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1.
Houser JR Hudson NE Ping L O'Brien ET Superfine R Lord ST Falvo MR 《Biophysical journal》2010,99(9):3038-3047
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. 相似文献
2.
Helms CC Ariëns RA Uitte de Willige S Standeven KF Guthold M 《Biophysical journal》2012,102(1):168-175
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. 相似文献
3.
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. 相似文献
4.
Nathan E. Hudson Feng Ding Igal Bucay E. Timothy O’Brien III Oleg V. Gorkun Richard Superfine Susan T. Lord Nikolay V. Dokholyan Michael R. Falvo 《Biophysical journal》2013
Fibrin fibers form the structural scaffold of blood clots. Thus, their mechanical properties are of central importance to understanding hemostasis and thrombotic disease. Recent studies have revealed that fibrin fibers are elastomeric despite their high degree of molecular ordering. These results have inspired a variety of molecular models for fibrin’s elasticity, ranging from reversible protein unfolding to rubber-like elasticity. An important property that has not been explored is the timescale of elastic recoil, a parameter that is critical for fibrin’s mechanical function and places a temporal constraint on molecular models of fiber elasticity. Using high-frame-rate imaging and atomic force microscopy-based nanomanipulation, we measured the recoil dynamics of individual fibrin fibers and found that the recoil was orders of magnitude faster than anticipated from models involving protein refolding. We also performed steered discrete molecular-dynamics simulations to investigate the molecular origins of the observed recoil. Our results point to the unstructured αC regions of the otherwise structured fibrin molecule as being responsible for the elastic recoil of the fibers. 相似文献
5.
Nathan E. Hudson E. Timothy O'Brien III Richard Superfine Michael R. Falvo 《Biophysical journal》2010,98(8):1632-1640
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. 相似文献
6.
Nathan?E. Hudson Feng Ding Igal Bucay E. Timothy O’Brien III Oleg?V. Gorkun Richard Superfine Susan?T. Lord Nikolay?V. Dokholyan Michael?R. Falvo 《Biophysical journal》2013,104(12):2671-2680
Fibrin fibers form the structural scaffold of blood clots. Thus, their mechanical properties are of central importance to understanding hemostasis and thrombotic disease. Recent studies have revealed that fibrin fibers are elastomeric despite their high degree of molecular ordering. These results have inspired a variety of molecular models for fibrin’s elasticity, ranging from reversible protein unfolding to rubber-like elasticity. An important property that has not been explored is the timescale of elastic recoil, a parameter that is critical for fibrin’s mechanical function and places a temporal constraint on molecular models of fiber elasticity. Using high-frame-rate imaging and atomic force microscopy-based nanomanipulation, we measured the recoil dynamics of individual fibrin fibers and found that the recoil was orders of magnitude faster than anticipated from models involving protein refolding. We also performed steered discrete molecular-dynamics simulations to investigate the molecular origins of the observed recoil. Our results point to the unstructured αC regions of the otherwise structured fibrin molecule as being responsible for the elastic recoil of the fibers. 相似文献
7.
Studies suggest that patients with deep vein thrombosis and diabetes often have hypercoagulable blood plasma, leading to a higher risk of thromboembolism formation through the rupture of blood clots, which may lead to stroke and death. Despite many advances in the field of blood clot formation and thrombosis, the influence of mechanical properties of fibrin in the formation of thromboembolisms in platelet-poor plasma is poorly understood. In this paper, we combine the concepts of reactive molecular dynamics and coarse-grained molecular modeling to predict the complex network formation of fibrin clots and the branching of fibrin monomers. The 340-kDa fibrinogen molecule was converted into a coarse-grained molecule with nine beads, and using our customized reactive potentials, we simulated the formation and polymerization process of a fibrin clot. The results show that higher concentrations of thrombin result in higher branch-point formation in the fibrin clot structure. Our results also highlight many interesting properties, such as the formation of thicker or thinner fibers depending on the thrombin concentration. To the best of our knowledge, this is the first successful molecular polymerization study of fibrin clots to focus on thrombin concentration. 相似文献
8.
Weisel JW 《Biophysical chemistry》2004,112(2-3):267-276
In this review, I set forth some basic information about the mechanical properties of fibrin clots and attempt to identify the big questions remaining. The intent is to make this topic understandable to both basic scientists who are interested in blood clotting and to hematologists or cardiologists, since I believe that this is something everyone working in these fields should know. The viscoelastic properties of fibrin are remarkable and unique among polymers. Moreover, these properties are essential to the physiology of blood clotting and are important for understanding and therefore preventing and treating thrombosis. 相似文献
9.
Wei Li Justin Sigley Marlien Pieters Christine?Carlisle Helms Chandrasekaran Nagaswami John?W. Weisel Martin Guthold 《Biophysical journal》2016,110(6):1400-1410
The major structural component of a blood clot is a mesh of fibrin fibers. Our goal was to determine whether fibrinogen glycation and fibrin fiber diameter have an effect on the mechanical properties of single fibrin fibers. We used a combined atomic force microscopy/fluorescence microscopy technique to determine the mechanical properties of individual fibrin fibers formed from blood plasma. Blood samples were taken from uncontrolled diabetic patients as well as age-, gender-, and body-mass-index-matched healthy individuals. The patients then underwent treatment to control blood glucose levels before end blood samples were taken. The fibrinogen glycation of the diabetic patients was reduced from 8.8 to 5.0 mol glucose/mol fibrinogen, and the healthy individuals had a mean fibrinogen glycation of 4.0 mol glucose/mol fibrinogen. We found that fibrinogen glycation had no significant systematic effect on single-fiber modulus, extensibility, or stress relaxation times. However, we did find that the fiber modulus, Y, strongly decreases with increasing fiber diameter, D, as Y ∝ D?1.6. Thin fibers can be 100 times stiffer than thick fibers. This is unusual because the modulus is a material constant and should not depend on the sample dimensions (diameter) for homogeneous materials. Our finding, therefore, implies that fibrin fibers do not have a homogeneous cross section of uniformly connected protofibrils, as is commonly thought. Instead, the density of protofibril connections, ρPb, strongly decreases with increasing diameter, as ρPb ∝ D?1.6. Thin fibers are denser and/or have more strongly connected protofibrils than thick fibers. This implies that it is easier to dissolve clots that consist of fewer thick fibers than those that consist of many thin fibers, which is consistent with experimental and clinical observations. 相似文献
10.
Adam?R. Wufsus Kuldeepsinh Rana Andrea Brown John?R. Dorgan Matthew?W. Liberatore Keith?B. Neeves 《Biophysical journal》2015,108(1):173-183
Fibrin is a biopolymer that gives thrombi the mechanical strength to withstand the forces imparted on them by blood flow. Importantly, fibrin is highly extensible, but strain hardens at low deformation rates. The density of fibrin in clots, especially arterial clots, is higher than that in gels made at plasma concentrations of fibrinogen (3–10 mg/mL), where most rheology studies have been conducted. Our objective in this study was to measure and characterize the elastic regimes of low (3–10 mg/mL) and high (30–100 mg/mL) density fibrin gels using shear and extensional rheology. Confocal microscopy of the gels shows that fiber density increases with fibrinogen concentration. At low strains, fibrin gels act as thermal networks independent of fibrinogen concentration. Within the low-strain regime, one can predict the mesh size of fibrin gels by the elastic modulus using semiflexible polymer theory. Significantly, this provides a link between gel mechanics and interstitial fluid flow. At moderate strains, we find that low-density fibrin gels act as nonaffine mechanical networks and transition to affine mechanical networks with increasing strains within the moderate regime, whereas high-density fibrin gels only act as affine mechanical networks. At high strains, the backbone of individual fibrin fibers stretches for all fibrin gels. Platelets can retract low-density gels by >80% of their initial volumes, but retraction is attenuated in high-density fibrin gels and with decreasing platelet density. Taken together, these results show that the nature of fibrin deformation is a strong function of fibrin fiber density, which has ramifications for the growth, embolization, and lysis of thrombi. 相似文献
11.
Visualization and mechanical manipulations of individual fibrin fibers suggest that fiber cross section has fractal dimension 1.3
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Guthold M Liu W Stephens B Lord ST Hantgan RR Erie DA Taylor RM Superfine R 《Biophysical journal》2004,87(6):4226-4236
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. 相似文献
12.
Fibrinogen Dusart: electron microscopy of molecules, fibers and clots, and viscoelastic properties of clots. 总被引:5,自引:0,他引:5
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J P Collet J L Woodhead J Soria C Soria M Mirshahi J P Caen J W Weisel 《Biophysical journal》1996,70(1):500-510
Ultrastructural perturbations resulting from defects in polymerization of fibrinogen Dusart, a congenital dysfibrinogenemia with the amino acid substitution A alpha 554 arginine to cysteine, were investigated by a variety of electron microscope studies. Polymerization of this mutant fibrinogen on addition of thrombin is impaired, producing clots with decreased porosity and increased resistance to fibrinolysis, resulting in thrombotic complications in the family members with this dysfibrinogenemia. Electron microscopy of rotary-shadowed individual molecules revealed that, in contrast to control fibrinogen, most of the alpha C domains of fibrinogen or fibrin Dusart appeared to be free-swimming appendages that do not exhibit intra- or intermolecular interactions either with each other or with the central domains. The location of albumin on the alpha C domains was demonstrated by electron microscopy using anti-albumin antibodies. Electron microscopy of negatively contrasted fibrin Dusart fibers indicated that they were less ordered than control fibers and had additional mass visible. Electron microscopy of freeze-dried, unidirectionally shadowed fibers showed that they were twisted with a shorter pitch. Scanning electron microscopy revealed that intact clots were made up of thin fibers with many branch points and very small pore sizes. The viscoelastic properties of Dusart fibrin clots measured with a torsion pendulum indicated a marked increase in stiffness consistent with the structural observations. 相似文献
13.
Katie?M. Weigandt Nathan White Dominic Chung Erica Ellingson Yi Wang Xiaoyun Fu Danilo?C. Pozzo 《Biophysical journal》2012,103(11):2399-2407
Using a combination of structural and mechanical characterization, we examine the effect of fibrinogen oxidation on the formation of fibrin clots. We find that treatment with hypochlorous acid preferentially oxidizes specific methionine residues on the α, β, and γ chains of fibrinogen. Oxidation is associated with the formation of a dense network of thin fibers after activation by thrombin. Additionally, both the linear and nonlinear mechanical properties of oxidized fibrin gels are found to be altered with oxidation. Finally, the structural modifications induced by oxidation are associated with delayed fibrin lysis via plasminogen and tissue plasminogen activator. Based on these results, we speculate that methionine oxidation of specific residues may be related to hindered lateral aggregation of protofibrils in fibrin gels. 相似文献
14.
Stéphanie M. C. Bruekers Maarten Jaspers José M. A. Hendriks Nicholas A. Kurniawan Gijsje H. Koenderink Paul H. J. Kouwer 《Cell Adhesion & Migration》2016,10(5):495-504
ABSTRACTThe mechanical and structural properties of the extracellular matrix (ECM) play an important role in regulating cell fate. The natural ECM has a complex fibrillar structure and shows nonlinear mechanical properties, which are both difficult to mimic synthetically. Therefore, systematically testing the influence of ECM properties on cellular behavior is very challenging. In this work we show two different approaches to tune the fibrillar structure and mechanical properties of fibrin hydrogels. Addition of extra thrombin before gelation increases the protein density within the fibrin fibers without significantly altering the mechanical properties of the resulting hydrogel. On the other hand, by forming a composite hydrogel with a synthetic biomimetic polyisocyanide network the protein density within the fibrin fibers decreases, and the mechanics of the composite material can be tuned by the PIC/fibrin mass ratio. The effect of the changes in gel structure and mechanics on cellular behavior are investigated, by studying human mesenchymal stem cell (hMSC) spreading and differentiation on these gels. We find that the trends observed in cell spreading and differentiation cannot be explained by the bulk mechanics of the gels, but correlate to the density of the fibrin fibers the gels are composed of. These findings strongly suggest that the microscopic properties of individual fibers in fibrous networks play an essential role in determining cell behavior. 相似文献
15.
16.
Proteolytic degradation of fibrin, the major structural component in blood clots, is critical both during normal wound healing and in the treatment of ischemic stroke and myocardial infarction. Fibrin-containing clots experience substantial strain due to platelet contraction, fluid shear, and mechanical stress at the wound site. However, little is understood about how mechanical forces may influence fibrin dissolution. We used video microscopy to image strained fibrin clots as they were degraded by plasmin, a major fibrinolytic enzyme. Applied strain causes up to 10-fold reduction in the rate of fibrin degradation. Analysis of our data supports a quantitative model in which the decrease in fibrin proteolysis rates with strain stems from slower transport of plasmin into the clot. We performed fluorescence recovery after photobleaching (FRAP) measurements to further probe the effect of strain on diffusive transport. We find that diffusivity perpendicular to the strain axis decreases with increasing strain, while diffusivity along the strain axis remains unchanged. Our results suggest that the properties of the fibrin network have evolved to protect mechanically loaded fibrin from degradation, consistent with its function in wound healing. The pronounced effect of strain upon diffusivity and proteolytic susceptibility within fibrin networks offers a potentially useful means of guiding cell growth and morphology in fibrin-based biomaterials. 相似文献
17.
Zhmurov A Brown AE Litvinov RI Dima RI Weisel JW Barsegov V 《Structure (London, England : 1993)》2011,19(11):1615-1624
Fibrinogen, upon enzymatic conversion to monomeric fibrin, provides the building blocks for fibrin polymer, the scaffold of blood clots and thrombi. Little has been known about the force-induced unfolding of fibrin(ogen), even though it is the foundation for the mechanical and rheological properties of fibrin, which are essential for hemostasis. We determined mechanisms and mapped the free energy landscape of the elongation of fibrin(ogen) monomers and oligomers through combined experimental and theoretical studies of the nanomechanical properties of fibrin(ogen), using atomic force microscopy-based single-molecule unfolding and simulations in the experimentally relevant timescale. We have found that mechanical unraveling of fibrin(ogen) is determined by the combined molecular transitions that couple stepwise unfolding of the γ chain nodules and reversible extension-contraction of the α-helical coiled-coil connectors. These findings provide important characteristics of the fibrin(ogen) nanomechanics necessary to understand the molecular origins of fibrin viscoelasticity at the fiber and whole clot levels. 相似文献
18.
Effects of thrombospondin on fibrin polymerization and structure 总被引:4,自引:0,他引:4
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. 相似文献
19.
The nanostructure of the fibrin fibers in fibrin clots is investigated by using spectrometry and small angle x-ray scattering measurements. First, an autocoherent analysis of the visible light spectra transmitted through formed clots is demonstrated to provide robust measurements of both the radius and density of the fibrin fibers. This method is validated via comparison with existing small-angle and dynamic light-scattering data. The complementary use of small angle x-ray scattering spectra and light spectrometry unambiguously shows the disjointed nature of the fibrin fibers. Indeed, under quasiphysiological conditions, the fibers are approximately one-half as dense as their crystalline fiber counterparts. Further, although the fibers are locally crystalline, they appear to possess a lateral fractal structure. 相似文献
20.
《The Journal of cell biology》1982,93(3):775-787
We explored the retraction or contraction of platelet-fibrin clots under isometric conditions. In the presence of micromolar calcium clots of normal platelet-rich plasma developed tension at an initial rate of 0.1 to 0.2 g/min per cm2 (initial cross-sectional area). Electron microscopy of clots fixed after attaining a force of 1.6 g/cm2 revealed platelets with elongated bodies and pseudopods in close apposition to fibrin strands which were oriented in cablelike fashion in the direction of tension. The development of tension could not be explained simply on the basis of platelet-platelet association and interaction alone. First, factor XIII-dependent cross-linking of fibrin fibers was critical to normal isometric contraction. Second, tension decreased linearly, rather than exponentially, when the platelet count in the platelet-fibrin clot was decreased, suggesting that platelets must be interacting with another component (i.e. fibrin). Thrombasthenic platelets, deficient in fibrinogen receptors, failed to develop tension or to align fibrin strands or pseudopods in the clot. Platelet-fibrin clots treated with vincristine to disassemble microtubules or cytochalasin B to disrupt microfilaments failed to develop tension and relaxed if these agents were added after tension had developed. Relaxation under these conditions, however, was not associated with loss of orientation of fibrin strands. Our findings suggest that platelet-fibrin interaction in clots under isometric conditions leads to orientation of fibrin strands and platelets in the direction of force generation. Tension develops as platelets simultaneously attach to and spread along fibrin strands, and contract. The contraction draws some fibrin into platelet-fibrin clumps and aligns other strands in the long axis of tension. The achievement and maintenance of maximum tension appears to depend on the development of platelet-fibrin attachments and extension of platelet bodies and long pseudopods containing bundles of microfilaments and microtubules along the oriented fibrin fibers. 相似文献