Rigidity of fibrin gels as measured by quasielastic light scattering

The strength of fibrin gels has been investigated by a recently developed laser light scattering technique for determining the shear modulus of soft gels. By this method, changes in the modulus were monitored as a function of time without perturbing the material. Fibrin gels were crosslinked with blood coagulation factor XIII. Rigidity measurements and SDS–polyacrylamide gel electrophoresis were used to correlate gel strength with the number of covalently bonded subunit chains. The modulus was found to vary linearly with the number of crosslinks until maximum rigidity was achieved.     

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.     

A poroviscoelastic description of fibrin gels

The mechanical induction of specific cell phenotypes can only be properly controlled if the local stimuli applied to the cells are known as a function of the external applied loads. Finite element analysis of the cell carriers would be one method to calculate these local conditions. Furthermore, the constitutive model of the construct material should be able to describe mechanical events known to be responsible for cell stimulation, such as interstitial fluid flow. The aim of this study was to define a biphasic constitutive model for fibrin, a natural hydrogel often used for tissue engineering but not yet thoroughly characterized. Large strain poroelastic and poroviscoelastic constitutive equations were implemented into a finite element model of a fibrin gel. The parameter values for both formulations were found by either analytically solving equivalent low strain equations, or by optimizing directly the large strain equations based on experimental stress relaxation data. No poroelastic parameters that satisfactorily described the fibrin carrier behaviour could be found, suggesting that network viscoelasticity and fluid-flow time-dependent behaviour must be separately accounted for. It was demonstrated that fibrin can be described as a poroviscoelastic material, but a large strain characterization of the parameter values was necessary. The analytical resolution of the low strain poroviscoelastic equations was, however, accurate enough to serve as a reliable initial condition for further optimization of the parameter values with the large strain formulation.     

Decorin synthesized by arterial smooth muscle cells is retained in fibrin gels and modulates fibrin contraction

Fibrin serves as a provisional extracellular matrix (ECM) for arterial smooth muscle cells (ASMC) after vascular injury, yet little is known about the effect of fibrin on ECM remodeling by these cells. To address this question, monkey ASMC were grown on fibrin gels and tissue culture (TC) plastic, and proteoglycan synthesis and accumulation were assessed by radiolabeling. Initial rates of (35)S-sulfate incorporation into proteoglycans were identical for both groups, but increased proteoglycan accumulation was observed in cultures grown for 48 h on fibrin. This increased accumulation on fibrin was due to reduced proteoglycan turnover and retention within the fibrin gel. Decorin and biglycan constituted 40 and 14% of the total proteoglycan in the fibrin gels, whereas their combined contribution was only 12% in control matrices. To explore whether the retention of decorin in fibrin had any influence on the properties of the fibrin gel, ASMC-mediated fibrin contraction assays were performed. Both de novo synthesis of decorin as well as decorin added during polymerization inhibited the ability of the cells to contract fibrin. In contrast, decorin added exogenously to mature fibrin matrices had no effect on fibrin gel contraction. This study illustrates that decorin derived from ASMC selectively accumulates in fibrin and modifies fibrin architecture and mechanical properties. Such an accumulation may influence wound healing and the thrombotic properties of this provisional pro-atherosclerotic ECM.     

Fibrin autography of plasminogen activator by electrophoretic transfer into fibrin agar gels

An electrophoretic modification of the conventional fibrin autography that can be used for the detection of plasminogen activators (urokinase type and tissue type) and fibrin-degrading enzymes in complex biological fluids is described. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins and the substrate plasminogen are transferred electrophoretically into the fibrin indicator gel, resulting in an efficient transfer of proteinases as well as high resolution and contrast of fibrinolytic zones caused by plasminogen activator activity. Picogram amounts of human urokinase type plasminogen activator (about 0.002 International Unit) are still detectable. The technique is also applicable to reversed fibrin autography for plasminogen activator inhibitors.     

Gold labeling of thrombin and ultrastructural studies of thrombin-gold conjugate binding by fibrin

Monodispersed thrombin-gold (T-Au) conjugates were prepared by the absorption of a monolayer (3.8 nm thick) of human alpha-thrombin around individual monodispersed colloidal gold particles (16.5 +/- 1.8 nm). Like free molecular thrombin, T-Au conjugates can cause platelet aggregation, plasma clotting, and the release of fibrinopeptides A and B from fibrinogen. At the same thrombin concentration, T-Au conjugates have only one-tenth the fibrinogen-clotting activity of free thrombin and one-third the amidolytic activity of free thrombin. Hirudin can completely inhibit the fibrinogen-clotting activity of both T-Au conjugates and free thrombin, but can inhibit only half of the amidolytic activity of the conjugates. Diisopropyl fluorophosphonate can completely inhibit the fibrinogen-clotting activity and the amidolytic activity of both T-Au conjugates and free thrombin. T-Au conjugates were further characterized by studying the mechanism of their binding to fibrin and the location of the binding site on fibrin. The results of electron microscopic studies showed that T-Au conjugates, but not albumin-Au conjugates, are bound by fibrin. Increasing T-Au conjugate concentrations are associated with an increase in the number of T-Au conjugates binding to fibrin. At 0.1 microM thrombin, 73% of the T-Au conjugates are bound to branch points of the fibrin network with 27% of the T-Au conjugates present in the fibrin strands. At higher thrombin concentration (e.g., 0.5 microM) the percentage of T-Au conjugates bound to locations other than branch points increases to 62%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anisotropic mechanical properties of magnetically aligned fibrin gels measured by magnetic resonance elastography

The anisotropic mechanical properties of magnetically aligned fibrin gels were measured by magnetic resonance elastography (MRE) and by a standard mechanical test: unconfined compression. Soft anisotropic biomaterials are notoriously difficult to characterize, especially in vivo. MRE is well-suited for efficient, non-invasive, and non-destructive assessment of shear modulus. Direction-dependent differences in shear modulus were found to be statistically significant for gels polymerized at magnetic fields of 11.7 and 4.7 T compared to control gels. Mechanical anisotropy was greater in the gels polymerized at the higher magnetic field. These observations were consistent with results from unconfined compression tests. Analysis of confocal microscopy images of gels showed measurable alignment of fibrils in gels polymerized at 11.7 T. This study provides direct, quantitative measurements of the anisotropy in mechanical properties that accompanies fibril alignment in fibrin gels.     

Rheological characterization and dissolution kinetics of fibrin gels crosslinked by a microbial transglutaminase

Various fibrin gels were prepared with a microbial transglutaminase under miscellaneous conditions. The gels were characterized through their rheological properties. The influence of fibronectin addition and that of covalent bonding on the viscoelastic characteristics were evaluated. Gel elasticity is proportional to fibrinogen concentration but shows a nonlinear dependence on transglutaminase concentration. Additional crosslink of fibronectin in fibrin gels has no effect on the rheological character of the matrix. Dissolution kinetics in concentrated urea solutions evidences the role of covalent bonds on gel stability. The rheological properties and gel stability are discussed in relation with the enzyme-catalyzed covalent bonding. The microbial enzyme reactions are compared to those of FXIII and tissue transglutaminases.