Substitution of the human αC region with the analogous chicken domain generates a fibrinogen with severely impaired lateral aggregation: fibrin monomers assemble into protofibrils but protofibrils do not assemble into fibers

Fibrin polymerization occurs in two steps: the assembly of fibrin monomers into protofibrils and the lateral aggregation of protofibrils into fibers. Here we describe a novel fibrinogen that apparently impairs only lateral aggregation. This variant is a hybrid, where the human αC region has been replaced with the homologous chicken region. Several experiments indicate this hybrid human-chicken (HC) fibrinogen has an overall structure similar to normal. Thrombin-catalyzed fibrinopeptide release from HC fibrinogen was normal. Plasmin digests of HC fibrinogen produced fragments that were similar to normal D and E; further, as with normal fibrinogen, the knob 'A' peptide, GPRP, reversed the plasmin cleavage associated with addition of EDTA. Dynamic light scattering and turbidity studies with HC fibrinogen showed polymerization was not normal. Whereas early small increases in hydrodynamic radius and absorbance paralleled the increases seen during the assembly of normal protofibrils, HC fibrinogen showed no dramatic increase in scattering as observed with normal lateral aggregation. To determine whether HC and normal fibrinogen could form a copolymer, we examined mixtures of these. Polymerization of normal fibrinogen was markedly changed by HC fibrinogen, as expected for mixed polymers. When the mixture contained 0.45 μM normal and 0.15 μM HC fibrinogen, the initiation of lateral aggregation was delayed and the final fiber size was reduced relative to normal fibrinogen at 0.45 μM. Considered altogether, our data suggest that HC fibrin monomers can assemble into protofibrils or protofibril-like structures, but these either cannot assemble into fibers or assemble into very thin fibers.     

Decreased lateral aggregation of a variant recombinant fibrinogen provides insight into the polymerization mechanism

We analyzed the polymerization of BbetaA68T fibrinogen, the recombinant counterpart of fibrinogen Naples, a variant known to have decreased thrombin binding. When polymerized with equal thrombin concentrations, BbetaA68T fibrinogen had a longer lag time and lower rate of lateral aggregation, V(max), than normal recombinant fibrinogen, but a similar final turbidity. At thrombin concentrations that equalized the rates of fibrinopeptide A release, BbetaA68T fibrinogen polymerized with a lag time and V(max) similar to normal, but reached a significantly lower final turbidity. Similar results were produced when BbetaA68T was polymerized with Ancrod, which cleaves fibrinopeptide A at the same rate from either fibrinogen, and when BbetaA68T desA monomers were polymerized. The polymerization of desAB fibrin monomers, which circumvents fibrinopeptide release, was the same for both fibrinogens. We confirmed that turbidity was indicative of fiber thickness by scanning electron microscopy of fibrin clots. Here, we present the first experimental evidence of fibrin polymerization with a normal period of protofibril formation and rate of lateral aggregation, but with a significantly decreased extent of lateral aggregation. We conclude that the decreased lateral aggregation seen in BbetaA68T fibrinogen is due to an altered step in the enzymatic phase of its polymerization process. We propose that during normal polymerization a subtle conformational change in the E domain occurs, between the release of FpA and FpB, and that this change modulates the mechanism of lateral aggregation. Without this change, the lateral aggregation of BbetaA68T fibrinogen is impaired such that variant clots have thinner fibers than normal clots.     

A new concept of fibrin formation based upon the linear growth of interlacing and branching polymers and molecular alignment into interlocked single-stranded segments

In a previous electron microscopic study of early fibrin polymers processed by freeze drying and rotatory shadowing, a large proportion of loosely constructed, frequently branching linear molecular chains was observed; their structural organization was inconsistent with a half-staggered double-stranded model for fibrin polymerization. These conflicting results prompted us to investigate the structure of early fibrin polymers prepared according to a large variety of methods currently used for electron microscopy of macromolecules. By use of a systematic random sampling procedure, fibrin polymers were photographically recorded. They were classified according to their morphological form, and the frequency of occurrence of each configuration was determined. Half-staggered double-stranded forms accounted for less than 1% of all types encountered. Interpretation of the structural organization manifested in the diverse polymer forms observed necessitated the construction of a new interlocked single-strand model for fibrin polymerization. The fibrin polymerization process combines simultaneous propagation of linear growth, branching, and lateral interlocking (leading to lateral association), resulting in the rapid formation of a fibrin network. The structural pattern developing during growth of fibrin polymers appears to be determined principally by the enzymatic mechanism and not solely by the intrinsic molecular structure of fibrinogen. The validity of the interlocked single-strand model was tested by selective fibrinopeptide-B-releasing experiments. Under such activation conditions, the polymer forms predicted according to this and the half-staggered double-strand models should differ; the structures observed were indeed consistent with the interlocked single-strand hypothesis. The compatibility of existing data with this model is discussed.     

Fibrinogen beta-chain tyrosine nitration is a prothrombotic risk factor

Elevated levels of circulating fibrinogen are associated with an increased risk of atherothrombotic diseases although a causative correlation between high levels of fibrinogen and cardiovascular complications has not been established. We hypothesized that a potential mechanism for an increased prothrombotic state is the post-translational modification of fibrinogen by tyrosine nitration. Mass spectrometry identified tyrosine residues 292 and 422 at the carboxyl terminus of the beta-chain as the principal sites of fibrinogen nitration in vivo. Immunoelectron microscopy confirmed the incorporation of nitrated fibrinogen molecules in fibrin fibers. The nitration of fibrinogen in vivo resulted in four distinct functional consequences: increased initial velocity of fibrin clot formation, altered fibrin clot architecture, increased fibrin clot stiffness, and reduced rate of clot lysis. The rate of fibrin clot formation and clot architecture was restored upon depletion of the tyrosine-nitrated fibrinogen molecules. An enhanced response to the knob "B" mimetic peptides Gly-His-Arg-Pro(am) and Ala-His-Arg-Pro(am) suggests that incorporation of nitrated fibrinogen molecules accelerates fibrin lateral aggregation. The data provide a novel biochemical risk factor that could explain epidemiological associations of oxidative stress and inflammation with thrombotic complications.     

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.     

Oxidation-induced destabilization of the fibrinogen αC-domain dimer investigated by molecular dynamics simulations

Upon activation, fibrinogen is converted to insoluble fibrin, which assembles into long strings called protofibrils. These aggregate laterally to form a fibrin matrix that stabilizes a blood clot. Lateral aggregation of protofibrils is mediated by the αC domain, a partially structured fragment located in a disordered region of fibrinogen. Polymerization of αC domains links multiple fibrin molecules with each other enabling the formation of thick fibrin fibers and a fibrin matrix that is stable but can also be digested by enzymes. However, oxidizing agents produced during the inflammatory response have been shown to cause thinner fibrin fibers resulting in denser clots, which are harder to proteolyze and pose the risk of deep vein thrombosis and lung embolism. Oxidation of Met⁴⁷⁶ located within the αC domain is thought to hinder its ability to polymerize disrupting the lateral aggregation of protofibrils and leading to the observed thinner fibers. How αC domains assemble into polymers is still unclear and yet this knowledge would shed light on the mechanism through which oxidation weakens the lateral aggregation of protofibrils. This study used temperature replica exchange molecular dynamics simulations to investigate the αC-domain dimer and how this is affected by oxidation of Met⁴⁷⁶. Analysis of the trajectories revealed that multiple stable binding modes were sampled between two αC domains while oxidation decreased the likelihood of dimer formation. Furthermore, the side chain of Met⁴⁷⁶ was observed to act as a docking spot for the binding and this function was impaired by its conversion to methionine sulfoxide.     

Nanoscale Probing Reveals that Reduced Stiffness of Clots from Fibrinogen Lacking 42 N-Terminal Bβ-Chain Residues Is Due to the Formation of Abnormal Oligomers

Removal of Bβl-42 from fibrinogen by Crotalus atrox venom results in a molecule lacking fibrinopeptide B and part of a thrombin binding site. We investigated the mechanism of polymerization of desBβ1-42 fibrin. Fibrinogen trinodular structure was clearly observed using high resolution noncontact atomic force microscopy. E-regions were smaller in desBβ1-42 than normal fibrinogen (1.2 nm ± 0.3 vs. 1.5 nm ± 0.2), whereas there were no differences between the D-regions (1.7 nm ± 0.4 vs. 1.7 nm ± 0.3). Polymerization rate for desBβ1-42 was slower than normal, resulting in clots with thinner fibers. Differences in oligomers were found, with predominantly lateral associations for desBβ1-42 and longitudinal associations for normal fibrin. Clot elasticity as measured by magnetic tweezers showed a G′ of ∼1 Pa for desBβ1-42 compared with ∼8 Pa for normal fibrin. Spring constants of early stage desBβ1-42 single fibers determined by atomic force microscopy were ∼3 times less than normal fibers of comparable dimensions and development. We conclude that Bβ1-42 plays an important role in fibrin oligomer formation. Absence of Bβ1-42 influences oligomer structure, affects the structure and properties of the final clot, and markedly reduces stiffness of the whole clot as well as individual fibrin fibers.     

Influence of fibrinogen on fibrin polymerization. Ultracentrifugation studies

During the transformation of fibrinogen to fibrin, excess fibrinogen suppresses further polymerization of fibrin, thereby enabling the nascent fibrin to be transported in a soluble form in blood. The question of possible complex formation between fibrin and fibrinogen was addressed by analyzing fibrin/fibrinogen (1:20, mol/mol) mixtures in the presence of calcium ions in stable linear sucrose density gradients by ultracentrifugation at 37 degrees C. During the period of ultracentrifugation in independent experiments, 40% of desAA-fibrin and 30% of desAABB-fibrin, respectively, precipitated without the participation of fibrinogen. The desAABB-fibrin, recovered in the gradient fractions, appeared as high-molecular-weight polymers (22 S), whereas the recovered desAA-fibrin exhibited only a slight increase in molecular weight (9 S) compared to fibrinogen (8 S). In contrast to this finding, both types of fibrin were totally recovered in gradient fractions provided that fibrinogen was present in the gradient at a uniform concentration of 2 mg/ml. In addition, the presence of fibrinogen but not human serum albumin reduced the size of desAABB-fibrin polymers (17 S). However, stable fibrin-fibrinogen complexes could not be demonstrated, since cosedimentation of differently labelled desAABB-fibrin and fibrinogen was not detectable. These studies suggest a specific but weak interaction of the solubilizing fibrinogen with the soluble fibrin polymers as demonstrated by a rapid exchange of both macromolecules.     

Structural variability of collagen fibers in the calcareous axial rod of a sea pen

Previous studies revealed that the organic matrix of the skeletal rod of the sea pen, Veretillum cynomorium, contained about 50% collagenous protein. The present ultrastructural study, based upon conventional staining methods, shows the existence of an abundant, longitudinally arranged nonbanded and fibrillar material separated by a reticular matrix. After incubation with ³H-proline, labeling is specifically localized on the fibrillar material. Some fibers occasionally display a transverse striation with a period of 11 to 14 nm which can be associated with a chevron striation. Infrequently, some other fibers display a more distinct banding of 55 to 70 nm or even yield a checkerboard pattern. However, a majority of fibers remain without a regular structure comparable to the periodic striations observed in the collagen of other animals. After treatment with 1% PTA in 70% ethanol, all the fibers show a clear banding of 14 nm and some of them possess two types of striations. The same result is obtained on fibers mechanically dissociated and negatively stained. As these methods show a periodic banding pattern on all the fibers, it is likely that all the fibers (striated or not) observed after routine electron microscopy correspond to collagen material. This collagen appears to be both polymorphic and completely new in comparison to that which is characteristic of the mesoglea. The polymorphic aspect is compared to that obtained from vertebrate collagens.     

Reconstruction of the musculature of <Emphasis Type="Italic">Magelona</Emphasis> cf. <Emphasis Type="Italic">mirabilis</Emphasis> (Magelonidae) and <Emphasis Type="Italic">Prionospio cirrifera</Emphasis> (Spionidae) (Polychaeta,Annelida) by phalloidin labeling and cLSM

Recent investigations have suggested that a lack of circular muscle fibers may be a common situation rather than a rare exception in polychaetes. As part of a comparative survey of polychaete muscle systems, the F-actin musculature subset of Magelona cf. mirabilis and Prionospio cirrifera were labeled with phalloidin and three-dimensionally analyzed and reconstructed by means of cLSM. Obvious similarities are sublongitudinal lateral, circumbuccal, palp retractor, dominating dorsal longitudinal, perpendicular lateral and ventral transverse muscles. Differences between M. cf. mirabilis and P. cirrifera are: (1) two types of prostomial muscles (transversal and longitudinal) in M. cf. mirabilis versus one type (diagonal) in P. cirrifera; (2) one type of palp muscles (longitudinal) in M. cf. mirabilis versus three types (longitudinal, diagonal, circular) in P. cirrifera; (3) five ventral longitudinal muscles (ventromedian, paramedian, ventral) in M. cf. mirabilis versus four (two paramedian, two ventral) in P. cirrifera. Ventral and lateral transverse fibers are present in the thorax, but absent in the abdomen of M. cf. mirabilis. The triangular lumen of the pharynx in M. cf. mirabilis is surrounded by radial muscle fibers; three sets of pharynx diductors attach to its dorsal side. The unique features of P. cirrifera are one pair of brain muscles and segmentally arranged dorsal transverse muscles, the latter located outside the longitudinal muscles. The transverse lateral muscles are restricted to the sides and lie beneath the longitudinal muscles, a pattern described here for the first time. A true, outer layer of circular fibers is absent in both species of Spionida that were investigated.     

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.     

High-resolution visualization of fibrinogen molecules and fibrin fibers with atomic force microscopy

We report an atomic force microscopy (AFM) study of fibrinogen molecules and fibrin fibers with resolution previously achieved only in few electron microscopy images. Not only are all objects triads, but the peripheral D regions are resolved into the two subdomains, apparently corresponding to the βC and γC domains. The conformational analysis of a large population of fibrinogen molecules on mica revealed the two most energetically favorable conformations characterized by bending angles of ～100 and 160 degrees. Computer modeling of the experimental images of fibrinogen molecules showed that the AFM patterns are in good agreement with the molecular dimensions and shapes detected by other methods. Imaging in different environments supports the expected hydration of the fibrinogen molecules in buffer, whereas imaging in humid air suggests the 2D spreading of fibrinogen on mica induced by an adsorbed water layer. Visualization of intact hydrated fibrin fibers showed cross-striations with an axial period of 24.0 ± 1.6 nm, in agreement with a pattern detected earlier with electron microscopy and small-angle X-ray diffraction. However, this order is clearly detected on the surface of thin fibers and becomes less discernible with the fiber's growth. This structural change is consistent with the proposal that thinner fibers are denser than thicker ones, that is, that the molecule packing decreases with the increasing of the fibers' diameter.     

Altered structure and function of fibrinogen after cleavage by Factor VII Activating Protease (FSAP)

Factor VII Activating Protease (FSAP) is a plasma protease affecting both coagulation and fibrinolysis. Although a role in hemostasis is still unclear, the identification of additional physiologic substrates will help to elucidate its role in this context. FSAP has been reported to cleave fibrinogen, but the functional consequences of this are not known. We have therefore undertaken this study to determine the implications of this cleavage for fibrin-clot formation and its lysis. Treatment of human fibrinogen with FSAP released an N-terminal peptide from the Bβ chain (Bβ1-53) and subsequently the fibrinopeptide B; within the Aα chain a partial truncation of the αC-region by multiple cleavages was seen. The truncated fibrinogen showed a delayed thrombin-catalyzed polymerization and formed fibrin clots of reduced turbidity, indicative of thinner fibrin fibers. Confocal laser scanning and scanning electron microscopy of these clots revealed a less coarse fibrin network with thinner fibers and a smaller pore size. A lower pore size was also seen in permeability studies. Unexpectedly, FSAP-treated fibrinogen or plasma exhibited a significantly faster tPA-driven lysis, which correlated exclusively with cleavage of fibrinogen and not with activation of plasminogen activators. Similar observations were also made in plasma after activation of endogenous zymogen FSAP, but not in plasma of carrier of the rare Marburg I single nucleotide polymorphism. In conclusion, altering fibrin clot properties by fibrinogenolysis is a novel function of FSAP in the vasculature, which facilitates clot lysis and may in vivo contribute to reduced fibrin deposition during thrombosis.     

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.     

Aggregates Dramatically Alter Fibrin Ultrastructure

Among the many factors influencing fibrin formation and structure (concentration, temperature, composition, pH, etc.), it has been suggested that the polydispersity of fibrinogen may play an important role. We propose here a detailed investigation of the influence of this parameter on fibrin multiscale structure. Two commercial fibrinogen preparations were used, a monodisperse and a polydisperse one. First, the respective compositions of both fibrinogen preparations were thoroughly determined by measuring the fibrin-stabilizing factor; fibronectin; α, β, and γ intact chain contents; the γ/γ′ chains ratio; the N-glycosylation; and the post-translational modifications. Slight variations between the composition of the two fibrinogen preparations were found that are much smaller than the compositional variations necessary to alter significantly fibrin multiscale structure as observed in the literature. Conversely, multiangle laser light scattering-coupled size exclusion chromatography and dynamic light scattering measurements showed that the polydisperse preparation contains significant amounts of aggregates, whereas the other preparation is essentially monodisperse. The multiscale structure of the fibrins produced from those two fibrinogen preparations was determined by using x-ray scattering, spectrophotometry, and confocal microscopy. Results show that fibers made from the aggregate-free fibrinogen present a crystalline longitudinal and lateral structure and form a mikado-like network. The network produced from the aggregates containing fibrinogen looks to be partly built around bright spots that are attributed to the aggregate. The multiscale structure of mixtures between the two preparations shows a smooth evolution, demonstrating that the quantity of aggregates is a major determining factor for fibrin multiscale structure. Indeed, the effect of a few percent in the mass of aggregates is larger than any other effect because of compositional differences under the same reaction conditions. Finally, we propose a mechanistic interpretation of our results, which points at a direct role of the aggregates during polymerization, which disrupts the ideal ordering of monomers inside fibrin protofibrils and fibers.     

Position of gamma-chain carboxy-terminal regions in fibrinogen/fibrin cross-linking mixtures

There are conflicting ideas regarding the location of the carboxyl-terminal regions of cross-linked gamma-chain dimers in double-stranded fibrin fibrils. Some investigators believe that the chains are always oriented longitudinally along each fibril strand and traverse the contacting ends of abutting fibrin D domains ("DD-long" cross-linking). Other investigations have indicated instead that the chains are situated transversely between adjacent D domains in opposing fibril strands (transverse cross-linking). To distinguish between these two possibilities, the gamma dimer composition of factor XIIIa-cross-linked fibrin/fibrinogen complexes that had been formed through noncovalent D/E interactions between fibrinogen D domains and fibrin E domains was examined. Two factor XIIIa-mediated cross-linking conditions were employed. In the first, fibrin/fibrinogen complexes were formed between (125)I-labeled fibrinogen 2 ("peak 2" fibrinogen), each heterodimeric molecule containing one gamma(A) and one larger gamma' chain, and nonlabeled fibrin 1 molecules ("peak 1" fibrin), each containing two gamma(A) chains. If DD-long cross-linking occurred, (125)I-labeled gamma(A)-gamma(A), gamma(A)-gamma', and gamma'-gamma'dimers in a 1:2:1 ratio would result. Transverse cross-linking would yield a 1:1 mixture of (125)I-labeled gamma(A)-gamma(A) and gamma(A)-gamma' dimers, without any gamma'-gamma' dimers. Autoradiographic analyses of reduced SDS-PAGE gels from protocol 1 revealed (125)I-labeled gamma(A)-gamma(A) and gamma(A)-gamma' dimers at a ratio of approximately 1:1. No labeled gamma'-gamma' dimers were detected. Protocol 2 used a converse mixture, (125)I-fibrin 2 and nonlabeled fibrinogen 1. DD-long cross-linking of this mixture would yield only nonradioactive gamma(A)-gamma(A) dimers, whereas transverse cross-linking would yield a 1:1 mixture of (125)I-labeled gamma(A)-gamma(A) and gamma(A)-gamma' dimers. Autoradiographic analyses of this mixture yielded (125)I-labeled gamma(A)-gamma(A) and gamma(A)-gamma' dimers in a 1:1 ratio. These findings provide no evidence that longitudinal (DD-long) gamma chain positioning occurs in cross-linked fibrin and indicate instead that most, if not all, gamma-chain positioning in an assembled fibrin polymer is transverse.     

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.     

Self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation

The mechanism of self-assembly of fibrin monomers and fibrinogen aggregation during ozone oxidation has been studied by the methods of elastic and dynamic light-scattering and viscosimetry. Fibrin obtained from oxidized fibrinogen exhibits higher average fiber mass/length ratio compared with native fibrin. Fibrinogen ozonation sharply reduced the latent period preceding aggregation of protein molecules; however, the mechanism of self-assembly of ozonated and non-ozonated fibrinogen cluster was identical. In both cases flexible polymers are formed and reaching a certain critical length they form densely packed structures and aggregate. Using infrared spectroscopy, it has been shown that free radical oxidation of amino acid residues of fibrinogen polypeptide chains catalyzed by ozone results in formation of carbonyl, hydroxyl, and ether groups. It is concluded that fibrinogen peripheral D-domains are the most sensitive to ozonation, which causes local conformational changes in them. On one hand, these changes inhibit the reaction of longitudinal polymerization of monomeric fibrin molecules; on the other hand, they expose reaction centers responsible for self-assembly of fibrinogen clusters.     

Department of Protein Structure and Function--Volodimir Oleksandrovich Bielitser School of the Ukrainian Academy of Sciences. Studies on structure of fibrin fiber structure and mechanism (1975-1987

In this short historical review the records about foundation and research activity of the Department of Structure and Function of Protein--school of V. A. Belitser, Member of the National Academy of Sciences of Ukraine are presented. V. A. Belitser was the founder and indispensable chief of the department since the date of its creation (1944) till 1987. The main research interests (1975-1987) of the department were focused at the investigation of structure, biological function of the fibrinogen-fibrin system, mechanisms of the network assembly and of the fibrin fibers structure. Studying the molecular mechanisms of the fibrin fiber assembly, it was shown that the specificity of the building structure was shown is determined by the specific reactive sites with strong affinity of the molecules. The activity of the sites was investigated on protein molecules as well as the fragments. The physical nature of the bonds created by the active sites, that appearing during in the process of fibrinogen activation by thrombin, was revealed. Examination of the fibrin assembly in cooperation with electronmicroscopists and studies of the complex formation between active fragments and fibrin monomer were summarized. Both the fibrin monomer polymerization and protofibril lateral association are presented as two stages in the assembly of the fibrin network. In the research of the domain fibrinogen structure the specific sites of the fibrin assembly in each of the domains were found. COOH-terminal regions of the A alpha-chains play independent part in the fibrinogen and fibrin. That is why it is relevant to consider them as alpha C-domains. In the free fibrinogen molecules (in solution) these domains are responsible for globular shape, they are linked to domains D intramolecularly. When fibrin assembly takes place, alpha C-domains play significant carriage role in fibrin molecules interaction, linking to domains D intermolecularly. The model of the fibrinogen molecule structure and the general scheme of the fibrin fibers network formation were proposed. Physico-chemical basics of a biological structure assembly were elucidated using the process of the fibrin self-assembly as an example. Much attention was devoted to the problems of practical medicine. The quantitative methods of fibrinogen, soluble fibrin and active fibrin/fibrinogen fragments estimation in blood plasma were developed.     

The effect of fibrin structure on fibrinolysis.

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