Chromatography and electron microscopy of cross-linked fibrin polymers--a new model describing the cross-linking at the DD-trans contact of the fibrin molecules

Polymerization of fibrin molecules results in the formation of a double-stranded protofibril. Although convincing data have not been presented, it is classically believed that γ-chain cross-linking of fibrin molecules occurs between the longitudinal end-to-end contacts (DD-long contacts) of the molecules within each of the two strands of a protofibril (intrastrand cross-linking). In this investigation the question addressed was whether γ-chain cross-linking takes place across the two strands (interstrand cross-linking) between the transversal half-staggered contacts of the molecules. Demonstration of double-stranded protofibrils in the presence of urea would indicate an interstrand cross-linking, whereas in the case of intrastrand cross-linking, the chaotropic agent urea would dissociate the double-stranded structure to form single-stranded fibrils. Protofibrils were obtained by generating soluble cross-linked fibrin polymers (sXLFbP): After incubation of souble fibrin polymers with Factor XIIIa at 37°C, the polymerization and cross-linking reaction was stopped by the addition of 6M urea and EDTA. Gel filtration of the reaction mixture in the presence of 3M urea was effect in separating sXLFbP from monomeric molecules. The sXLFbP-containing fractions were adsorbed onto mica in the presence of different concentrations of urea and investigated by electron microscopy after rotary shadowing. In the presence of 3M urea the sXLFbP appeared as double-stranded protofibrils. In the presence of 4M urea some parts of the double-stranded structure were found to be unfolded whereas in the presence of 6M urea multiple-bended single-stranded fibrils were observed. SDS-polyacrylamide gel electrophoresis of the sXLFbP demonstrated no γ-chain cross-linking within the protofibrils. Ultracentrifugation of the sXLFbP showed that in the presence of 3M urea noncross-linked fibrin polymers dissociated to monomeric molecules. When sXLFbP was centrifuged into 6M urea on sucrose density gradients, no reduction of the polymer size could be observed. The data indicate that γ-chain cross-linking occurs between the transversal contacts of the fibrin molecules within a protofibril, thus generating interstrand cross-linking. A model of the cross-linking of polymerized fibrin molecules is developed and the term DD-trans contact is proposed for this specific alignment of the D-domains.     

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.     

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.     

Structure, stability, and interaction of fibrin αC-domain polymers

Our previous studies revealed that in fibrinogen the αC-domains are not reactive with their ligands, suggesting that their binding sites are cryptic and become exposed upon its conversion to fibrin, in which these domains form αC polymers. On the basis of this finding, we hypothesized that polymerization of the αC-domains in fibrin results in the exposure of their binding sites and that these domains adopt the physiologically active conformation only in αC-domain polymers. To test this hypothesis, we prepared a recombinant αC region (residues Aα221-610) including the αC-domain (Aα392-610), demonstrated that it forms soluble oligomers in a concentration-dependent and reversible manner, and stabilized such oligomers by covalently cross-linking them with factor XIIIa. Cross-linked Aα221-610 oligomers were stable in solution and appeared as ordered linear, branching filaments when analyzed by electron microscopy. Spectral studies revealed that the αC-domains in such oligomers were folded into compact structures of high thermal stability with a significant amount of β-sheets. These findings indicate that cross-linked Aα221-610 oligomers are highly ordered and mimic the structure of fibrin αC polymers. The oligomers also exhibited functional properties of polymeric fibrin because, in contrast to the monomeric αC-domain, they bound tPA and plasminogen and stimulated activation of the latter by the former. Altogether, the results obtained with cross-linked Aα221-610 oligomers clarify the structure of the αC-domains in fibrin αC polymers and confirm our hypothesis that their binding sites are exposed upon polymerization. Such oligomers represent a stable, soluble model of fibrin αC polymers that can be used for further structure-function studies of fibrin αC-domains.     

Assembly of lamins in vitro

After lamins A,B and C were isolated and purified from rat liver,their assembly properties were examined by electron microscopy and scanning tunneling microscopy by electron microscopy and scanning tunneling microscopy using negative staining and the glycerol coating method,respectively.By varying the assembly time or the ionic conditions under which polymerization takes place,we have observed different stages of lamin assembly,which may provide clues on the structure of the 10 nm lamin filaments.At the first level of structural organization,two lamin polypeptides associate laterally into dimers with the two domains being parallel and in register.At the second level of structural organization,two dimers associate in a half-staggered and antiparallel fashion to form a tetramer 75 nm in length.At the third level of structural organization,4-10 lamin tetramers associate laterally in register to form 75 nm long 10nm filaments,which in turn combine head to head into long,fully assembled lamin filaments.The assembled lamin filaments are nonpolar.     

The incipient stage in thrombin-induced fibrin polymerization detected by FCS at the single molecule level.

We used fluorescence correlation spectroscopy (FCS) to study the activation of fibrinogen by thrombin and the subsequent aggregation of fibrin monomers into fibrin polymers at a very low and at physiological fibrinogen concentrations. In the labeling procedure used the fibrinogen was randomly labeled and the label was bound to the fibrinopeptide A and/or to the part of fibrinogen which after activation takes part in fibrin formation. We measured a diffusion coefficient for fibrinogen of 2.48 x 10(-7) +/- 0.10 x 10(-7) cm2/s. After activation with thrombin both fibrinopeptide A and fibrin polymerization products could be demonstrated. From our findings we suggest a model for the formation of a three-dimensional network as two parallel processes, elongation and branching and that fibrin oligomers are not only intermediates in the polymerization process but also are substrates for branching.     

Expression of chicken lamin B2 in Escherichia coli: characterization of its structure, assembly, and molecular interactions

Chicken lamin B2, a nuclear member of the intermediate-type filament (IF) protein family, was expressed as a full-length protein in Escherichia coli. After purification, its structure and assembly properties were explored by EM, using both glycerol spraying/low-angle rotary metal shadowing and negative staining for preparation, as well as by analytical ultracentrifugation. At its first level of structural organization, lamin B2 formed "myosin-like" 3.1S dimers consisting of a 52-nm-long tail flanked at one end by two globular heads. These myosin-like molecules are interpreted to represent two lamin polypeptides interacting via their 45-kD central rod domains to form a segmented, parallel and unstaggered 52-nm-long two-stranded alpha-helical coiled-coil, and their COOH-terminal end domains folding into globular heads. At the second level of organization, lamin B2 dimers associated longitudinally to form polar head-to-tail polymers. This longitudinal mode of association of laminin dimers is in striking contrast to the lateral mode of association observed previously for cytoplasmic IF dimers. At the third level of organization, these polar head-to-tail polymers further associated laterally, in an approximately half-staggered fashion, to form filamentous and eventually paracrystal-like structures revealing a pronounced 24.5-nm axial repeat. Finally, following up on recent studies implicating the mitotic cdc2 kinase in the control of lamin polymerization (Peter, M., J. Nakagawa, M. Dorée, J. C. Labbé, and E. A. Nigg. 1990. Cell. 61:591-602), we have examined the effect of phosphorylation by purified cdc2 kinase on the assembly properties and molecular interactions of the bacterially expressed lamin B2. Phosphorylation of chicken lamin B2 by cdc2 kinase interferes with the head-to-tail polymerization of the lamin dimers. This finding supports the notion that cdc2 kinase plays a major, direct role in triggering mitotic disassembly of the nuclear lamina.     

Characterization of polymers of adenosine diphosphate ribose generated in vitro and in vivo

Methods have been developed and applied to determine the size and branching frequency of polymers of ADP-ribose synthesized in nucleotide-permeable cultured mouse cells and in intact cultured cells. Polymers were purified by affinity chromatography with a boronate resin and were fractionated according to size molecular sieve high-performance liquid chromatography. Fractions were enzymatically digested to nucleotides, which were separated by strong anion exchange high-performance liquid chromatography. From these data, average polymer size and branching frequency were calculated. A wide range of polymer sizes was observed. Polymers as large as 190 residues with at least five points of branching per molecule were generated in vitro. Polymers of up to 67 residues containing up to two points of branching per molecule were isolated from intact cells following treatment with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Cells treated with hyperthermia prior to DNA damage contained polymers of an average maximum size of 244 residues containing up to six points of branching per molecule. The detection of large polymers of ADP-ribose in intact cells suggests that alterations in chromatin organization effected by poly(ADP-ribosylation) may extend beyond the covalently modified proteins and very likely involve noncovalent interactions of poly(ADP-ribose) with other components of chromatin.     

A novel mode of polymerization of alpha1-proteinase inhibitor

Patients homozygous for the Z mutant form of alpha1-proteinase inhibitor (alpha1-PI) have an increased risk for the development of liver disease because of the accumulation in hepatocytes of inclusion bodies containing linear polymers of mutant alpha1-PI. The most widely accepted model of polymerization proposes that a linear, head-to-tail polymer forms by sequential insertion of the reactive center loop (RCL) of one alpha1-PI monomer between the central strands of the A beta-sheet of an adjacent monomer. This model derives primarily from two observations: peptides that are homologous with the RCL insert into the A beta-sheet of alpha1-PI monomer and this insertion prevents alpha1-PI polymerization. Normal alpha1-PI monomer does not spontaneously polymerize; however, here we show that the disulfide-linked dimer of normal alpha1-PI spontaneously forms linear polymers in buffer. The monomers within this dimer are joined head-to-head. Thus, the arrangement of monomers in these polymers must be different from that predicted by the loop-A sheet model. Therefore, we propose a new model for alpha1-PI polymer. In addition, polymerization of disulfide-linked dimer is not inhibited by the presence of the peptide even though dimer appears to interact with the peptide. Thus, RCL insertion into A beta-sheets may not occur during polymerization of this dimer.     

Higher order assembling of the mycobacterial polar growth factor DivIVA/Wag31

DivIVA or Wag31, which is an essential pole organizing protein in mycobacteria, can self-assemble at the negatively curved side of the membrane at the growing pole to form a higher order structural scaffold for maintaining cellular morphology and localizing various target proteins for cell-wall biogenesis. The structural organization of polar scaffold formed by polymerization of coiled-coil rich Wag31, which is implicated in the anti-tubercular activities of amino-pyrimidine sulfonamides, remains to be determined. A single-site phosphorylation in Wag31 regulates peptidoglycan biosynthesis in mycobacteria. We report biophysical characterizations of filaments formed by mycobacterial Wag31 using circular dichroism, atomic force microscopy and small angle solution X-ray scattering. Atomic force microscopic images of the wild-type, a phospho-mimetic (T73E) and a phospho-ablative (T73A) form of Wag31 show mostly linear filament formation with occasional curving, kinking and apparent branching. Solution X-ray scattering data indicates that the phospho-mimetic forms of the Wag31 polymers are on average more compact than their phospho-ablative counterparts, which is likely due to the extent of bending/branching. Observed structural features in this first view of Wag31 filaments suggest a basis for higher order Wag31 scaffold formation at the pole.     

The fibrinogen molecule: its size,shape, and mode of polymerization

Improved electron micrographs of the shadow-cast bovine fibrinogen molecule have been obtained establishing its general morphology and dimensions in the dry state. It consists of a linear array of 3 nodules held together by a very thin thread which is estimated to have a diameter of from 8 to 15 A, though it is not clearly resolved. The two end nodules are alike but the center one is slightly smaller. Measurements of shadow lengths indicate that nodule diameters are in the range 50 to 70 A. The length of the dried molecule is 475 +/- 25 A. Adopting the molecular volume from previous physical chemical data and the general morphological features and length from electron microscopy, we calculate the diameters of the end nodules to be 65 A and the center one as 50 A. The model of the molecule so obtained is consistent with the electron microscopical observations and the data from physical chemistry. The intermediate polymers formed when fibrinogen is activated with thrombin were also examined and found to be end-to-end aggregates of altered fibrinogen molecules which shrink in length during the process. Intermediate polymer lengths are from 1000 to 5000 A. The nodular nature of fibrinogen, its shrinkage and end-to-end aggregation on polymerization permits us to deduce an explanation for the system of cross-bands previously observed in stained fibrin fibrils.     

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.     

The Fibrinogen Molecule: Its Size, Shape, and Mode of Polymerization

Improved electron micrographs of the shadow-cast bovine fibrinogen molecule have been obtained establishing its general morphology and dimensions in the dry state. It consists of a linear array of 3 nodules held together by a very thin thread which is estimated to have a diameter of from 8 to 15 A, though it is not clearly resolved. The two end nodules are alike but the center one is slightly smaller. Measurements of shadow lengths indicate that nodule diameters are in the range 50 to 70 A. The length of the dried molecule is 475 ± 25 A. Adopting the molecular volume from previous physical chemical data and the general morphological features and length from electron microscopy, we calculate the diameters of the end nodules to be 65 A and the center one as 50 A. The model of the molecule so obtained is consistent with the electron microscopical observations and the data from physical chemistry. The intermediate polymers formed when fibrinogen is activated with thrombin were also examined and found to be end-to-end aggregates of altered fibrinogen molecules which shrink in length during the process. Intermediate polymer lengths are from 1000 to 5000 A. The nodular nature of fibrinogen, its shrinkage and end-to-end aggregation on polymerization permits us to deduce an explanation for the system of cross-bands previously observed in stained fibrin fibrils.     

Experimental tests of a geometrical abstraction of fibrin polymerization

By combining measurements of the enzymatic release of fibrinopeptide A (FPA) with measurements of intensity and linewidth of Rayleigh scattering from fibrin polymer solutions prior to gelation, we have systematically tested a variety of predictions that can be made on the basis of a simple geometrical abstraction of fibrin polymerization. The experimental investigations include FPA content of fibrin polymers, aggregation of fibrin with fibrinogen, enzyme kinetics, shift of the chemical equilibrium by adding Gly-Pro-Arg-Pro or fibrinogen to the polymer solution, evolution of the polymerization, and influence of fibrinopeptide B release. Among the considered geometrical abstractions there is only one that survives the experimental tests and at the same time is compatible with the electron micrographs by other authors. The main conclusions that can be drawn are (1) the location of binding sites A must be taken from the structure of the fibrinogen molecule proposed by Hoeprich and Doolittle [Biochemistry (1983) 22 , 2049–2055], (2) The fibrinogen monomer is basically centrosymmetric, (3) the state of polymerization is reversible and corresponds to a chemical equilibrium, and (4) Michaelis–Menten enzyme kinetics can be applied.     

Allosteric models for cooperative polymerization of linear polymers

In the cytoskeleton, unfavorable nucleation steps allow cells to regulate where, when, and how many polymers assemble. Nucleated polymerization is traditionally explained by a model in which multistranded polymers assemble cooperatively, whereas linear, single-stranded polymers do not. Recent data on the assembly of FtsZ, the bacterial homolog of tubulin, do not fit either category. FtsZ can polymerize into single-stranded protofilaments that are stable in the absence of lateral interactions, but that assemble cooperatively. We developed a model for cooperative polymerization that does not require polymers to be multistranded. Instead, a conformational change allows subunits in oligomers to associate with high affinity, whereas a lower-affinity conformation is favored in monomers. We derive equations for calculating polymer concentrations, subunit conformations, and the apparent affinity of subunits for polymer ends. Certain combinations of equilibrium constants produce the sharp critical concentrations characteristic of cooperative polymerization. In these cases, the low-affinity conformation predominates in monomers, whereas virtually all polymers are composed of high-affinity subunits. Our model predicts that the three routes to forming HH dimers all involve unstable intermediates, limiting nucleation. The mathematical framework developed here can represent allosteric assembly systems with a variety of biochemical interpretations, some of which can show cooperativity, and others of which cannot.     

Polymorphism of fibrin equilibrium oligomers in the presence of fragment D

The methods of viscosimetry, the Rayleigh light-scattering and analytical ultracentrifugation were applied to study the physicochemical mechanism of the effect of fragment D on the structure of fibrin equilibrium oligomers. Using the values of intrinsic viscosity, weight average molecular masses and mass/length ratio it was shown that when producing an antipolymerization effect the fragment D retains the three-dimensional organization of fibrin polymers, i.e. rigid rod-like single- and double-stranded protofibrillas. The paper has proved that along with the traditional mechanism of inhibiting self-assembly of of the double-stranded structure due to the competition of fragment D with fibrin monomer for central domain E there is an alternative attributed to its attachment to a peripheral region of the fibrin monomer. The second mechanism is the only one which occurs in the region of single-stranded pseudoprotofibrillas existence. The role of alpha C-domains in protein-protein interactions is also 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.     

Effects of hyaluronic acid and other glycosaminoglycans on fibrin polymer formation

Previously, we reported that the glycosaminoglycan (GAG) hyaluronic acid (HA) specifically bound to the plasma protein fibrinogen [LeBoeuf, R. D., Raja, R. R., Fuller, G. M., & Weigel, P. H. (1986) J. Biol. Chem. 261, 12586]. The binding of other macromolecules to fibrinogen could influence the conversion of fibrinogen to fibrin. Therefore, we tested whether HA and other GAGs could alter the kinetics of fibrin polymer formation and the physical structure of the resulting gel. In this study, we present data showing that the GAGs HA and chondroitin sulfate (CS) affect fibrin formation in three specific ways: (i) they decreased the clotting time of fibrinogen 3-10-fold; (ii) both GAGs increase significantly the rate of fibrin polymer formation; and (iii) fibrin gels containing HA or CS had a final A450 that was greater than controls, indicating that these two glycosaminoglycans influence either the final size of fibrin fibrils or the extent of the lateral association between fibrils. These results demonstrate that the interactions of HA and CS with forming fibrin polymers can alter both the kinetics of formation and may produce structural changes in fibrin gels.     

Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled.

Although much is known about fibrin polymerization, because it is complex, the effects of various modifications are not intuitively obvious and many experimental observations remain unexplained. A kinetic model presented here that is based on information about mechanisms of assembly accounts for most experimental observations and allows hypotheses about the effects of various factors to be tested. Differential equations describing the kinetics of polymerization were written and then solved numerically. The results have been related to turbidity profiles and electron microscope observations. The concentrations of intermediates in fibrin polymerization, and fiber diameters, fiber and protofibril lengths have been calculated from these models. The simplest model considered has three steps; fibrinopeptide A cleavage, protofibril formation, and lateral aggregation of protofibrils to form fibers. The average number of protofibrils per fiber, which is directly related to turbidity, can be calculated and plotted as a function of time. The lag period observed in turbidity profiles cannot be accurately simulated by such a model, but can be simulated by modifying the model such that oligomers must reach a minimum length before they aggregate. Many observations, reported here and elsewhere, can be accounted for by this model; the basic model may be modified to account for other experimental observations. Modeling predicts effects of changes in the rate of fibrinopeptide cleavage consistent with electron microscope and turbidity observations. Changes only in the rate constants for initiation of fiber growth or for addition of protofibrils to fibers are sufficient to account for a wide variety of other observations, e.g., the effects of ionic strength or fibrinopeptide B removal or thrombospondin. The effects of lateral aggregation of fibers has also been modeled: such behavior has been observed in turbidity curves and electron micrographs of clots formed in the presence of platelet factor 4. Thus, many aspects of clot structure and factors that influence structure are directly related to the rates of these steps of polymerization, even though these effects are often not obvious. Thus, to a large extent, clot structure is kinetically determined.     

Modification of fibrin network ultrastructure by Fab fragments specific for different domain of fibrinogen

Kinetics of inhibition of fibrin monomer polymerization produced by Fab fragments prepared from immunochemically purified monospecific antibodies to the surface epitopes of different domains of fibrinogen molecule has been correlated with electron microscopic observations of resulting specimens. Fab fragments prepared from anti FgD antisera were the most efficient inhibitors of thrombin-catalysed conversion of fibrinogen to fibrin; polymerization of fibrin monomers as detected spectrophotometrically was abolished at 2:1 molar ratio of anti FgD Fab fragments to fibra monomer. These Fab fragments acting as a steric hindrance of polymerization sites inhibited the first stage of fibrin monomer aggregation. Interaction of Fab fragments derived from antibodies specific for alpha 239-476 with corresponding segment of fibrinogen molecule resulted in a weak inhibition of fibrin monomer polymerization. However, fibrin obtained in the presence of these Fab fragments was significantly modified and showed no periodicity. This observation may suggest that anti alpha 239-476 Fab impaired the course of the second stage of fibrin monomer polymerization, i.e. lateral association of fibrin fibrils.