Analysis of soluble fibrin complexes by agarose gel chromatography and protamine sulfate gelation.

The molecular makeup of soluble fibrin complexes was studied by gel exclusion chromatography using radio-labelling to characterize individual components in protein mixtures. Products of limited plasmin degradation of fibrinogen and mixtures of fibrinogen and "early" fibrinogen digests formed high molecular weight soluble fibrin complexes upon incubation with thrombin. Purified, nonclottable fragment Y did not incorporate into soluble fibrin complexes, nor could we demonstrate incorporation of fragments D and E as previously described from our laboratory. Thus, under the conditions of these experiments, soluble fibrin complexes have two identifiable components, fibrin monomer and clottable fragment X monomer, although incorporation of native fibrinogen or fragment X unreacted by thrombin into soluble fibrin complexes cannot be excluded. Individual fractions of thrombin-treated early fibrinogen digests isolated by agarose gel chromatography were treated with protamine sulfate at 37 degrees C resulting in precipitation-gelation of greater than 90 per cent of high molecular weight soluble fibrin complexes; whereas, less than 10 per cent of lower molecular weight fibrinogen degradation products precipitated by protamine sulfate. These findings do not support the widely held concept that soluble fibrin complexes incorporate nonclottable degradation products of fibrinogen proteolysis, nor do they support the notion that the so-called paracoagulation reaction induced by protamine sulfate results from the splitting of complexes between fibrin monomer and nonclottable fibrinogen degradation products.     

Electronmicroscopic study on the molecular shape of non-stabilized fibrin break-down products produced by complex heparin compounds

Experimental data indicate that products develop from non-stabilized fibrin because of nonfermentative splitting by complex heparin connections. These products have a globular form with a diameter of 10-250 A and are similar to morphological fibrinogen molecules. The identified products show no marked lytic effect towards non-stabilized fibrin. The application of S35 labelled heparin in combination with preparative ultracentrifugation and electron microscopy enabled the determination to be established that heparin complexes will combine with particles of fibrinomeres, thus causing their transfer from the fibrillary to the globular condition. The destruction of the connections of heparin complexes with their globular molecule structures of fibrinmonomere, e.g. by protamine sulfate, guarantees their development and primary polymerization. With factor XIIIa being present, the structurally reconstructed fibrin will form a stabilized coagulum of full value which is similar in its ultrastructure to fibrin obtained in control tests.     

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.     

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.     

Equilibria in the fibrinogen-fibrin conversion. IX. Effects of calcium ions on the reversible polymerization of fibrin monomer

An investigation was made of the role of calcium ions in the reversible stage of fibrin polymerization, using a direct and relatively simple approach. Purified fibrin monomer in solution (7.5 mg/ml) in 1.0 m NaBr (pH 5.3) was polymerized by raising the pH to 5.7–7.7 by the addition of aliquots of standard NaOH solution and the rate and total extent of proton release during polymerization were measured potentiometrically. In the presence of added CaCl₂ (10⁻⁵-10⁻²m) the rate of proton release was increased and the clotting time was decreased. The profile of equilibrium proton release vs pH of polymerization was also shifted, the maximum being increased and occurring at a lower pH. Sedimentation velocity studies in the intermediate pH range (5.7–6.0) showed that the altered profile of equilibrium proton release was due to a broadening of the pH range of polymerization, and that polymerization remained reversible in the presence of CaCl₂. At pH 5.3, where fibrin is essentially monomeric, addition of CaCl₂ resulted in the release of protons and small increases in sedimentation coefficient and reduced viscosity. Under the same conditions, a similar release of protons was observed from fibrinogen, but there was no effect on its sedimentation coefficient. It was concluded that the proton release at pH 5.3 was due mainly to binding of calcium ions to fibrinogen and fibrin monomer. The effect of CaCl₂ on the sedimentation coefficient of fibrin at pH 5.3 was found to decrease with decreasing protein concentration, indicating that it was the result of a small extent of polymerization, rather than a conformational change. Added MgCl₂ had no effect on fibrin monomer at pH 5.3 and no significant effect on the rate or extent of proton release during polymerization at higher pH, indicating that there are specific binding sites for calcium ions in fibrinogen and fibrin. The observed effects of bound calcium ions on reversible fibrin polymerization are explained most simply in electrostatic terms.     

A re-examination of the cleavage of fibrinogen and fibrin by plasmin.

Three Fragment D species (D1, D2, D3) were isolated with time from a plasmin digest of fibrinogen and had molecular weights of 92,999, 86,000 and 82,000 by summation of subunit molecular weights from sodium dodecyl sulfate polyacrylamide gel electrophoresis. Their molecular weights by sedimentation equilibrium ultracentrifugation were 94,000 t87,000, 88,000 to 82, 000, and 76,000 to 70,000 depending on the values calculated for the partial specific volumes. Each of the Fragment D species contained three disulfide-linked subunits derived from the Aalpha, Bbeta, and gamma chains of fibrinogen and differed only in the extent of COOH-terminal degradation of their gamma chain derivatives. Plasmin cleaved Fragment D1 to release the cross-link sites from its gamma' subunit of 38,000 molecular weight; however, the beta' subunit of 42,000 molecular weight and the alpha' subunit of 12,000 molecular weight were resistant to further digestion by plasmin. Fragment D isolated from highly cross-linked fibrin had a dimeric structure due to cross-link formation between the gamma' subunits of two fibrinogen Fragment D species. The molecular weight of fibrin Fragment D was 184,000 by summation of subunit molecular weights and 190,000 to 175,000 by sedimentation equilibrium. Cross-linking the gamma chain, as well as incorporating the site-specific fluorescent label monodansyl cadaverine into the gamma chain cross-link acceptor site, prevented its COOH-terminal degradation by plasmin. Therefore, only one species of fibrin Fragment D, as well as only one species of monodansyl cadaverine-labeled fibrin Fragment D monomer, was generated during plasmin digestion. These results show unequivocally that each fibrinogen Fragment D contains only three subunit chains and therefore the digestion of fibrinogen by plasmin must result in the production of two Fragment D molecules from each fibrinogen molecule. The recently proposed model of fibrinogen cleavage that postulates the generation of a single Fragment D with three pairs of subunit chains from each fibrinogen molecule is incorrect. Incorporation of monodansyl cadaverine into the cross-link acceptor sites of the alpha chain did not alter its cleavage by plasmin detectably. A series of monodansyl cadaverine-labeled peptides, which ranged in molecular weight from 40,000 to 23,000, were cleaved from the alpha chain of monodansyl cadaverine-labeled fibrin monomer during the early stages of plasmin digestion. These peptides were degraded progressively to a brightly fluorescent plasmin-resistant peptide of 21,000 molecular weight and a weakly fluorescent peptide of 2,500 molecular weight. Thus both alpha chain cross-link acceptor sites are contained within a peptide segment of 23,000 molecular weight.     

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.     

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.     

Inhibition of the enzymatic activity of thrombin by concanavalin A

Concanavalin A, a carbohydrate lectin derived from the jack bean, prolongs the thrombin clotting time of human plasma or purified fibrinogen. Prolongation is due to delay in peptide release from fibrinogen. The rate of fibrin monomer polymerization is not affected. Hydrolysis of protamine sulfate by thrombin is inhibited by concanavalin A. All inhibitory effects are prevented by α-methyl-D-mannoside. Concanavalin A does not delay clotting of fibrinogen by reptilase (releases fibrinopeptide A only) or by Ancistrodon contortrix contortrix (releases fibrinopeptide B initially followed by a small amount of A). It is concluded that concanavalin A binds to a carbohydrate on the thrombin molecule thus inhibiting its enzymatic activity.     

Molecular organization of copolymers of fibrinogen-fibrin fragment

In this study we have produced for the first time a native fibrinogen copolymer with a fragment of fibrin E. and the molecular mechanism of its formation was studied by different physicochemical methods. Based on the data of angular dependency of the Debay scattering factor, the average molecular mass, coefficients of translational diffusion and the intrinsic viscosity it was shown that the primary interaction comprised the "end-to-end" fibrinogen dimerization through the D-D contacts with the following fragment E specific binding. It resulted in the stable three-domain D-E-D knot formation. The structural flexibility of the copolymer determines the tendency to their folding and the strong intermolecular hydrodynamic interaction indicates the structural compactization. This correlates as we think, with the presence of the centers of lateral binding in the fibrinogen molecule. Single-strand copolymers aggregate when they reach their critical sweep length resulting in microgel formation with the raise of the molecular mass. We came to the conclusion that fibrinogen molecules are capable to associate due to the stable native conformation shift into the active state, thus demasking the reaction groups in the D-domain. Possible reasons for the lack of fibrinogen heteropolymer rigidity characteristic for the fibrin polymers are discussed.     

Effect of peptides--structural analogs of NH2-terminal sites of fibrin alpha- and beta-chains--on specific binding of the NH2-terminal disulfide bond of fibrin with fibrinogen

Peptides Gly-Pro-Arg-Pro and Gly-His-Arg-Pro (fibrin alpha- and beta-chain NH2-terminal analogs, respectively) are studied for their effect on fibrinogen (F) and fibrin NH2-terminal disulphide knot (N-DSK) specific binding. Both peptides are found to inhibit the formation of soluble and insoluble F-N-DSK-complexes through inhibition of the interdomain D-E-binding. Gly-Pro-Arg-Pro is much more potent inhibitor than Gly-His-Arg-Pro. Lowering the insoluble F-N-DSK-copolymer quantity by concentration-dependent way these peptides do not change its composition described by the formula [F(N-DSK)2]n. Invariability of fibrinogen and N-DSK copolymer structure is asserted. In this structure neighbouring fibrinogen molecules are bound by two N-DSK molecules via the D1-E1 and D2-E2 binding sites.     

Complexes of the N-terminal disulfide branch point of fibrin with fibrinogen

Investigation of fibrin N-terminal disulphide knot (N-DSK) binding with fibrinogen (F) showed, that the F-N-DSK-complex represents growing polymer structure which is soluble at early polymerization stage and forms a solid phase during the further growth. This complex is characterized by constant stoichiometry expressed by formula F (N-DSK)2. A model of the complex structure as a regular copolymer of fibrinogen and N-DSK is proposed, in which neighbouring fibrinogen molecules are clamped with two N-DSK molecules. Such copolymer was never described. Since its formation is caused by specific D-E interdomain binding, it may be considered as a peculiar analogue of fibrin polymer.     

A unique recognition site mediates the interaction of fibrinogen with the leukocyte integrin Mac-1 (CD11b/CD18)

Mac-1 (CD11b/CD18), a leukocyte-restricted integrin receptor, mediates neutrophil/monocyte adhesion to vascular endothelium and phagocytosis of complement-opsonized particles. Recent studies have shown that Mac-1 also functions as a receptor for fibrinogen in a reaction linked to fibrin deposition on the monocyte surface. In this study, we have used extended proteolytic digestion of fibrinogen to identify the region of this molecule that interacts with Mac-1. We found that an Mr approximately 30,000 plasmic fragment D of fibrinogen (D30) produced dose-dependent inhibition (IC50 = 1.6 microM) of the interaction of intact 125I-fibrinogen with stimulated neutrophils and monocytes. 125I-D30 bound saturably to these cells with specific association of 136,200 +/- 15,000 molecules/cell in a reaction inhibited by OKM1 and M1/70, monoclonal antibodies specific for the alpha subunit of Mac-1. Direct microsequence analysis and an epitope-mapped monoclonal antibody showed that D30 lacks the COOH-terminal dodecapeptide of the gamma chain as well as the Arg-Gly-Asp sequences in the A alpha chain. We conclude that fibrinogen interacts with the leukocyte integrin Mac-1 through a novel recognition site that is not shared with other known integrins that function as fibrinogen receptors.     

Fibrinolytic action of an enzyme preparation conjugated with specific antibodies

The authors have modified alpha-chymotrypsin covalently bound via aldehyde dextran with polyclonal antibodies to fibrinogen. Modification does not lead to any substantial changes in the catalytic properties of the enzymes or to appreciable losses in specific activity of antibodies. At the same time in experimental thrombolysis in vitro, the derivatives obtained exhibit a higher fibrinolytic action as compared with control preparations. It is assumed that the effect observed is accounted for by an increase in tropism of the conjugate obtained to the fibrin clot.     

Fibrinogen and fibrin interaction with concanavalin a dimer and its influence on coagulation

Concanavalin A dimer interacts with fibrinogen and soluble fibrin at pH 5.2 Analysis of the binding data shows that there are in both cases four binding sites per molecule and that the dissociation constant does not change by removal of fibrinopeptides A and B. Ultracentrifugal studies shows that no aggregates of fibrinogen or fibrin are formed through concanavalin A binding and that up to four molecules of concanavalin A dimer can be bind to one molecule of fibrinogen or fibrin. These results imply that the four carbohydrate chains in the molecule are accessible to concanavalin A dimer. There is a diminution in the coagulation of fibrinogen by thrombin at low relative lectin concentrations and an increase at high concentrations. However, the lectin always favours the aggregation of fibrin monomers and does not have any inhibitory effect on the release of fibrinopeptides. We conclude that the electric charge in the neighbourhood of the carbohydrate in both chains, Bβ and γ plays an important role in the attraction between monomeric fibrin and fibrinogen-monomeric fibrin. The different effect of concanavalin A on the coagulation, depending on the relative concentration of the lectin, would be the result of the screening of this electric charge favouring either the interaction of fibrinogen-monomeric fibrin or the polymerization of monomeric fibrin.     

Calix[4]arene methylenebisphosphonic acids as inhibitors of fibrin polymerization

Calix[4]arenes bearing two or four methylenebisphosphonic acid groups at the macrocyclic upper rim have been studied with respect to their effects on fibrin polymerization. The most potent inhibitor proved to be calix[4]arene tetrakis-methylene-bis-phosphonic acid (C-192), in which case the maximum rate of fibrin polymerization in the fibrinogen + thrombin reaction decreased by 50% at concentrations of 0.52 × 10(-6) M (IC(50)). At this concentration, the molar ratio of the compound to fibrinogen was 1.7 : 1. For the case of desAABB fibrin polymerization, the IC(50) was 1.26 × 10(-6) M at a molar ratio of C-192 to fibrin monomer of 4 : 1. Dipropoxycalix[4]arene bis-methylene-bis-phosphonic acid (C-98) inhibited fibrin desAABB polymerization with an IC(50) = 1.31 × 10(-4) M. We hypothesized that C-192 blocks fibrin formation by combining with polymerization site 'A' (Aα17-19), which ordinarily initiates protofibril formation in a 'knob-hole' manner. This suggestion was confirmed by an HPLC assay, which showed a host-guest inclusion complex of C-192 with the synthetic peptide Gly-Pro-Arg-Pro, an analogue of site 'A'. Further confirmation that the inhibitor was acting at the initial step of the reaction was obtained by electron microscopy, with no evidence of protofibril formation being evident. Calixarene C-192 also doubled both the prothrombin time and the activated partial thromboplastin time in normal human blood plasma at concentrations of 7.13 × 10(-5) M and 1.10 × 10(-5) M, respectively. These experiments demonstrate that C-192 is a specific inhibitor of fibrin polymerization and blood coagulation and can be used for the design of a new class of antithrombotic agents.     

Localization of the domains of fibrin involved in binding to platelets

The molecular basis of platelet-fibrin interactions has been investigated by using synthetic peptides as potential inhibitors of fibrin protofibril and fibrinogen binding to ADP-stimulated platelets, adhesion of fibrin fibers to the platelet surface, and platelet-mediated clot retraction. Synthetic peptides of sequence RGDS and HHLGGAKQAGDV, corresponding to regions of the fibrinogen alpha- and gamma-chains previously identified as platelet recognition sites, inhibited the binding of radiolabelled soluble fibrin oligomers to ADP-stimulated platelets with IC50 values of 10 and 40 microM, respectively. Synthetic GPRP and GHRP, corresponding to the N-terminal tripeptide sequence of the fibrin alpha-chains and the tetrapeptide sequence of the beta-chains, respectively, were minimally effective in blocking soluble fibrin polymer binding to ADP-stimulated platelets. Platelet functions which are unique to the three-dimensional fibrin network were examined by measurements of the extent of adhesion of fluorophore-labelled fibrin to platelets with a microfluorimetric technique and by light scattering measurements of the time course of clot retraction. Inhibition of fibrin-platelet adhesion by RGDS, HHLGGAKQAGDV and GHRP exhibited a similar, linear dependence reaching 1/2 maximum at about 200 microM, suggesting nonspecific effects. GPRP inhibited fibrin assembly but did not appear to have specific effects on fibrin-platelet adhesion. Only RGDS effected clot retraction, causing a 4-6-fold decrease in rate at 230 microM. These results indicate that fibrinogen and fibrin protofibrils, which are obligatory intermediates on the fibrin assembly pathway, share a set of common platelet recognition sites located at specific regions of the alpha- and gamma-chains of the multinodular fibrin(ogen) molecules. The RGDS site is also involved in mediating interactions between the three-dimensional fibrin network and stimulated platelets.     

Separation of human plasma fibrin stabilizing factor (factor XIII) from fibrinogen by fibrinogen polymer formation

Human plasma fibrin stabilizing factor (factor XIII) may be separated from fibrinogen through reversible fibrinogen polymer formation at pH 6.6, gamma/2 0.3, 0 degrees C, and subsequent Bio-Gel A 1.5m filtration. Factor XIII activity is eluted after the monomer fibrinogen peak. Polymer fractions from eight preparations, processed in duplicate, contain a mean 0.002 units factor XIII per mg fibrinogen, or about 0.7% the factor XIII content of standard plasma. Factor XIII-free fibrinogen polymers are easily dissociated (greater than 98%) to the monomer form by incubation at 37 degrees C, 18 hours. The fibrinogen preparations utilized were devoid of plasma fibronectin; thus these studies also show that reversible human fibrinogen polymer formation occurs in its absence.     

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

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

Characterization of the inhibition of fibrin assembly by fibrinogen fragment D.

Fragment D (Mr 100 000) prepared from a terminal plasmin digest of fibrinogen was isolated and used to study its effect on fibrin formation. Increasing amounts of fragment D added to a solution of fibrinogen and thrombin decrease the rigidity of the resultant gel (10% of control at 2 mol of fragment D/mol of fibrinogen). Half-maximal inhibition is achieved at 1 mol of fragment D/mol of fibrinogen for non-cross-linked clots and at 1/2 mol of fragment D/mol of fibrinogen for cross-linked clots. "Clottability' decreases concomitantly with the rigidity. Only small amounts of fragment D (less than 10% for non-cross-linked gels) are incorporated into the gel. Light-scattering shows an increase in the final fibre thickness at fragment D concentrations up to 2 mol of fragment D/mol of fibrinogen, from 60 molecules/cross-section for the control to 120 molecules/cross-section. Higher fragment D concentrations lead to a decrease in the final fibre thickness. The limit fibre thickness is 8 nm, with a length of 80 nm, which is equivalent to a fibrin trimer. On the basis of results of synthetic-substrate and fibrinopeptide-release assays, it is clear that thrombin inactivation is not responsible for this effect. These data suggest that fragment D may inhibit fibrin formation by blocking the bimolecular polymerization of activated fibrin monomer molecules to form protofibrils, although additional effects on subsequent assembly steps may also be involved.