Decorin modulates fibrin assembly and structure

Emerging evidence indicates that fibrin clotting is regulated by different external factors. We demonstrated recently that decorin, a regulator of collagen fibrillogenesis and transforming growth factor-beta activity, binds to the D regions of fibrinogen (Dugan, T.A., Yang, V. W.-C., McQuillan, D.J., and H??k, M. (2003) J. Biol. Chem. 278, 13655-13662). We now report that the decorin-fibrinogen interaction alters the assembly, structure, and clearance of fibrin fibers. Relative to fibrinogen, substoichiometric amounts of decorin core protein modulated clotting, whereas an excess of an active decorin peptide was necessary for similar activity. These concentration-dependent effects suggest that decorin bound to the D regions sterically modulates fibrin assembly. Scanning electron microscopy images of fibrin clotted in the presence of increasing concentrations of decorin core protein showed progressively decreasing fiber diameter. The sequestration of Zn(2+) ions from the N-terminal fibrinogen-binding region abrogated decorin incorporation into the fibrin network. Compared with linear thicker fibrin fibers, the curving thin fibers formed with decorin underwent accelerated tissue-type plasminogen activator-dependent fibrinolysis. Collectively, these data demonstrate that decorin can regulate fibrin organization and reveal a novel mechanism by which extracellular matrix components can participate in hemostasis, thrombosis, and wound repair.     

Gel formation by fibrin oligomers without addition of monomers

Soluble fibrin oligomers were formed by reacting fibrinogen with thrombin under fine clotting conditions where the action of thrombin is the rate-determining step for polymerization, and by inhibiting the reaction shortly before gelation. Oligomeric fibrin was separated from unreacted fibrinogen and small oligomers by gel permeation chromatography. Electron microscopy revealed that the largest soluble fibrin oligomers resemble the protofibrils present in fine clots, but are somewhat shorter and entirely lack the twisted, trifunctional junctions that contribute to the elastic properties of fine clots. When thrombin was added to the soluble fibrin oligomers, polymerization resumed and clots were formed at a more rapid rate than from fibrinogen at the same concentration and resulted in a less-opaque clot under coarse clotting conditions. The results confirm a prediction of a theory for the polymerization of fibrin and provide additional evidence that the final state of a coarse fibrin clot depends on the mobility of protofibrils during its formation.     

Effects of thrombospondin on fibrin polymerization and structure

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

The conversion of fibrinogen to fibrin. XIII. Dissolution of fibrin and inhibition of clotting by various neutral salts

1. Fibrin clots prepared in the absence of calcium can be dissolved in solutions of lithium chloride and bromide and sodium bromide and iodide, as well as of guanidine hydrochloride and urea. These salts do not denature fibrinogen under the same conditions of concentration, temperature, and time. Sedimentation experiments on the fibrin solutions show in each case a single sharp peak with a sedimentation constant close to that of fibrinogen. 2. At lower concentrations, these salts inhibit the clotting of fibrinogen by thrombin, but in the case of lithium bromide and sodium iodide, at least, allow an intermediate polymer to accumulate whose sedimentation constant is close to that of the polymer observed in systems inhibited by hexamethylene glycol or urea.     

Effect of fibronectin on fibrinogen conversion into fibrin

The effects of fibronectin on fibrinogen clotting induced by thrombin or reptilase and on fibrin monomer polymerization in a pure system in the absence of factor XIIIa were studied. It was shown that within a broad range of concentrations and molar ratios of the mixed proteins, fibronectin does not alter significantly the fibrinogen clotting time either under thrombin or under reptilase action. The effect of fibronectin on the fibrin self-assembly consists in a slight acceleration of this process, whose degree is directly dependent on the fibronectin/fibrin monomer molar ratio as well as on the absolute fibrin monomer content at a constant molar ratio. The stimulating effect of fibronectin is amplified by Ca2+. The experimental results suggest that fibronectin can noncovalently bind the fibrin monomer and/or intermediate polymers in the non-enzymatic phase of fibrinogen conversion to fibrin.     

Characterization of the fibrin polymer structure that accelerates thrombin cleavage of plasma factor XIII

The effect of plasmin-derived fibrin(ogen) degradation products on alpha-thrombin cleavage of plasma Factor XIII was studied to identify the fibrin polymer structure that promotes Factor XIIIa formation. Fibrin polymers derived from fibrinogen and Fragment X enhanced the rate of thrombin cleavage of plasma Factor XIII in plasma or buffered solutions. The concentrations of fibrinogen and Fragment X that promoted half-maximal rates of Factor XIIIa formation were 5 and 40 micrograms/ml, respectively. Fragments Y, D, E, D-dimer, and photooxidized fibrinogen did not enhance thrombin cleavage of Factor XIII. Although purified Fragment D1 inhibited fibrin gelation, the soluble protofibrils promoted thrombin activation of Factor XIII. Noncrosslinked fibrin fibers failed to enhance thrombin cleavage of Factor XIII. In conclusion, soluble fibrin oligomers function to promote thrombin cleavage of plasma Factor XIII during blood clotting.     

Fibronectin and fibrin gel structure

Plasma fibronectin is covalently incorporated into alpha-chains of fibrin gels in the presence of Factor XIII activated by thrombin (FXIIIaT) but not by Factor XIII activated by the snake venom enzyme batroxobin (FXIIIaB). FXIIIaB catalyzes introduction of gamma-gamma cross-links in fibrin but cross-linked alpha-chains are not formed. In the presence of FXIIIaT, fibrin gels formed by batroxobin incorporated fibronectin and the alpha-chains are cross-linked indicating that FXIIIaB has a different substrate specificity from FXIIIaT. In the presence of FXIIIaT the incorporation of fibronectin approaches 1 mol/340 kDa unit weight of fibrin. Fibronectin when present in a fibrinogen thrombin mixture containing FXIII does not influence the clotting time of the system nor the release of fibrinopeptides. Incorporation of fibronectin is not appreciable before the gel point. This indicates that the polymerization and gelation of fibrinogen is essentially not perturbed by the presence of fibronectin and that fibrin in the gel matrix rather than the fibrin polymers formed prior to gel point is the preferred structure for fibronectin incorporation. Incorporation of fibronectin into fibrin gels during formation leads to an increase in turbidity and a small decrease in Ks (permeability coefficient). This suggests that the width of the strands in the gel increases as a result of fibronectin incorporation. Fibronectin is also incorporated into preformed gels having completely cross-linked gamma- and alpha-chains perhaps indicating that the sites in fibrin involved in fibronectin incorporation are different from those involved in fibrin cross-linking. FXIIIaT appeared to be adsorbed to fibrin gel matrix in the presence but not in the absence of calcium ions.     

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.     

Structural origins of fibrin clot rheology

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

In vitro angiogenesis in fibrin matrices containing fibronectin or hyaluronic acid.

Angiogenesis involved numerous interactions between extracellular matrix and endothelial cells which may exhibit changes in actin filament distribution. Using an in vitro model, capillary endothelial cells were grown in fibrin matrix containing fibronectin or hyaluronic acid. Actin filament distribution, nucleus localization and cell morphology were observed. Preliminary study showed the formation of tube-, branche- and capillary-like structures within fibrin. In the presence of both fibrin and fibronectin, cells with actin filament stress fibers were more spreading than those in fibrin. In the presence of hyaluronic acid, tubes were limited in extension into the fibrin. In addition, the study of co-localization of nucleus and actin filaments showed different cell behaviours. Migratory cells seem to arrange in parallel to each other and a capillary-like structure may be formed at the proximal extremity of this cell pattern.     

Effect of methylmercuric chloride (MMC) on fibrin polymerization

Methylmercuric chloride (MMC) in concentrations 0.1–10μM reduces the amount of fibrinopeptides released from thrombinactivated human fibrinogen. However, the fibrin clot formation is not discriminated and the turbidity of the fibrin gel is even augmented. MMC does not cause such changes in the process of repolymerization of fibrin monomers. The addition of fibrinopeptides to the fibrin monomers results in a similar increase of turbidity of the repolymerizing sample in the presence of MMC as in the case of fibrinogen clotting. These experiments indicate that MMC modifies the structure of fibrin in the presence of fibrinopeptides.     

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...     

Lateral packing of protofibrils in fibrin fibers and fibrinogen polymers

The distinctive transverse banding pattern of fibrin fibers clearly indicates ordering of molecules in the longitudinal direction. In this study we examined the fibers of fibrin clots, as well as two types of fibrinogen polymers, by thin-section electron microscopy. The fibrinogen polymers have a transverse banding pattern identical to that of fibrin fibers—clearly indicating a regular longitudinal repeat—but they are larger in diameter, and show little or no branching. We therefore expected their overall ordering to be better than that of fibrin fibers. Several different fixation protocols were used. We readily observed the typical transverse banding seen previously by negative stain and metal replication techniques. However, only very rarely was any regular lateral lattice seen in any of the samples. X-ray diffraction was used to examine unfixed specimens of the two fibrinogen polymers and, once again, although a longitudinal repeat was evident, only rarely was evidence for lateral crystallinity seen. The electron-microscope and x-ray results showed that the needles and pellet fibers of fibrinogen have essentially the same internal architecture as thick fibrin fibers, and that all three types of polymer, although clearly transversely banded, have almost no crystallinity in their lateral protofibril packing.     

Coarse-grained molecular dynamics simulations of fibrin polymerization: effects of thrombin concentration on fibrin clot structure

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.     

Characterization of soluble polymerized fibrin formed in the presence of excess fibrinogen fragment D

Polymerization of fibrin is inhibited in the presence of excess fibrinogen fragment D. This study was performed in order to test the proposal that these inhibited solutions contain short linear polymers of fibrin (protofibrils) whose further polymerization is prevented as a result of attachment of a molecule of fragment D at each end. Negative-stain electron micrographs, intrinsic viscosities, angular dependence of light scattering intensity, and kinetics of the increase of the scattered intensity with polymerization all were found to support the above model of the inhibited polymer and to reflect the presence of a broad distribution of the lengths of the inhibited fibrin polymers. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of polymers stabilized with gamma-dimer cross-links introduced by factor XIIIa demonstrates cross-linking of fragment D to fibrin oligomers. Cross-linked polymers have been separated from excess fragment D by gel exclusion chromatography in 1 M urea. (In the absence of urea, the purified polymers very slowly associate to fibers.) The observation of the relative stability of short isolated inhibited protofibrils and the decrease or absence of inhibition of fibrin gelation when fragment D was added to solutions in which fibrin had been given time to polymerize to long protofibrils demonstrate that the inhibitory effect of fragment D occurs as a result of inhibition of the first fibrin polymerization step.     

Mechanism of fibrin coagulation based on selective, cation-driven, protofibril association

The cation sensitivity of linear and lateral assembly processes of thrombin- and reptilase-activated fibrinogen was examined. Analytic ultracentrifugation shows that the linear assembly of fibrin oligomers (protofibrils) is neither cation dependent nor sensitive to chelating agents. Protofibrils generated with thrombin–hirudin gelate with either 1–2 mM Ca(II) or 15–100 μM Zn(II). By contrast, protofibril B, generated with reptilase–diisopropylfluorophosphonate, gelates only with Ca(II) but is insensitive to Zn(II). These results indicate that the release of fibrinopeptides A and B (FPA and FPB) expose two types of lateral binding sites that are sensitive to Ca(II) and Zn(II) respectively. Transmission electron (TEM) micrographs of negatively stained gels indicate that the linear packing of the monomers within the fibrin- and cation-induced protofibrin fibers is essentially identical. Scanning electron (SEM) micrographs show that the Ca(II)-induced protofibrin B gel is similar to fibrin. In all, it seems that branching and gelation derive from two types of cation-sensitive, lateral associative processes. Based on these findings, a new paradigm for fibrin coagulation is proposed.     

Formation of fibrin gel in fibrinogen-thrombin system: static and dynamic light scattering study

The dynamics of thrombin-induced fibrin gel formation was investigated by means of static and dynamic light scattering. The decay time distribution function, obtained by the dynamic light scattering, clearly revealed a stepwise gelation process: the formation of fibrin and protofibril from fibrinogen followed by the lateral aggregation of protofibrils to form fibrin fibers and the formation of a three-dimensional network consisting of fibers. This conversion process was correlated with the angular dependence of the scattered light intensity (static light scattering). The correlation function of dynamic light scattering was analyzed in terms of sol-gel transition and gel structure. The correlation function showed a stretched exponential type behavior before the sol to gel transition point, and it showed a power law behavior at the gelation point.     

Mechanisms involved in fibrin formation

That the role of thrombin in the conversion of fibrinogen to fibrin is essentially enzymatic, is established not only by the minute amounts of thrombin which are effective but also by the complete independence of fibrin yields and thrombin concentrations over a very wide range of thrombin dilutions and clotting times. The thrombin-fibrinogen reaction, in the phase beyond the "latent period" at least, seems fundamentally "first order." Technical requirements of the experiments leading to these conclusions include: (1) a highly purified (e.g. 97 per cent "clottable") fibrinogen, (2) absence of traces of thrombic impurities in the fibrinogen, (3) absence of fibrinolytic protease contaminant of the thrombin and the fibrinogen, and (4) sufficient stability of the thrombin even at very high dilutions. Four conditions affecting thrombin stability have been investigated. Fibrin yields are not significantly modified by numerous experimental circumstances that influence the clotting time, such as (1) temperature, (2) pH, (3) non-specific salt action due to electrical (ionic) charges, which alter the Coulomb forces involved in the fibrillar aggregation, (4) specific ion effects, whether clot-accelerating (e.g. Ca⁺⁺) or clot-inhibitory (e.g. Fe(CN)₆'), (5) occluding (adsorptive) colloids, which have a "fibrinoplastic" action, e.g. (a) acacia and probably (b) fibrinogen which has been mildly "denatured" by salt-heating, acidification, etc. The data with which several European workers have attempted to substantiate the idea of a two-stage thrombin-fibrinogen reaction with an intermediary "profibrin" (allegedly partly "denatured") have been reanalyzed with controls which lead us to very different conclusions, viz. (1) denaturation and fibrin formation are independent; (2) partial denaturation is "fibrinoplastic" (see above); and (3) conditions of strong salinity and acid pH (5.1) usually do not completely prevent the thrombin-fibrinogen reaction but merely prolong the "latent" phase and lessen the time required for completion of essentially the same reaction (fibrin polymerization) when more favorable clotting conditions are restored. Thus, our experiments advance the modern concepts concerning the coagulation mechanisms along lines that, for the most part, agree with those of the Harvard physical chemists, and we oppose the European views concerning a two-stage reaction, "profibrin," and "the denaturase theory" of clotting.     

Peptide-derivatized albumins that inhibit fibrin polymerization

Synthetic peptides patterned on sequences that appear during thrombin proteolysis of fibrinogen are known to influence fibrin formation in very different ways. A-Knob sequences (GPR-) inhibit polymerization, but B-knob sequences (GHR-) can actually enhance the process. We now report that when such peptides are attached to albumin carriers, both knob conjugates inhibit fibrin formation. In contrast, the 2-aminoethylthiol-albumin conjugate control enhances the polymerization to the same degree as albumin. The peptide AHRPam, which is known to bind exclusively to the βC holes of fibrinogen/fibrin, nullifies the inhibitory effects of the GHRPYGGGCam-albumin conjugate on fibrin polymerization, indicating that the inhibition was exclusively due to interactions with βC holes. AHRPam was much less effective in countering inhibition by the GPRPGGGGCam-albumin conjugate, suggesting that the observed effects with this conjugate involve mainly the γC holes of fibrin/fibrinogen. This study demonstrates that peptides modeled on fibrin polymerization knobs tethered to albumin retain their capacity to interact with fibrinogen/fibrin and may prove useful as inhibitors of clotting in vivo.     

Dynamic changes of fibrin architecture during fibrin formation and intrinsic fibrinolysis of fibrin-rich clots

Clotting and fibrinolysis are initiated simultaneously in vivo, and fibrinolysis usually occurs without any individualized lysis front (intrinsic fibrinolysis). We have developed a novel model to assess whether morphological changes resulting from intrinsic fibrinolysis are similar to those previously reported at the lysis front using externally applied lytic agents. Fibrin assembly and fibrinolysis were followed in real-time by confocal microscopy using gold-labeled fibrinogen molecules. An increase in fiber absorbance (30%, p < 0.01) and a decrease in fiber diameter (60%, p < 0.01) due to the ongoing accumulation and packing of fibrin molecules were the most significant detectable features occurring during fibrin assembly. Similar features with a similar magnitude were observed during fibrin dissolution, but in the reverse order and with a 3-fold increase in duration. Then, lysing fibers were progressively transected laterally, and thinner fibers were cleaved at a 2.5-fold faster rate than thicker fibers (p < 0.001). Frayed lysing fibers were seen to interact progressively with adjoining fibers (agglomeration), leading to a 76 and 88% increase in the network pore diameter (p < 0.05) and fiber diameter (p < 0.01), respectively. At the maximum decrease in fiber absorbance (46%, p < 0.05), the network suddenly collapsed with the release of large fragments that gradually vanished. Morphological changes of fibrin that occur during intrinsic fibrinolysis are similar as those observed next to the lysis front, although they are not restricted spatially to the clot/surrounding milieu interface but are observed through the entire clot.