Native fibrin gel networks observed by 3D microscopy, permeation and turbidity

Native fully hydrated fibrin gels formed at different fibrinogen and thrombin concentrations and at different ionic strengths were studied by confocal laser 3D microscopy, liquid permeation and turbidity. The gels were found to be composed of straight rod-like fiber elements that often came together at denser nodes. In gels formed at high fibrinogen concentrations, or with high amounts of thrombin, the spaces between the fibers decreased, indicating a decrease of gel porosity. The fiber strands were also shorter. Gel porosity decreased dramatically in gels formed at the high ionic strengths. Shorter fibers were observed and fiber swelling occurred at ionic strengths above 0.24. Quantitative parameters for gel porosity, fiber mass/length ratio and diameter were also derived by liquid permeation and turbidometric analyses of the gels. Permeation analysis showed that gel porosity (measured as Ks) decreased in gels formed at higher fibrin and thrombin concentrations in agreement with the porosity observed by microscopy. The turbidometric analysis showed good agreement with the permeation data for gels formed at various thrombin concentrations, but supported the permeation data more poorly in gels formed at different fibrinogen concentrations, especially above 2.5 mg/ml. Turbidometric analysis showed that the fiber mass/length ratio and diameter decreased in gels formed at ionic strength up to 0.24, as was seen in the permeation study. However, at higher ionic strengths swelling of the fibers was suggested from the gel turbidity data and this was also indicated by microscopy. These findings are discussed in relation to previous hydrodynamic and electron microscopic studies of fibrin gels.     

Ligation of fibrinogen by factor XIIIa with dithiothreitol: mechanical properties of ligated fibrinogen gels

Human fibrinogen (concentration 8.4 mg/mL) was ligated (cross-linked) with factor XIIIa and dithiothreitol (DTT) at pH 8.5, ionic strength 0.45. With 7.5 μg/mL of factor XIIIa alone, there was almost no γ-γ ligation, but with 2 mM DTT added, oligomers appeared, and γ-γ and Aα-Aα ligation was nearly complete after 3 days. At 38 μg/mL of factor XIIIa, some γ-γ and Aα-Aα ligation occurred even without DTT. For fibrinogen concentrations of 4.0 and 8.4 mg/mL, 38 μ/mL factor XIIIa, 2.0 mM DTT, clot-like gels formed and the shear modulus of elasticity increased slowly over several days to a constant value. The final modulus was similar in magnitude to those of ligated clots of α-fibrin (clotted by thrombin) and α-fibrin (clotted by batroxobin) under the same conditions. However, the opacity was somewhat higher; whereas in fine fibrin clots there is minimal lateral association of the protofibrils, in fibrinogen gels at the same pH and ionic strength the protofibrils (which are presumably single chains of fibrinogen monomers joined end to end at their D domains) are evidently associated in bundles (although not to the degree seen in coarse fibrin clots). Creep and creep recovery measurements showed almost perfect elastic behavior, with essentially no creep under stress and complete recovery after removal of stress. The modulus was scarcely affected by introduction of lithium bromide by diffusion to a concentration of 0.6M, which in unligated fibrin clots causes substantial softening. Whereas in fine fibrin clots (both αβ-fibrin and α-fibrin) factor XIIIa causes only γ-γ ligation, addition of 2 mM DTT produced some α-α ligation in these also.     

“Ordered” structure in solutions and gels of a globular protein as studied by small angle neutron scattering

Small-angle neutron scattering measurements were used for structural investigation of β-lactoglobulin solutions and heat-set gels in conditions of strong double layer repulsions. At pH 9 and low ionic strength, a correlation peak was observed on the scattering curves of the solutions whatever the protein concentration C used (in the range C = 0.02–0.10 g/mL). The wave vector value q_max corresponding to these maxima scaled as C^0.25. This exponent value is in agreement with those reported in the literature for other globular proteins in the same concentration range. Increasing the ionic strength decreased the peak which vanished without changing position at 0.1M NaCl. This polyelectrolyte-like behaviour suggests a local structure in the protein solution due to double layer repulsions. In the case of heat-set aggregates and gels (0.02–0.13 g/mL) formed at pH 9 and low ionic strength, a peak in the scattering curves was also observed indicating that even after gelation a correlation is still present; q_max varied as C^0.5. As in the case of the solutions, the correlation peak decreased with increasing ionic strength, and it vanished at 0.06M NaCl. The dilution of the aggregates in order to determine their intraparticle structure factor showed that the correlations were lost and that the aggregates displayed the same internal structure as the elementary subunit in the gels. At high ionic strength, fractal structures of the aggregates down to a length scale of about 40 Å were observed with d_f = 1.3–1.75 ± 0.05, increasing with protein concentration. Subsequent dilution didn't change the fractal dimension of these structures. © 1996 John Wiley & Sons, Inc.     

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.     

Elastic Behavior and Platelet Retraction in Low- and High-Density Fibrin?Gels

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.     

PFG-NMR diffusometry: a tool for investigating the structure and dynamics of noncommercial purified pig gastric mucin in a wide range of concentrations

For the first time, Pulsed Field Gradient-Nuclear Magnetic Resonance, a powerful noninvasive tool for studying the dynamics and structure of complex gels, has been used to measure diffusion of probe molecules in aqueous solutions/gels of noncommercial purified pig gastric mucin (PGM), in a concentration range up to 5 wt %. Complementary data were obtained from rheology measurements. The combination of techniques revealed a strong pH dependency of the structure of the PGM samples while changes in concentration, ionic strength, and temperature appeared to induce less pronounced alterations. Viscosity was found to vary in a nonmonotonous way with pH, with the more viscous solutions found at intermediate pH. We propose that this finding is due to a reduced charge density at lower pH, which is expected to continuously increase the relative importance of hydrophobic associations. The results suggest a loose network of expanded fully charged PGM molecules with considerable mobility at neutral pH (pH 7.4). At intermediate pH (pH 4), a three-dimensional expanded network is favored. At pH 1, the charge density is low and microphase separation occurs since hydrophobic associations prevail. This leads to the formation of clusters concentrated in PGM molecules separated by regions depleted in PGM. The results obtained increase our knowledge about the gastric mucosal layer, which in vivo contains mucin in the same concentration range as that of the samples investigated here.     

Darcy permeability of agarose-glycosaminoglycan gels analyzed using fiber-mixture and donnan models

Agarose-glycosaminoglycan (GAG) membranes were synthesized to provide a model system in which the factors controlling the Darcy (or hydraulic) permeability could be assessed in composite gels of biological relevance. The membranes contained a GAG (chondroitin sulfate) that was covalently bound to agarose via terminal amine groups, and the variables examined were GAG concentration and solution ionic strength. The addition of even small amounts of GAG (0.4 vol/vol %) resulted in a twofold reduction in the Darcy permeability of 3 vol/vol % agarose gels. Electrokinetic coupling, caused by the negative charge of the GAG, resulted in an additional twofold reduction in the open-circuit permeability when the ionic strength was decreased from 1 M to 0.01 M. A microstructural hydrodynamic model was developed, based on a mixture of neutral, coarse fibers (agarose fibrils), and fine, charged fibers (GAG chains). Heterogeneity within agarose gels was modeled by assuming that fiber-rich, spherical inclusions were distributed throughout a fiber-poor matrix. That model accurately predicted the Darcy permeability when the ionic strength was high enough to suppress the effects of charge, but underestimated the influence of ionic strength. A more macroscopic approach, based on Donnan equilibria, better captured the reductions in Darcy permeability caused by GAG charge.     

Polymerization and gelation of fibrinogen in D2O

The solution properties of fibrinogen and the thrombin-induced activation and gelation of fibrinogen in 95% D2O at pH 7.4 were compared to those in H2O under similar conditions. The initial release rates of fibrinopeptides A and B in D2O were slightly slower than those in H2O. However, the values of the Michaelis-Menten parameters Km and V for the release of the two peptides in D2O and H2O in the presence of 0.5 M NaCl were about the same. From turbidity measurements at 450 nm it is obvious that fibrinogen is soluble in a slightly more narrow range of NaCl concentration and that the fibrin gels have a higher degree of lateral aggregation in D2O than in H2O. The variation of fibrinogen concentration, thrombin concentration, pH and ionic a strength have a similar dependence on the final gel structure and clotting time in D2O and H2O. SDS-gel electrophoresis on fibrin samples, which were cross-linked by factor XIII, yielded results where the cross-linking of the gamma-chain appeared to be the same in D2O and H2O. The alpha-chain cross-linking was somewhat faster in D2O than in H2O. When fibrinogen solutions in 95% D2O were incubated at 20 mM CaCl2, a slow gelation of fibrinogen was observed, which was found to be induced by trace amounts of factor XIII. The final gel turbidity appeared to be about the same for this gelation as for that induced by thrombin. The differences in solubility for fibrinogen, kinetics for the enzyme reaction and optical properties for the fibrin gels in D2O and H2O may be explained by differences in electrostatic interactions, hydrogen bonding and hydration of fibrinogen in these two media.     

Influence of Ca2+ on the structure of reptilase-derived and thrombin-derived fibrin gels.

The effects of Ca2+ ion on the structure of thrombin-derived and reptilase-derived fibrin gels formed at various ionic strengths were studied turbidimetrically. For both enzymes clotting times were shorter, final gel turbidities were higher and fibre mass/length ratios were increased as the ionic strength was lowered. The addition of 5 mM-Ca2+ augmented each of these effects for any given ionic strength. In the thrombin system, Ca2+ increased the final gel turbidity from 0.04 to 0.26 A632.8 at ionic strength 0.15. Under identical conditions in the reptilase system, the final gel turbidity increased from 0.03 A632.8 in the absence of Ca2+ to 0.345 A632.8 in the presence of 5 mM-Ca2+. In the thrombin system, fibre mass/length ratios increased from 0.4 X 10(12) to 6.9 X 10(12) Da/cm in the absence of Ca2+, and from 4.4 X 10(12) to 7.9 X 10(12) Da/cm in the presence of Ca2+, as the ionic strengths were decreased from 0.15 to 0.08 and to 0.11 respectively. In the reptilase system, the mass/length ratios increased from 0.9 X 10(12) to 5.8 X 10(12) Da/cm in the absence of Ca2+, and from 4.8 X 10(12) to 8.7 X 10(12) Da/cm in the presence of Ca2+, as the ionic strengths were decreased from 0.15 to 0.08 and to 0.10 respectively. At ionic strengths below 0.10, the presence of 5 mM-Ca2+ caused precipitation and macroscopic aggregation of fibrinogen upon the addition of either enzyme. In the presence of 5 mM-Ca2+, the fibres composing thrombin-induced and reptilase-induced gels were virtually identical.     

DNA orientation in shear flow

The linear dichroism (LD) has been measured for DNA molecules 239–164,000 base pairs long oriented in shear flow over a large range of velocity gradients (30–3,000 s ^?1) and ionic strengths (2–250 mM). At very low gradients, the degree of DNA orientation increases quadratically with the applied shear as predicted by the Zimm theory [J. Zimm, (1956) Chemical Physics, Vol. 24, p. 269]. At higher gradients, the orientation of fragments ≥ 7 kilobase pairs (kbp) increases linearly with increasing shear, whereas the orientation of fragments ≥ 15 kbp shows a more complicated dependence. In general, the orientation decreases with increasing ionic strength throughout the studied ionic strength interval, owing to a decrease in the persistence length of the DNA. The effect is most dramatic at ionic strengths below 10 mM, and is more pronounced for longer DNA fragments. For fragments ≥ 15 kbp and velocity gradients ≥ 100 s^?1, the orientation can be adequately described by the empirical relation: LD^r= –(k₁-G)/(k₂ + G), where k₁is a linear function of the square root of the ionic strength and k₂ depends on the DNA contour length. Since the DNA persistence length can be represented as a linear function of the reciprocal square root of the ionic strength [D. Porschke, (1991) Biophysical Chemistry, Vol. 40, p. 169], extrapolation of the empirical relation provides information about the stiffness of the DNA fibers. © 1993 John Wiley & Sons, Inc.     

Concentration of protein in fibrin fibers and fibrinogen polymers determined by refractive index matching

We have used refractive index matching to determine the concentration of protein in the fibers in fibrin clots and of needlelike crystals of native fibrinogen. Our results are in agreement with those of Carr and Hermans [(1978) Macromolecules 11 , 46–50], as determined by light scattering—namely, that protein makes up about 20% of the volume of the fiber. However, we have found that the protein concentration is strongly dependent on ionic strength. An increase in ionic strength caused a substantial drop in the protein concentration. In a buffer containing 100 mM NaCl, the protein concentration was 26.6–29.8 g of protein per 100 cm³ of polymer, and at 200 mM NaCl it was reduced to 22.1–23.1 g/100 cm³.     

Albumin indirectly modulates fibrin and protofibrin ultrastructure

Albumin modulation of fibrin and protofibrin coagulation parameters was studied. Cation-depleted, fatty acid free, human and bovine albumins decrease fibrin clot turbidity in a concentration-dependent manner. Albumin also inhibits the formation of protofibrin gels, induced by addition of 25 microM Zn(II) to protofibrils, though it does not bind to (proto)fibrin. In order to verify that competition for cations underlies the influence of albumin, fibrinogen was dialyzed against cation-depleted albumin. Elemental analysis indicates a redistribution of Zn(II) from the fibrinogen to the albumin compartment, and the resultant fibrin clots are less turbid. Apparently, cation-depleted albumin acts as a competitor for divalent cations. The ability of albumin to compete for available Zn(II) was also expressed in gels formed by pH-jump experiments, in which fibrin monomer, maintained soluble at pH 4.9, is induced to change phase by addition of NaOH to pH 7.4. While turbidimetric evidence indicates that individual fibrin fibers simply become thinner with albumin, scanning electron micrographs (SEM) reveal a more complex effect on ultrastructure. Though albumin does not bind to the gels, fibrin gels produced with albumin show major changes in fiber ultrastructure, particularly evident in gels formed in the presence of Zn(II). These structural modifications are discussed within the context of the "excluded volume" effect, in which "crowding" by albumin alters (proto)fibrin reactivity and ultrastructure.     

Cl- regulates the structure of the fibrin clot.

The differences between coarse and fine fibrin clots first reported by Ferry have been interpreted in terms of nonspecific ionic strength effects for nearly 50 years and have fostered the notion that fibrin polymerization is largely controlled by electrostatic forces. Here we report spectroscopic and electron microscopy studies carried out in the presence of different salts that demonstrate that this long-held interpretation needs to be modified. In fact, the differences are due entirely to the specific binding of Cl- to fibrin fibers and not to generic ionic strength or electrostatic effects. Binding of Cl- opposes the lateral aggregation of protofibrils and results in thinner fibers that are also more curved than those grown in the presence of inert anions such as F-. The effect of Cl- is pH dependent and increases at pH > 8.0, whereas fibers grown in the presence of F- remain thick over the entire pH range from 6.5 to 9.0. From the pH dependence of the Cl- effect it is suggested that the anion exerts its role by increasing the pKa of a basic group ionizing around pH 9.2. The important role of Cl- in structuring the fibrin clot also clarifies the role played by the release of fibrinopeptide B, which leads to slightly thicker fibers in the presence of Cl- but actually reduces the size of the fibers in the presence of F-. This effect becomes more evident at high, close to physiological concentrations of fibrinogen. We conclude that Cl- is a basic physiological modulator of fibrin polymerization and acts to prevent the growth of thicker, stiffer, and straighter fibers by increasing the pKa of a basic group. This discovery opens new possibilities for the design of molecules that can specifically modify the clot structure by targeting the structural domains responsible for Cl- binding to fibrin.     

Influence of the ionic strength on the heat-induced aggregation of the globular protein beta-lactoglobulin at pH 7

The influence of the ionic strength on the structure of beta-lactoglobulin aggregates formed after heating at pH 7 has been studied using static and dynamic light scattering. The native protein depletion has been monitored using size exclusion chromatography. Above a critical association concentration (CAC) well-defined clusters are formed containing about 100 monomers. The CAC increases with decreasing ionic strength. The so-called primary aggregates associate to form self similar semi-flexible aggregates with a large scale structure that is only weakly dependent on the ionic strength. The local density of the aggregates increases with increasing ionic strength. At a critical gel concentration, Cg, the size of the aggregates diverges. Cg decreases from 100 g/l without added salt to 1 g/l at 0.4M NaCl. For C > Cg the system gels except at high ionic strength close to Cg where the gels collapse under gravity and a precipitate is formed.     

The effect of fibrin structure on fibrinolysis.

Fibrin structure contributes to the regulation of the fibrinolytic rate. As the fibrin fiber size is decreased, the fibrinolytic rate also decreases. Fibrin structure was altered by either changing the ratio of thrombin to fibrinogen, i.e. altering the assembly rate or by adding a fibrin assembly inhibitor, iopamidol. Changes in the fibrinolytic rate were followed by measuring the time dependence of the decrease in the fiber mass/length ratio during fibrinolysis. A measure of the overall fibrinolytic rate was determined from the decrease in the mass/length ratio versus time. An 8-fold reduction in the fibrinolytic rate was seen on decreasing the mass/length ratio from 2.7 x 10(12) daltons/cm to 0.5 x 10(12) daltons/cm. It is shown that thin fibrin fibers have a decreased rate of conversion of plasminogen to plasmin by tissue plasminogen activator and that thin fibrin fibers are lysed more slowly than thick fibrin fibers.     

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.     

Effect of homo poly(L-amino acids) on fibrin assembly: role of charge and molecular weight

Positively charged molecules such as protamine, leukocyte cationic protein, and the carboxyl terminus of platelet factor 4 have been shown to increase fibrin fiber thickness. Synthetic homo poly(L-amino acids) were used to explore the role of charge and molecular weight of cationic molecules on fibrin assembly. The effects of poly(L-lysine) (PLL), poly(L-glutamic acid) (PLG), poly(L-aspartic acid) (PLA), poly(L-histidine) (PLH), and poly(L-arginine) (PLArg) on the assembly and structure of fibrin gels were studied by using light-scattering techniques. At a PLG (Mr 60,000) concentration of 80 micrograms/mL and a PLA (Mr 20,000) concentration of 64 microgram/mL, neither of these negatively charged polymers produced a detectable change in either fibrin assembly kinetics or final structure. Positively charged PLArg (16 micrograms/mL) caused a 30% increase in fibrin fiber mass/length ratio without calcium. In contrast, PLH (16 micrograms/mL), also positively charged, had no effect in the absence of CaCl2 but produced a 40% increase in fiber mass/length ratio with 5 mM CaCl2. At concentrations as low as 1 microgram/mL, positively charged PLL increased the initial fibrin assembly kinetics and led to larger fiber mass/length ratio. The impact on fibrin mass/length ratio was equivalent for three different molecular weight preparations of PLL (Mr 25,000, 90,000, and 240,000). The lack of a molecular weight effect on fiber thickness and the low polymer concentrations required to produce the perturbation argue against an excluded volume effect as the mechanism by which lateral fiber growth is augmented. Mechanisms by which poly(L-amino acids) may perturb fibrin assembly are discussed.     

Protofibrin clots induced by calcium and zinc

During the course of studies with fibrin protofibrils, produced by adding hirudin to thrombin-activated fibrinogen prior to the onset of gelation, turbid clots were observed to be generated merely by adding Ca(II) or Zn(II) to protofibrils. The rate of gelation (CT) and turbidity of the “protofibrin” clots increases with cation levels in a concentration-dependent manner, with Zn(II) much more potent than Ca(II). For example, 50 μM Zn(II) generated a more turbid protofibrin clot than 0.5 mM Ca(II). In combination, levels of Zn(II) and Ca(II), which individually have no effect, induce protofibril gelation. The generation of protofibrin clots by Zn(II) is decreased at increasing ionic strength. Apparently, the underlying electrostatic forces that bind the monomers in fibrin and protofibrin gels are similar. SEM micrographs show that Ca(II)- or Zn(II)-induced protofibrin clots (600–1500Å thick) are essentially indistinguishable from those formed directly from fibrinogen and thrombin with divalent cation. The protofibrin fibers induced by the cations are thicker than the fibers formed directly from fibrinogen and thrombin in the absence of divalent cation. Branching appears brought about the the divalent cation-sensitive lateral association of different protofibril strands. These findings describe simple experimental methods for separately studying the early and late stages of fibrin gelation.     

Assembly of fibrin. A light scattering study.

Using stopped flow light scattering, we show that assembly of fibrin following activation with non-rate-limiting amounts of thrombin or reptilase occurs in two steps, of which the first is end-to-end polymerization of fibrin monomers to protofibrils and the second is lateral association of protofibrils to fibers, in agreement with Ferry's original proposal. Polymerization is found to proceed as a bimolecular association of bifunctional monomers; the overall rate varies as the inverse first power of the concentration; end-to-end association of two monomers, of a monomer and an oligomer, and of two oligomers occurs with the same rate constant. The value of the rate constant is 8.2 C 10(5) M-1 s-1 in 0.5 M NaCl, is three times larger in 0.1 M NaCl (0.05 M Tris, pH 7.4), and is the same following activation by reptilase and by thrombin. The onset of growth of fibers from protofibrils takes 12 times longer in 0.5 than in 0.1 M salt, i.e. thick fibers ("coarse" gels) form from short protofibrils, and thin fibers ("fine" gels) form from longer protofibrils. Jumps of salt concentration at times when protofibrils, but not fibers, have formed result in immediate growth of thick fibers at low salt from long protofibrils formed at high salt. The rate of fiber growth in these experiments varies as the inverse first power of the concentration. 3the instant of gelation (formation of a network of fibers) falls in the later half of the time during which the scattering rises due to fiber growth; the rise of gel rigidity after gelation is found to continue beyond the end of this period. Jumps from low to high salt result in retention of whatever fibers have formed at low salt and a very small additional increase of the scattering due to further fiber growth at high salt. From a variety of evidence, we conclude that the properties of fibrin are determined by kinetics and not equilibria of assembly steps. Results obtained here agree with the following scheme of fibrin assembly: monomers polymerize to protofibrils; long protofibrils associate laterally to fibers; occasionally a long protofibril associates with two different fibers to form an interfiber connection; fiber growth does not reverse to yield stabler, more compact, structures and terminates in formation of a network of fibers. The typical delay of fiber growth is the time during which protofibrils form from monomers. Measurements at rate-limiting concentrations of thrombin have allowed estimation of turnover rates of fibrinopeptides that agree with kinetic parameters obtained with direct assay of fibrinopeptide. Release of fibrinopeptide B causes more rapid fiber formation. Addition of thrombin after activation by reptilase, at a time when protofibrils, but not fibers, have formed, is followed rapidly by fiber formation; this proves that thrombin readily removes fibrinopeptide B from protofibrils. On the basis of these new results and earlier work (in particular, Blomb?ck, B., Hessel, B., Hogg, D., and Therkildsen, L...     

Structure of the histone tetramer (H3-H4)2: 1. Peculiarities of optical absorption,fluorescence and light scattering in response to changes in ionic strength and pH of the medium

Using the methods of spectrophotometry, spectrofluorimetry, light scattering and gel filtration, it was shown that, at pH 5.6 and 7.4 and various ionic strengths, the histone tetramer (H3-H4)₂ may have several structural states with different packing of the polypeptide chains of histones H3 and H4. Two structural changes of the tetramer (H3-H4)₂ at pH 7.4 in the ranges 0.1–0.3 m and 0.7–0.9 m NaCl were observed. In the high ionic strength solution, the tetramer (H3-H4)₂ had a more compact structure at pH 7.4 than at pH 5.6. At pH 3.0 destruction of the histone tetramer (H3-H4)₂ and formation of non-specific aggregates took place.