A combined elastic, fibrin and collagen stain

A method is described which demonstrates nuclei, elastic fibers, red blood cells, collagen and fibrin. Nuclei and elastic fibers are stained by a modified Verhoeff's elastic tissue stain which was previously developed and used in the elastic-Masson combination. Both early fibrin and red blood cells are shown by lissamine fast yellow. Mature fibrin, some types of collagen and other cytoplasmic changes are stained by a combination of acid fuchsin, Biebrich scarlet and ponceau 2R, while old fibrin is demonstrated by the collagen stain. This method takes about 1 hr to perform and has the added advantage that several entities are clearly shown in a single slide.     

Stain Technology: A Combined Elastic, Fibrin and Collagen Stain

A method is described which demonstrates nuclei, elastic fibers, red blood cells, collagen and fibrin. Nuclei and elastic fibers are stained by a modified VerhoefPs elastic tissue stain which was previously developed and used in the elastic-Masson combination. Both early fibrin and red blood cells are shown by Hssamine fast yellow. Mature fibrin, some types of collagen and other cytoplasmic changes are stained by a combination of acid fuchsia, Biebrich scarlet and ponceau 2R, while old fibrin is demonstrated by the collagen stain. This method takes about 1 hr to perform and has the added advantage that several entities are clearly shown in a single slide.     

Orbitometer flow and thrombus formation

Orbitometry (Hartert) is a rheological ex-vivo method to follow up physical assembly of a coagulum in layers during natural intensity of flow by orbital movement. Fibrin elasticity in the Orbitometers mode of Resonance Thrombography is differentiated from platelet activity as well as e.g. from the effects of disseminated coagulation-minimal in liver disease and maximal during disturbances of delivery. Transition into the mode of dynamic Tendography (Hartert) will e.g. register all fast going tests lasting minutes or seconds. It is comparable to an accelerated form of Thrombelastography (Hartert), the intercourse of which with coagulum yet is an exclusively static operation. Another category is measurement of blood and plasma viscosity. In concentrated blood it seizes plasticity of blood cells as well as their intensity of aggregation in orbital flow. The latest methodical development of Orbitometry is control of platelet activity in its function of adhesion. This is realized by measurement of specific physical effects released in platelet containing coagulum. They generate a structural degradation of fibrin elasticity modul as well as a tendency for coagulum adhesion. The practical use of Adhesiography is control of anticoagulants and platelet protecting substances in their quantitative influence on coagulum structure and on the mentioned platelet activities. A special disturbance of these platelet depending mechnisms obviously is getting evidence in case of v. Willebrand's syndrome.     

Surface plasmon resonance and free oscillation rheometry in combination: a useful approach for studies on haemostasis and interactions between whole blood and artificial surfaces

In haemostatic and biomaterial research biological processes at surfaces and in the bulk phase of the surface-contacting medium are important. The present work demonstrates the usefulness of the combination of surface plasmon resonance (SPR), sensitive to changes in refractive index at surfaces, and free oscillation rheometry (FOR), sensitive to rheological properties of the bulk, for simultaneous real-time measurements on coagulation and fibrinolysis of blood plasma and coagulation of whole blood. SFLLRN stimulated coagulation of native whole blood presented a higher SPR signal with different appearance than plasma coagulation, while the FOR signals corresponding to plasma and whole blood coagulation were similar. This indicated that the SPR technique was more sensitive to cell-surface interactions than to fibrin formation in whole blood during coagulation, while the FOR technique were equally sensitive to coagulation in whole blood and plasma. Spontaneous coagulation of native whole blood in contact with methyl- and hydroxyl-terminated self-assembled monolayers (SAM) on gold and gold surfaces regenerated after coagulation were also studied. The regenerated gold surfaces displayed the shortest coagulation times, although the contact-activation of blood coagulation for these surfaces was low. The methylated and hydroxylated surfaces were comparable in terms of coagulation activation, while the hydroxylated surfaces presented FOR signals that indicated detaching of the coagulum from the surface. The combination of SPR and FOR is well suited for studies of cell- and protein-surface interactions and simultaneous bulk processes. Possible applications are investigations of blood cell defects in patients and monitoring of native whole blood interactions with artificial surfaces.     

Electronmicroscopic study on the molecular shape of non-stabilized fibrin break-down products produced by complex heparin compounds

Experimental data indicate that products develop from non-stabilized fibrin because of nonfermentative splitting by complex heparin connections. These products have a globular form with a diameter of 10-250 A and are similar to morphological fibrinogen molecules. The identified products show no marked lytic effect towards non-stabilized fibrin. The application of S35 labelled heparin in combination with preparative ultracentrifugation and electron microscopy enabled the determination to be established that heparin complexes will combine with particles of fibrinomeres, thus causing their transfer from the fibrillary to the globular condition. The destruction of the connections of heparin complexes with their globular molecule structures of fibrinmonomere, e.g. by protamine sulfate, guarantees their development and primary polymerization. With factor XIIIa being present, the structurally reconstructed fibrin will form a stabilized coagulum of full value which is similar in its ultrastructure to fibrin obtained in control tests.     

Cells Actively Stiffen Fibrin Networks by Generating Contractile Stress

During wound healing and angiogenesis, fibrin serves as a provisional extracellular matrix. We use a model system of fibroblasts embedded in fibrin gels to study how cell-mediated contraction may influence the macroscopic mechanical properties of their extracellular matrix during such processes. We demonstrate by macroscopic shear rheology that the cells increase the elastic modulus of the fibrin gels. Microscopy observations show that this stiffening sets in when the cells spread and apply traction forces on the fibrin fibers. We further show that the stiffening response mimics the effect of an external stress applied by mechanical shear. We propose that stiffening is a consequence of active myosin-driven cell contraction, which provokes a nonlinear elastic response of the fibrin matrix. Cell-induced stiffening is limited to a factor 3 even though fibrin gels can in principle stiffen much more before breaking. We discuss this observation in light of recent models of fibrin gel elasticity, and conclude that the fibroblasts pull out floppy modes, such as thermal bending undulations, from the fibrin network, but do not axially stretch the fibers. Our findings are relevant for understanding the role of matrix contraction by cells during wound healing and cancer development, and may provide design parameters for materials to guide morphogenesis in tissue engineering.     

Cells Actively Stiffen Fibrin Networks by Generating Contractile Stress

During wound healing and angiogenesis, fibrin serves as a provisional extracellular matrix. We use a model system of fibroblasts embedded in fibrin gels to study how cell-mediated contraction may influence the macroscopic mechanical properties of their extracellular matrix during such processes. We demonstrate by macroscopic shear rheology that the cells increase the elastic modulus of the fibrin gels. Microscopy observations show that this stiffening sets in when the cells spread and apply traction forces on the fibrin fibers. We further show that the stiffening response mimics the effect of an external stress applied by mechanical shear. We propose that stiffening is a consequence of active myosin-driven cell contraction, which provokes a nonlinear elastic response of the fibrin matrix. Cell-induced stiffening is limited to a factor 3 even though fibrin gels can in principle stiffen much more before breaking. We discuss this observation in light of recent models of fibrin gel elasticity, and conclude that the fibroblasts pull out floppy modes, such as thermal bending undulations, from the fibrin network, but do not axially stretch the fibers. Our findings are relevant for understanding the role of matrix contraction by cells during wound healing and cancer development, and may provide design parameters for materials to guide morphogenesis in tissue engineering.     

Plasminogen activation by tissue plasminogen activator on fibrin clots with different surface structures

Transformation of fibrinogen into fibrin with consequent formation of the fibrin clot trimeric structure is one of the final steps in the blood coagulation system. The plasminogen activation by the tissue plasminogen activator (t-PA) is one of the fibrinolysis system key reactions. The effect of different factors on transformation of plasminogen into plasmin is capable to change essentially the equilibrium between coagulation and fibrinolytic sections of haemostasis system. We have studied the plasminogen activation by tissue plasminogen activator on fibrin clots surface formed on the interface between two phases and in presence of one phase. The t-PA plasminogen activation rate on fibrin clots both with film and without it the latter has been analyzed. These data allow to assume that the changes of fibrin clot structure depend on its formations, as well as are capable to influence essentially on plasminogen activation process by means of its tissue activating agent.     

Numerical analysis of flow in an elastic artery model.

Oscillatory and pulsatile flows of Newtonian fluids in straight elastic tubes are simulated numerically with the aid of Ling and Atabek's "local flow" assumption for the nonlinear convective acceleration terms. For the first time, a theoretical assessment of the local flow assumption is presented, and the range of validity of the assumption is estimated by comparison with perturbation solutions of the complete flow problem. Subsequent simulations with the local flow model indicate that the flow field and associated wall shear stress are extremely sensitive to the phase angle between oscillatory pressure and flow waves (impedance phase angle). This phase angle, which is a measure of the wave reflection present in the system, is known to be altered by arterial disease (e.g., hypertension) and vasoactive drugs. Thus, the paper elucidates a mechanism by which subtle changes in systemic hemodynamics (i.e., phase angles) can markedly influence local wall shear stress values.     

Features of the structure of fibrin clots,connected with conditions of gel formation

Fibrinogen to fibrin conversion and then fibrin clot three-dimensional network formation is one of the final steps in the coagulation system activation. Different factors, such as the environment temperature and pH, ions, so on, render an effect on the fibrin gel formation. Recently, the presence or absences of interface between two phases influence on the fibrin gel structure during its formation have been shown. Studies of fibrin gel structure peculiarities formed at different conditions (between two phases and without one phase) are demonstrated in this article. The plasmin enzymatic hydrolysis of fibrin clots both with surface film and without it was investigated. Experimental data allow to make a conclusion that the fibrin clot structure changes depend on its essential influence on the plasmin hydrolysis process of these clots.     

Coagulation Factor XIIIa Substrates in Human Plasma: IDENTIFICATION AND INCORPORATION INTO THE CLOT

Coagulation factor XIII (FXIII) is a transglutaminase with a well defined role in the final stages of blood coagulation. Active FXIII (FXIIIa) catalyzes the formation of ϵ-(γ-glutamyl)lysine isopeptide bonds between specific Gln and Lys residues. The primary physiological outcome of this catalytic activity is stabilization of the fibrin clot during coagulation. The stabilization is achieved through the introduction of cross-links between fibrin monomers and through cross-linking of proteins with anti-fibrinolytic activity to fibrin. FXIIIa additionally cross-links several proteins with other functionalities to the clot. Cross-linking of proteins to the clot is generally believed to modify clot characteristics such as proteolytic susceptibility and hereby affect the outcome of tissue damage. In the present study, we use a proteomic approach in combination with transglutaminase-specific labeling to identify FXIIIa plasma protein substrates and their reactive residues. The results revealed a total of 147 FXIIIa substrates, of which 132 have not previously been described. We confirm that 48 of the FXIIIa substrates were indeed incorporated into the insoluble fibrin clot during the coagulation of plasma. The identified substrates are involved in, among other activities, complement activation, coagulation, inflammatory and immune responses, and extracellular matrix organization.     

A new method for measuring the yield stress in thin layers of sedimenting blood.

A new method is presented to describe the low shear rate behavior of blood. We observed the response of a thin layer of sedimenting blood to a graded shear stress in a wedge-shaped chamber. The method allows quantitation of the degree of phase separation between red cells and plasma, and extracts the yield stress of the cell phase as a function of hematocrit. Our studies showed that the behavior of normal human blood underwent a transition from a solid-like gel to a Casson fluid. This transition began at the Casson predicted yield stress. The viscoelastic properties of blood were examined at shear stresses below the yield stress. The measured Young's elastic moduli were in good agreement with published data. The yield stress of blood showed a linear dependence on hematocrit up to 60%, and increased more rapidly at higher hematocrit.     

Preparation of fibrin des-AA by thrombin

The active thrombin is formed in the blood stream when the blood coagulation system is activated. It attacks fibrinogen, splits off two fibrinopeptides A and fibrinogen is transformed into des-AA fibrin which is able to polymerize spontaneously forming protofibrils. At high thrombin concentration the enzyme splits off two fibrinopeptides B and des-AA fibrin units are transformed into des-AABB fibrin. These two forms of fibrin are widely used in the biological experiments. However des-AA fibrin is obtained usually from fibrinogen using the snake poisons (such as reptilase). Des-AA fibrin was obtained also by physiological enzyme thrombin, but that des-AA fibrin samples had the contamination of des-AABB fibrin. At the present paper we have described the method of the des-AA fibrin preparation by thrombin without any contamination of des-AABB fibrin.     

Numerical simulation of sinusoidal flow in a straight elastic tube: effects of phase angles

Numerical simulations of flow in straight elastic (moving wall) tubes subjected to a sinusoidal pressure gradient were performed for conditions prevailing in large and medium sized arteries. The effects of varying the phase angle between the pressure gradient and the tube radius, the amplitude of wall motion, and the unsteadiness parameter (alpha) on flow rate and wall shear stress were investigated. Mean and peak flow rates and shear stresses were found to be strongly affected by the phase angle between the pressure gradient and the tube radius with greater sensitivity at higher diameter variation and higher alpha. In large artery simulations (alpha = 12), means flow rate was found to be 60% higher and peak flow rate to be 73% higher than corresponding rigid tube values for certain phase angles, while a threefold increase in mean wall shear stress and sevenfold increase in peak wall shear stress were observed in a sensitive phase angle range. Significant reversal in the wall shear stress direction occurred in the sensitive phase angle range even when there was negligible flow rate reversal. All effects were greatly diminished in simulations of medium sized vessels (alpha = 4). Some experimental evidence to support the predictions of a strong effect of phase angle on wall shear stress in large vessels is presented. Finally, physiological implications of the present work are discussed from a basis of aortic input impedance data, and a physical explanation for the extreme sensitivity of the flow field to small amplitude wall motion at high alpha is given.     

Viscoelastic behaviour aids extrusion from and reabsorption of the liquid phase into the digesta plug: creep rheometry of hindgut digesta in the common brushtail possum Trichosurus vulpecula

Temporal variation in the rheometric properties of the proximal and distal colonic digesta of an arboreal marsupial folivore, the common brushtail possum, was examined to assess flow behaviour during peristalsis, segmentation and other aspects of intestinal motility. The time-dependent rheometric characteristics on application of a constant shear stress within the physiological range showed an initial elastic and subsequent viscoelastic phase, which fitted Burger’s model of creep compliance. Similarly, the time-dependent rheometric characteristics on recovery from shear stress fitted with a generalised two-component Maxwell model of elastic and viscoelastic components for creep recovery. Differences in the relative magnitudes of the viscoelastic components during recovery from those during shear indicated that the physical properties of the digesta plug changed with sustained shear stress, a phenomenon, which is likely to result from extrusion of the liquid phase from the solid elements of the digesta plug. There was significant viscoelastic recovery during the initial 4 s following cessation of stress, which would allow for prompt concomitant reabsorption of the liquid phase into the digesta plug. This supports a hypothesis of alternate extrusion and reabsorption of the liquid phase of the digesta plug. This would promote both nutrient absorption across the intestinal wall (from liquid extrusion) and enzyme permeation and digestion (from liquid absorption into the plug). However, the presence of a slower component of viscoelastic recovery indicates that liquid phase reabsorption into the digesta plug is incomplete if the interval before a subsequent contraction is less than 150 s, in which case unreabsorbed liquid may be driven either orally or aborally. This would at least partly account for differences in retention times of liquid and solid phase digesta markers reported for the gastrointestinal tracts of numerous vertebrate species.     

An investigation of biphasic failure criteria for impact-induced fissuring of articular cartilage.

Articular cartilage consists of both solid and fluid phases with fissures observed on the surface occurring in the solid portion. In order to determine which of the solid phase stresses provides the best predictor for the initiation of a fissure, elastic stresses from a series of in vitro impact experiments were used to derive stresses in the solid phase of the cartilage. This stress information was then analyzed using a logistic regression to identify the best predictor of fissuring. The mechanical analysis indicated that low-magnitude tensile solid hoop stress develops in the solid phase within the contact zone in impacts involving the two smaller radius interfaces. The logistic regression, however, indicated that maximum shear stress in the solid (which is equal to the shear stress from the elastic analysis) was the best predictor of the occurrence of a fissure. This study helps support the suggestion that in stress fields dominated by compression, the maximum shear stress from an elastic analysis may be used to predict fissure initiation in cartilage.     

Fluid shear stress and the vascular endothelium: for better and for worse

As blood flows, the vascular wall is constantly subjected to physical forces, which regulate important physiological blood vessel responses, as well as being implicated in the development of arterial wall pathologies. Changes in blood flow, thus generating altered hemodynamic forces are responsible for acute vessel tone regulation, the development of blood vessel structure during embryogenesis and early growth, as well as chronic remodeling and generation of adult blood vessels. The complex interaction of biomechanical forces, and more specifically shear stress, derived by the flow of blood and the vascular endothelium raise many yet to be answered questions:How are mechanical forces transduced by endothelial cells into a biological response, and is there a "shear stress receptor"?Are "mechanical receptors" and the final signaling pathways they evoke similar to other stimulus-response transduction systems?How do vascular endothelial cells differ in their response to physiological or pathological shear stresses?Can shear stress receptors or shear stress responsive genes serve as novel targets for the design of diagnostic and therapeutic modalities for cardiovascular pathologies?The current review attempts to bring together recent findings on the in vivo and in vitro responses of the vascular endothelium to shear stress and to address some of the questions raised above.     

Elastic characterization of transversely isotropic soft materials by dynamic shear and asymmetric indentation

The mechanical characterization of soft anisotropic materials is a fundamental challenge because of difficulties in applying mechanical loads to soft matter and the need to combine information from multiple tests. A method to characterize the linear elastic properties of transversely isotropic soft materials is proposed, based on the combination of dynamic shear testing (DST) and asymmetric indentation. The procedure was demonstrated by characterizing a nearly incompressible transversely isotropic soft material. A soft gel with controlled anisotropy was obtained by polymerizing a mixture of fibrinogen and thrombin solutions in a high field magnet (B?=?11.7 T); fibrils in the resulting gel were predominantly aligned parallel to the magnetic field. Aligned fibrin gels were subject to dynamic (20-40 Hz) shear deformation in two orthogonal directions. The shear storage modulus was 1.08?±?0. 42 kPa (mean?±?std. dev.) for shear in a plane parallel to the dominant fiber direction, and 0.58?±?0.21 kPa for shear in the plane of isotropy. Gels were indented by a rectangular tip of a large aspect ratio, aligned either parallel or perpendicular to the normal to the plane of transverse isotropy. Aligned fibrin gels appeared stiffer when indented with the long axis of a rectangular tip perpendicular to the dominant fiber direction. Three-dimensional numerical simulations of asymmetric indentation were used to determine the relationship between direction-dependent differences in indentation stiffness and material parameters. This approach enables the estimation of a complete set of parameters for an incompressible, transversely isotropic, linear elastic material.     

Foam-like compression behavior of fibrin networks

The rheological properties of fibrin networks have been of long-standing interest. As such there is a wealth of studies of their shear and tensile responses, but their compressive behavior remains unexplored. Here, by characterization of the network structure with synchronous measurement of the fibrin storage and loss moduli at increasing degrees of compression, we show that the compressive behavior of fibrin networks is similar to that of cellular solids. A nonlinear stress–strain response of fibrin consists of three regimes: (1) an initial linear regime, in which most fibers are straight, (2) a plateau regime, in which more and more fibers buckle and collapse, and (3) a markedly nonlinear regime, in which network densification occurs by bending of buckled fibers and inter-fiber contacts. Importantly, the spatially non-uniform network deformation included formation of a moving “compression front” along the axis of strain, which segregated the fibrin network into compartments with different fiber densities and structure. The Young’s modulus of the linear phase depends quadratically on the fibrin volume fraction while that in the densified phase depends cubically on it. The viscoelastic plateau regime corresponds to a mixture of these two phases in which the fractions of the two phases change during compression. We model this regime using a continuum theory of phase transitions and analytically predict the storage and loss moduli which are in good agreement with the experimental data. Our work shows that fibrin networks are a member of a broad class of natural cellular materials which includes cancellous bone, wood and cork.     

Engineered Molecular Therapeutics Targeting Fibrin and the Coagulation System: a Biophysical Perspective

The coagulation cascade represents a sophisticated and highly choreographed series of molecular events taking place in the blood with important clinical implications. One key player in coagulation is fibrinogen, a highly abundant soluble blood protein that is processed by thrombin proteases at wound sites, triggering self-assembly of an insoluble protein hydrogel known as a fibrin clot. By forming the key protein component of blood clots, fibrin acts as a structural biomaterial with biophysical properties well suited to its role inhibiting fluid flow and maintaining hemostasis. Based on its clinical importance, fibrin is being investigated as a potentially valuable molecular target in the development of coagulation therapies. In this topical review, we summarize our current understanding of the coagulation cascade from a molecular, structural and biophysical perspective. We highlight single-molecule studies on proteins involved in blood coagulation and report on the current state of the art in directed evolution and molecular engineering of fibrin-targeted proteins and polymers for modulating coagulation. This biophysical overview will help acclimatize newcomers to the field and catalyze interdisciplinary work in biomolecular engineering toward the development of new therapies targeting fibrin and the coagulation system.