber growth, perhaps by serving as a trifunctional branching unit during network formation. The properties of fibrin around aggregating platelets, therefore, may be influenced considerably by secreted TSP.     

Roots and calibers of the human coronary arteries

The anatomical structure of the coronary-aortic junctions in humans is studied by using corrosion casts of the coronary network. A model is proposed for the specification of these junctions in terms of vessel diameters and branching angles, and the model is used to produce morphological data on these junctions which hitherto have not been available. This anatomical model correlates poorly with the accepted theoretical model of arterial bifurcations in the cardiovascular system. The results suggest that the structure of the coronary-aortic junctions is very different from the structure of typical arterial bifurcations and, by implication, that the flow conditions under which they function are very different. A good understanding of these junctions is important in coronary bypass surgery, where the coronary-aortic junctions are emulated by creating a new anastomosis for the graft at the base of the ascending aorta, and in coronary artery disease, where atherosclerotic lesions occur not far from the coronary-aortic junctions.     

FXIII polymorphisms, fibrin clot structure and thrombotic risk

Fibrin clot structure is highly dependent on factor XIII activity. Activated FXIII catalyzes the formation of the peptide bonds between the gamma and alpha chains in noncovalently bound fibrin polymers and incorporates various adhesive and antifibrinolytic proteins into the final fibrin clot. In the absence of activated FXIII, clots are unstable and susceptible to fibrinolysis. Several studies have examined the effects of FXIII polymorphisms on final fibrin clot structure and clinical thrombotic risk. The Val34Leu FXIII polymorphism is associated with increased activation by thrombin. In the presence of saturating thrombin concentrations, however, FXIIIa specific enzyme activity is not affected by genetic polymorphisms. Fibrin clots formed in the presence of the FXIII 34Leu polymorphisms do tend to be thinner and less porous, however. The effects of prothrombin concentrations on clot structure have suggested that thinner clots are more resistant to fibrinolysis and associated with increased thrombotic risk. Most clinical studies of 34Leu FXIII carriers, however, have demonstrated a lower incidence of both venous and arterial thrombosis in carriers of the mutant allele compared to Val/Val carriers. One recent study has suggested that the interactions between FXIII phenotype and plasma fibrinogen concentrations significantly influence clinical thrombotic risk.     

Stiffening of Individual Fibrin Fibers Equitably Distributes Strain and Strengthens Networks

As the structural backbone of blood clots, fibrin networks carry out the mechanical task of stemming blood flow at sites of vascular injury. These networks exhibit a rich set of remarkable mechanical properties, but a detailed picture relating the microscopic mechanics of the individual fibers to the overall network properties has not been fully developed. In particular, how the high strain and failure characteristics of single fibers affect the overall strength of the network is not known. Using a combined fluorescence/atomic force microscope nanomanipulation system, we stretched 2-D fibrin networks to the point of failure, while recording the strain of individual fibers. Our results were compared to a pair of model networks: one composed of linearly responding elements and a second of nonlinear, strain-stiffening elements. We find that strain-stiffening of the individual fibers is necessary to explain the pattern of strain propagation throughout the network that we observe in our experiments. Fiber strain-stiffening acts to distribute strain more equitably within the network, reduce strain maxima, and increase network strength. Along with its physiological implications, a detailed understanding of this strengthening mechanism may lead to new design strategies for engineered polymeric materials.     

Polymerization of deoxy-sickle cell hemoglobin in high-phosphate buffer

Deoxy-sicklecell hemoglobin (HbS) polymerizes in 0.05 M phosphate buffer to form long helical fibers. The reaction typically occurs when the concentration of HbS is about 165 mg/ml. Polymerization produces a variety of polymorphic forms. The structure of the fibers can be probed by using site-directed mutants to examine the effect of altering the residues involved in intermolecular interactions. Polymerization can also be induced in the presence of 1.5 M phosphate buffer. Under these conditions polymerization occurs at much lower concentrations (ca. 5 mg/ml), which is advantageous when site-directed mutants are being used because only small quantities of the mutants are available. We have characterized the structure of HbS polymers formed in 1.5 M phosphate to determine how their structures are related to the polymers formed under more physiological conditions. Under both sets of conditions fibers are the first species to form. At pHs between 6.7 and 7.3 fibers initially form bundles and then crystals. At lower pHs fibers form macrofibers and then crystals. Fourier transforms of micrographs of the polymers formed in 1.5 M phosphate display the 32- and 64-A(-1) periodicity characteristic of fibers formed in 0.05 M phosphate buffer. The 64-A(-1) layer line is less prominent in Fourier transforms of negatively stained fibers formed in 1.5 M phosphate possibly because salt interferes with staining of the fibers. However, micrographs and Fourier transforms of frozen hydrated fibers formed in high and low phosphate display the same periodicities. Under both sets of reaction conditions HbS polymers form crystals with the same unit cell parameters as Wishner-Love crystals (a = 64 A, b = 185 A, c = 53 A). Some of the polymerization intermediates were examined in the frozen-hydrated state in order to determine whether their structures were significantly perturbed by negative staining. We have also carried out reconstructions of the frozen-hydrated fibers in high and low phosphate to compare their molecular coordinates. The helical projection of the reconstructions in low phosphate shows the expected 14-strand structure. In high phosphate the 14-strand fibers are also formed and their molecular coordinates are the same (within experimental error) as those of fibers formed in 0.05 M phosphate. In addition, the reconstructions of high-phosphate fibers reveal a new minor variant of fiber containing 10 strands. The polymerization products in 1.5 M phosphate buffer were generally indistinguishable from those formed in 0.05 M phosphate buffer. Micrographs of frozen hydrated specimens have facilitated the interpretation of previously published micrographs using negative staining.     

Electron microscopy of fine fibrin clots and fine and coarse fibrin films: Observations of fibers in cross-section and in deformed states

Fine fibrin clots and coarse and fine fibrin films (both ligated and unligated), formed by shrinkage of clots in one dimension, were examined by electron microscopy. Specimens of clots were prepared by critical point drying and by embedding and sectioning; specimens of films were prepared by embedding and sectioning only. In the fine clots, network junctions appeared to be formed by fiber segments in which two or more protofibrils were gently twisted around each other for distances of the order of 200 nm and then diverged to give trifunctional branch points. This topology appeared to be preserved in the fine films. It is proposed that the strength of the junctions is primarily provided by the twisting topology, though reinforced by non-covalent bonding involving the B sites uncovered by thrombin. In coarse films, bundles of protofibrils, lying primarily in the film plane, had diameters of 40 to 200 nm and were gently twisted around each other to form thicker cables. Uniaxial stretching, up to 100%, of either fine or coarse film before fixing caused suprisingly extensive orientation of the protofibrils or bundles. However, random orientation was recovered if a stretched ligated film was allowed to retract to its original dimensions before fixing. In a stretched coarse film sectioned perpendicular to the stretch direction, fiber bundles could be seen in cross-section; these were roughly circular with scalloped edges. The changes with stretching and recovery are discussed in relation to possible mechanisms of deformation and elastic energy storage.     

Structural Hierarchy Governs Fibrin Gel Mechanics

Fibrin gels are responsible for the mechanical strength of blood clots, which are among the most resilient protein materials in nature. Here we investigate the physical origin of this mechanical behavior by performing rheology measurements on reconstituted fibrin gels. We find that increasing levels of shear strain induce a succession of distinct elastic responses that reflect stretching processes on different length scales. We present a theoretical model that explains these observations in terms of the unique hierarchical architecture of the fibers. The fibers are bundles of semiflexible protofibrils that are loosely connected by flexible linker chains. This architecture makes the fibers 100-fold more flexible to bending than anticipated based on their large diameter. Moreover, in contrast with other biopolymers, fibrin fibers intrinsically stiffen when stretched. The resulting hierarchy of elastic regimes explains the incredible resilience of fibrin clots against large deformations.     

Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells.

The Arp2/3 complex and filamin A (FLNa) branch actin filaments. To define the role of these actin-binding proteins in cellular actin architecture, we compared the morphology of FLNa-deficient human melanoma (M2) cells and three stable derivatives of these cells expressing normal FLNa concentrations. All the cell lines contain similar amounts of the Arp2/3 complex. Serum addition causes serum-starved M2 cells to extend flat protrusions transiently; thereafter, the protrusions turn into spherical blebs and the cells do not crawl. The short-lived lamellae of M2 cells contain a dense mat of long actin filaments in contrast to a more three-dimensional orthogonal network of shorter actin filaments in lamellae of identically treated FLNa-expressing cells capable of translational locomotion. FLNa-specific antibodies localize throughout the leading lamellae of these cells at junctions between orthogonally intersecting actin filaments. Arp2/3 complex-specific antibodies stain diffusely and label a few, although not the same, actin filament overlap sites as FLNa antibody. We conclude that FLNa is essential in cells that express it for stabilizing orthogonal actin networks suitable for locomotion. Contrary to some proposals, Arp2/3 complex-mediated branching of actin alone is insufficient for establishing an orthogonal actin organization or maintaining mechanical stability at the leading edge.     

Visualization and mechanical manipulations of individual fibrin fibers suggest that fiber cross section has fractal dimension 1.3

We report protocols and techniques to image and mechanically manipulate individual fibrin fibers, which are key structural components of blood clots. Using atomic force microscopy-based lateral force manipulations we determined the rupture force, FR, f fibrin fibers as a function of their diameter, D, in ambient conditions. As expected, the rupture force increases with increasing diameter; however, somewhat unexpectedly, it increases as FR approximately D1.30+/-0.06. Moreover, using a combined atomic force microscopy-fluorescence microscopy instrument, we determined the light intensity, I, of single fibers, that were formed with fluorescently labeled fibrinogen, as a function of their diameter, D. Similar to the force data, we found that the light intensity, and thus the number of molecules per cross section, increases as I approximately D1.25+/-0.11. Based on these findings we propose that fibrin fibers are fractals for which the number of molecules per cross section increases as about D1.3. This implies that the molecule density varies as rhoD approximately D -0.7, i.e., thinner fibers are denser than thicker fibers. Such a model would be consistent with the observation that fibrin fibers consist of 70-80% water and only 20-30% protein, which also suggests that fibrin fibers are very porous.