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.     

Rheology of fibrin clots: III. Shear creep and creep recovery of fine ligated and coarse unligated clots

Creep and creep recovery of human fibrin clots in small shearing deformations have been investigated over a time scale from 24 to 10⁴ s. Coarse, unligated dots and fine dots ligated by fibrinoligase in the presence of calcium ions were studied to suppllement previous data on coarse ligated and fine unligated clots. Stress was found to be proportional to strain up to at least a maximum shear strain (in torsion geometry) of 2.6%. The initial modulus (25 s after imposition of stress) is proportional to approximately the 1.5 power of concentration for fine ligated and coarse unligated clots. For fine unligated clots, there is comparatively little creep subsequent to the initial deformation; ligation (in this case involving mostly the γ chains) reduces the creep to nearly zero. For coarse unligated dots, there is substantially more creep under constant stress, and creep recovery is not complete. legation (in this casa involving both γ and α chains) largely suppresses the creep and causes the recovery to be complete. If the structure is fully formed before creep begins, tests of creep recovery by the Boltzmann superposition principle show adherence to linear viscoelastic behavior for all four clot types. Otherwise, the Boltzmann test fails and the recovery is much less than calculated. For fine ligated clots, the observed recovery agrees well with that calculated on the basis of a dual structure model in which an additional independent structure is built up in the deformed state, so that the state of ease after removal of stress is a balance between two structures deformed in opposite senses, it is postulated that the coherence and elastic modulus of the fine ligated dot are largely due to steric blocking of long protofibrils with a high flexural stiffness. In the coarse clot, it is proposed that the structure involves extensive branching of thick bundles of protofibrils, which become permanently secured by the ligation of the α chains of the fibrin.     

Small-angle X-ray scattering studies of fibrin film: comparisons of fine and coarse films prepared with thrombin and ancrod

Measurements of small-angle x-ray scattering have been made on films prepared from fine and coarse (i.e., formed at high and low, respectively, pH and ionic strength) clots of bovine fibrin by osmotic shrinkage or compression in one dimension. Intensity profiles were obtained with pinhole geometry on films stretched up to a stretch ratio of 1.43. In unstretched coarse films, repeat spacings were seen at about 245, 120, and 77-80 A. These peaks can probably be identified with the first, second, and third orders of the well-known fibrin repeat of 225 A. In unstretched fine films, only the 77-80 A spacing was seen. In this case, the first two orders may be weak because the half-staggered arrangement of monomer units giving rise to the 225 A reflection is not reinforced by lateral aggregation of protofibrils; the third order may be strong since the molecular subdomains appear to divide the repeat roughly into thirds. After stretching, the 77-80 A spacing persisted in the meridional direction but almost disappeared in the equatorial. Experiments on unstretched films prepared with ancrod substituted for thrombin gave similar results.     

Stress relaxation in fine fibrin films: Comparison of films prepared with thrombin and ancrod

Measurements of stress relaxation in uniaxial extension have been made on fibrin film prepared from fine bovine fibrin clots (i.e., clots in which there is minimal lateral aggregation of protofibrils), both ligated and unligated, and polymerized with both thrombin and ancrod, plasticized with either aqueous buffer or glycerol. The stress 100 s after imposition of strain was approximately proportional to In λ, where λ is the stretch ratio. Ligated thrombin films showed comparatively little relaxation over a period of one day and almost complete recovery after release of stress. In unligated thrombin films, there was substantial relaxation in two stages, as previously observed for coarse films, and substantial irrecoverable deformation. The extent of relaxation and the proportion of strain that was irrecoverable increased with the magnitude of the strain. In ancrod films (unligated), there was much more relaxation (stress decaying by as much as a factor of 10) and much more irrecoverable deformation (about 70% of the initial deformation); these results did not depend on the magnitude of the strain. When an ancrod film was released after relaxation and submitted to a second stretch, the extent of the second relaxation was much less. These observations are discussed in relation to the structure of fine films and possible mechanisms for relaxation and irrecoverable deformation.     

Rheological studies of creep and creep recovery of unligated fibrin clots: comparison of clots prepared with thrombin and ancrod

Mechanical creep and creep recovery in small shearing deformations have been studied in unligated clots formed with both thrombin and ancrod. In thrombin clots, both A binding sites (which interact with “a” sites to link monomer units within a protofibril) and B sites (which interact with “b” sites to form links between protofibrils) are exposed to enable formation of linkages; in ancrod clots, only the A sites are exposed. Fine clots (with minimal lateral aggregation of protofibrils), coarse clots (with substantial aggregation of fibril bundles), and clots of intermediate coarseness were compared. Fine thrombin clots showed less creep at short times but more creep at long times than coarse or intermediate clots and had more irrecoverable deformation relative to the initial elastic deformation. Ancrod clots had greater irrecoverable deformation than the corresponding thrombin clots, both fine and coarse. The permanent deformation in fine ancrod clots was enormous, corresponding almost to fluid character; the rate of permanent deformation was larger than that in fine thrombin clots by more than two orders of magnitude. For all types of clots, differential measurements of compliance (or its reciprocal, elastic modulus), as well as the applicability of the Boltzmann superposition principle to calculation of creep recovery, showed that the overall density of structure remained constant throughout the mechanical history; i.e., if structural elements were breaking, they were reforming at the same rate in different configurations. The possibility that the weakness of ancrod clots is attributable to partial degradation of α-chains rather than absence of Bb linkages was eliminated by comparisons of clots made with thrombin, ancrod, and ancrod plus thrombin; the last two showed identical partial degradation of α-chains (by gel electrophoresis), but the first and third had essentially identical initial elastic moduli and creep behavior. Two alternative mechanisms for irrecoverable deformation in fine clots are discussed, involving rupture of protofibrils and slippage of twisted segments, respectively.     

Studies of fibrin film. I. Stress relaxation and birefringence

Measurements of stress relaxation in uniaxial extension and associated time-dependent birefringence have been made on bovine fibrin film, prepared by gentle compaction of coarse fibrin clots, containing 13–22% fibrin plasticized with either aqueous buffer or glycerol. Both unligated and ligated (i.e., with α-α and γ-γ ligation by fibrinoligase, factor XIIIa) films were studied. Both types showed two stages of stress relaxation, with time scales of approximately 10 and 10³–10⁴ s, respectively, with a plateau region between. In the plateau, the nominal (engineering) stress for ligated glycerol-plasticized film is proportional to In λ, where λ is the stretch ratio, up to λ ? 2, and it decreases with increasing temperature. For unligated glycerol-plasticized film, the stresses are smaller by a factor of one-half to one-third. For ligated film, the second stage of relaxation is relatively slight, and recovery after release of stress is often nearly complete. For unligated film, the second stage involves a substantial drop in stress, and after recovery there is a significant permanent set. A second relaxation for ligated film reproduces the first, but for unligated film it reproduces the first only if the initial relaxation is terminated before the second stage; otherwise, the second relaxation shows a weaker structure. The behavior of water-plasticized film is similar to that of glycerol-plasticized except that the second stage of relaxation occurs at shorter times. During the first stage of stress relaxation, up to about 100 s, the birefringence and the stress-optical coefficient increase; during the plateau zone of stress relaxation, the birefringence of ligated films is approximately constant and is proportional to 2λ²/(λ² + 1) ? 1, where λ is the stretch ratio. This dependence is predicted by a two-dimensional model in which rodlike elements in the plane of the film are oriented with independent alignment. During the final stage of stress relaxation, the birefringence of ligated films decreases slightly; that of unligated films decreases substantially, but less rapidly than the stress, corresponding to a further increase in the stress-optical coefficient. With additional information from small-angle x-ray scattering reported in an accompanying paper, the first stage of relaxation is attributed to partial release of bending forces in the fibers by orientation, accompanied by increased birefringence. The second stage is attributed, for ligated films, to an internal transition in the fibrin units accompanied by elongation of some of the fibers; and in the unligated films, to a combination of the latter transition with slippage of protofibrils lengthwise within the fiber bundles that causes some loss of orientation, which diminishes the birefringence.     

Identification of fibrin oligomers in sonicated fibrin clots

Clots of bovine fibrin, with both coarse and fine structure, and ligated to different extents by fibrinoligase, have been broken up by ultrasonic agitation and the sonicates have been examined by ultracentrifugal sedimentation. Sonication is followed by gross aggregation of the fragments unless guanidine hydrochloride is introduced (order of 1 M). In that case, sonicates of gamma-ligated fine clots contain two species whose sedimentation coefficients correspond to fibrin monomer and an oligomer with twice the monomer cross-section area and at least 20 monomer units, presumably with the structure of lateral dimerization with staggered overlapping. If the gamma ligation is incomplete, shorter oligomers are identified. The monomer and oligomer with degree of polymerization greater than 20 appear also in sonicates of coarse clots, but in smaller amounts, the principal product consisting of larger aggregates. The implications of these results with respect to metastability of the fine clot and the pattern of polymerization are discussed.     

Effects of semi-dilute actin solutions on the mobility of fibrin protofibrils during clot formation

Low concentrations of actin filaments (F-actin) inhibit the rate and extent of turbidity developed during polymerization of purified fibrinogen by thrombin. Actin incorporates into the fibrin clot in a concentration-dependent manner that does not reach saturation, indicating nonspecific trapping of actin filaments in the fibrin network. Actin does not retard activation of fibrinogen by thrombin, but rather the alignment of fibrin protofibrils into bundles which constitute the coarse clot. In contrast, equivalent F-actin concentrations have little or no effect on the turbidity of plasma clots. The difference is attributed to the presence of a plasma protein, gelsolin, that severs actin filaments. Purified gelsolin greatly reduces the effect of F-actin on the turbidity of a pure fibrin clot and decreases the fraction of actin incorporated by the clot. A calculation of the extent to which the gelsolin concentrations used in these experiments reduce the fraction of actin filaments which are long enough to impede each other's rotational diffusion indicates that it is the overlapping actin filaments which retard the association of fibrin protofibrils. The findings suggest that one role for the F-actin depolymerizing and particularly actin severing activities in blood is to prevent actin filaments released by tissue injury from interfering with the formation of coarse fibrin clots.     

Modification of shear modulus and creep compliance of fibrin clots by fibronectin

Shear moduli and creep compliances have been measured for four types of clots of human fibrin (about 7 $\text{mg}\text{ml}$) clotted with and without human plasma fibronectin (usually 1.2 $\text{mg}\text{ml}$). Fine clots (with little lateral aggregation of the fibrin protofibrils) were formed at pH 8.5, ionic strength 0.45 ; coarse clots (with substantial lateral aggregation) were formed at pH 7.5, ionic strength 0.15; in both cases with and without ligation by fibrinougase. In fine clots, the addition of fibronectin without ligation scarcely affected the shear modulus; with ligation, the modulus was decreased by a factor of 0.48. In coarse clots, the shear modulus was increased by addition of fibronectin. The increase was by a factor of 2.0 without ligation and by a factor of 2.4 with ligation. Creep and creep recovery in clots formed with and without fibronectin were similar except for the scale factor represented by the change in modulus.     

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.     

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.     

Rheology of fibrin clots. I. Dynamic viscoelastic properties and fluid permeation

The storage and loss shear moduli (G', G″) of human fibrin clots have been measured in small oscillating deformations over a frequency range of 0.01 to 160 Hz with the modified Birnboim transducer apparatus. Most clots were prepared by the action of thrombin on purified fibrinogen, under various conditions of pH and ionic strength to produce networks ranging from coarse to fine structure; some were liaated by fibrinoligase. The fine, unligated clot showed very little mechanical loss or frequency dependence of G' over the experimental frequency range, though loss mechanisms evidently appear at higher frequencies; G' was proportional to the 1.5 power of fibrin concentration. The coarse, unligated clot showed a slight increase of G' with frequency, reflecting some relaxation mechanisms with time constants whose reciprocals lie in the experimental frequency range. Ligation did not greatly affect the magnitude of G'. However, clots prepared by dilution of solutions of fibrin monomer in 1 M sodium bromide had smaller moduli by a factor of ten than corresponding clots prepared by the action of thrombin of fibrinogen. Oscillatory measurements in the Birnboim apparatus with closed-end (annular pumping) geometry revealed a low-frequency anomaly which was shown to be due to permeation of fluid through the clot structure, and from these measurements the Darcy constants for coarse clots were calculated. From the Darcy constants, the average thicknesses of the fibrous elements of the structures were estimated to be from 300 to 700 A.     

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.     

Interaction of fibrinogen-binding tetrapeptides with fibrin oligomers and fine fibrin clots

The polymerization of fibrin, at pH 8.5 and ionic strength 0.45, and under conditions where the action of thrombin on fibrinogen was the rate-determining step, was interrupted by inactivating thrombin with p-nitrophenyl-p′-guanidinobenzoate (NPGB). Addition of the tetrapeptide Gly-Pro-Arg-Pro (GPRP) partially dissociated the fibrin oligomers as shown by subsequent ligation with Factor XIIIa and calcium ion followed by denaturation and gel electrophoresis; polyacrylamide gel electrophoresis with reduction showed a decrease in the proportion of γ-γ ligation compared with controls untreated by GPRP, and agarose gel electrophoresis showed a shift in the distribution of oligomer sizes. The dissociation was accomplished within 15 min and its extent was consistent with establishment of an equilibrium in which two molecules of GPRP react to sever an oligomer. When GPRP was introduced into fine unligated fibrin clots by diffusion, there was some dissociation as shown by differences in the degree of γ-γ ligation after treatment by Factor XIIIa; but the action of GPRP was much slower and less complete than on soluble oligomers. However, even a small amount of dissociation affected the mechanical properties of fine clots profoundly. The shear modulus (measured 25 s after application of stress) decreased progressively with increasing concentration of GPRP introduced by diffusion. The rate of shear creep under constant stress and the proportion of irrecoverable deformation also increased enormously. If the steadystate creep rate is interpreted in terms of an effective viscosity, the latter is decreased by up to three orders of magnitude by the presence of GPRP. In terms of transient network theories of viscoelasticity, the average lifetime of a network strand is greatly diminished. However, the total density of strands remains constant during creep and creep recovery as shown by constancy of the differential modulus or compliance. Removal of GPRP by diffusion only partially restores the original shear modulus and creep behavior of the original clot. Some limited data on the effect of the tetrapeptide Gly-His-Arg-Pro are also reported.     

Rheology of fibrin clots. IV. Darcy constants and fiber thickness.

Measurements of small oscillatory deformations of a fibrin clot by axial motion of a rod in a closed tube reveal an anomalous mechanical loss due to permeation of fluid through the clot structure. The Darcy constant for permeation can be calculated from data at the frequency where the apparent storage and loss shear moduli are equal, without the necessity of measurements at much lower frequencies as previously employed. From the Darcy constant, the average number of fibrin monomer units (v) per cross-section of a fibrous element of the clot can be calculated; it ranges from 4 to several hundred. In the range of fibrin concentration(c) from 3 to 14 milligrams, v is approximately proportional to c-2 for clots of coarse structure and to c-0.5 for clots of fine structure.     

Three-dimensional reconstruction of fibrin clot networks from stereoscopic intermediate voltage electron microscope images and analysis of branching.

Fibrin polymerizes to produce branching fibers forming a three-dimensional network, which has been difficult to visualize by conventional microscopy. Three-dimensional images of whole clots at high resolution were obtained from stereo-pair intermediate-voltage electron micrographs. Computer software was developed to produce three-dimensional reconstructions of the networks in the form of a pattern of links that connect branching junctions. Network parameters were measured and analyzed to characterize the clots quantitatively. Models in which all links were moved to the origin, while preserving their orientation, allowed visualization of some network parameters and facilitated comparison of networks. Fibrin clots formed in three different conditions were analyzed and compared by these methods. Clots formed in 0.20 M saline buffer consist of fibers of uniform size, and most of the branching junctions consist of three links. Fibrin clots formed in 0.05 M saline buffer are made up of very large diameter fiber bundles with far fewer branching junctions and correspondingly longer links. Clots formed in 0.40 M saline buffer consist of very fine fibers with numerous branching junctions and very short links. In summary, the extent of lateral aggregation is directly related to the distance between branching junctions and inversely related to the total number of branching junctions. These observations must be considered in defining possible mechanisms of fibrin branching.     

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...     

Rheology of fibrin clots. v. shear modulus,creep, and creep recovery of fine unligated clots

Creep and creep recovery in small shearing deformations have been studied in fibrin clots at pH 8.5 and ionic strength 0.45, where the fine, transparent clot is formed with very little lateral aggregation of protofibrils. The initial shear modulus G₁ was measured 25 s after deformation on clots aged long enough for complete development of structure. For both human and bovine fibrin, the data were approximately described by log G₁ = 1.45 + 1.90 log c, where c is concentration in $\text{g}\text{l}$ and G₁ is in $\text{dyn}\text{cm}{}^2$, over a range of c from 4 to 13 $\text{g}\text{l}$. For bovine clots with completely developed structure, creep and creep recovery showed substantial irrecoverable deformation but the differential modulus G_Δ measured at intervals agreed with G₁ and did not change during the course of the experiment; it also agreed with the value calculated from the initial recovery after removal of stress. Moreover, several tests showed that the course of recovery conformed closely to the Boltzmann superposition principle. Thus the irrecoverable strain was associated with a structural rearrangement which caused no permanent damage. The irrecoverable deformation relative to the initial deformation was proportional to the elapsed time during creep in the early stages with a proportionality constant that decreased somewhat with increasing clot age prior to imposition of stress; it corresponded to a pseudo-viscosity of the order of 10⁷ poise. However, the irrecoverable deformation does not represent viscous flow and appears to approach a limiting value at long times. Experiments on clots without completely developed structure, i.e., with imposition of stress at an earlier clot age, showed an increase in the differential modulus G_Δ during creep. The irrecoverable deformation was greater and a portion of it could be attributed to the balance between two structures formed in the unstrained and strained states. However, unlike the case of ligated clots strained before complete development of structure, where the irrecoverable deformation is entirely due to a two-structure balance, there is also a contribution from structural rearrangement. Experiments with reverse creep and creep recovery showed that the structural rearrangement is symmetrical with respect to direction of deformation. The interpretation of these results in terms of clot structure and internal motions of protofibrils is discussed.     

Deglycosylation of fibrinogen accelerates polymerization and increases lateral aggregation of fibrin fibers

Fibrinogen, the major structural precursor of blood clots, was deglycosylated by peptide-N-(N-acetyl-beta-glucosaminyl)asparagine amidase without denaturation of the polypeptide chains. Deglycosylated fibrinogen behaved normally in clinical coagulation assays, although it is less soluble than normal fibrinogen. However, the turbidity of clots formed from deglycosylated fibrinogen always rose faster and higher than that of clots from normal fibrinogen. Scanning and transmission electron microscopy demonstrated that fibrin made from clots of deglycosylated fibrinogen consisted of thicker, less-branched fiber bundles in a more porous network. Moreover, the degree of lateral aggregation was directly related to clot turbidity and inversely related to branching. Deglycosylation promoted turbidity development, lateral aggregation, and porosity of clots under all conditions tested. All other steps in the coagulation pathways appeared to be unaffected by the absence of carbohydrate. These results suggest that carbohydrate constitutively affects the behavior of deglycosylated fibrinogens by 1) contributing a repulsive force that promotes fibrinogen solubility and limits fibrin assembly and 2) sensitizing fibrin to conditions that influence assembly and clot structure.