Effect of lectin-binding to fibrinogen D and E domains on coagulation and fibrinolysis

The effect on fibrinogen coagulation and fibrinolysis of the mannose-specific lectins concanavalin A, its acetyl derivative and Lens culinaris agglutinin was studied. Concanavalin A and acetyl-concanavalin A, which bind to the four carbohydrate chains of fibrinogen, and L. culinaris agglutinin, which only binds to the carbohydrate present in fibrinogen D domains, has the same effect on the coagulation rate: an inhibition at low lectin concentrations and an increase at high concentrations. On the other hand, L. culinaris agglutinin does not alter fibrin crosslinking while acetyl-concanavalin A produces a slight inhibition of both gamma-gamma and alpha-polymer formation. However, this effect is very small when compared with the clear inhibitory effect produced by concanavalin A. Concanavalin A and acetyl-concanavalin A have an inhibitory effect on the rate of fibrin clot lysis proportional to the lectin concentration. Nearly 100% inhibition was obtained when two lectin-binding sites were occupied by either concanavalin A or acetyl-concanavalin A. However, L. culinaris agglutinin has a clearly weaker effect and more than 50% inhibition was not observed. The comparative study of the effect of the three lectins on fibrinolysis as well as on the formation of fibrinogen aggregates suggests that the inhibitory effect of concanavalin A and acetyl-concanavalin A is primarily due to their binding to the carbohydrate chains of fibrinogen E domain.     

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.     

Differential binding of mannose-specific lectins to the carbohydrate chains of fibrinogen domains D and E

The interaction of fibrinogen with the mannose-specific lectins concanavalin A (ConA), its acetyl derivative (Ac-ConA) and Lens culinaris agglutinin (LcH) was studied. Both ConA and LcH interact specifically with individual fibrinogen B beta and gamma chains and with denatured fragments D and E. However, analysis of the binding data shows that four moles of Ac-ConA are bound per mole of fibrinogen with two sets of binding sites (Kd1 = 2.4 microM and Kd2 = 16.6 microM; n1 = n2 = 2) while only two moles of LcH are bound per mole of fibrinogen (Kd = 2.6 microM). Ultracentrifugation studies are also in agreement with the presence in the fibrinogen molecule of two and four binding sites for LcH and Ac-ConA, respectively. No aggregates of fibrinogen formed through LcH or Ac-ConA linkages are observed. The use of a crosslinking reagent and ultracentrifugal analysis of the lectin-fibrinogen fragments D1 and E complexes indicated that ConA, as well as Ac-ConA, interact with both fragments D and E while LcH interacts only with fragment D. Furthermore, the binding of ConA to both D and E domains in the intact fibrinogen molecule is clearly demonstrated by using a bifunctional reagent. The bivalent character of ConA tetramers may be misinterpreted as a lack of accessibility of the lectin to two of the four carbohydrate chains of fibrinogen. The differential binding of LcH and ConA to the carbohydrate chains of fibrinogen can be related to a different exposure of the oligosaccharide in D and E fragments and domains and to the different requirements of both lectins for their binding to glycoproteins.     

Effect of methylmercuric chloride (MMC) on fibrin polymerization

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

Preparation of fibrin des-AA by thrombin

The active thrombin is formed in the blood stream when the blood coagulation system is activated. It attacks fibrinogen, splits off two fibrinopeptides A and fibrinogen is transformed into des-AA fibrin which is able to polymerize spontaneously forming protofibrils. At high thrombin concentration the enzyme splits off two fibrinopeptides B and des-AA fibrin units are transformed into des-AABB fibrin. These two forms of fibrin are widely used in the biological experiments. However des-AA fibrin is obtained usually from fibrinogen using the snake poisons (such as reptilase). Des-AA fibrin was obtained also by physiological enzyme thrombin, but that des-AA fibrin samples had the contamination of des-AABB fibrin. At the present paper we have described the method of the des-AA fibrin preparation by thrombin without any contamination of des-AABB fibrin.     

Localization of a fibrin polymerization site

The formation of a fibrin clot is initiated after the proteolytic cleavage of fibrinogen by thrombin. The enzyme removes fibrinopeptides A and B and generates fibrin monomer which spontaneously polymerizes. Polymerization appears to occur though the interaction of complementary binding sites on the NH2-terminal and COOH-terminal (Fragment D) regions of the molecule. A peptide has been isolated from the gamma chain remnant of fibrinogen Fragment D1 which has the ability to bind to the NH2-terminal region of fibrinogen as well as to inhibit fibrin monomer polymerization. The peptide reduces the maximum rate and extent of the polymerization of thrombin or batroxobin fibrin monomer and increases the lag time. The D1 peptide does not interact with disulfide knot, fibrinogen, or Fragment D1, but it binds to thrombin-treated disulfide knot with a Kd of 1.45 X 10(-6) M at approximately two binding sites per molecule of disulfide knot. Fibrin monomer formed either by thrombin or batroxobin binds approximately two molecules of D1 peptide per molecule of fibrin monomer, indicating that the complementary site is revealed by the loss of fibrinopeptide A. The NH2-terminal sequence (Thr-Arg-Trp) and COOH-terminal sequence (Ala-Gly-Asp-Val) of the D1 peptide were determined. Therefore the gamma 373-410 region of fibrinogen contains a polymerization site which is complementary to the thrombin-activated site on the NH2-terminal region of fibrinogen.     

Comparison of structures of various human fibrinogens and a derivative thereof by a study of the kinetics of release of fibrinopeptides

The kinetics of the thrombin-induced release of fibrinopeptides from several variants of human fibrinogen, and from the plasmin digestion fragment E thereof, have been studied by using an HPLC technique to separate the reaction products. The data were analyzed in terms of a Michaelis-Menten mechanism in which the A alpha and B beta chains compete for thrombin. Phosphorylation of Ser-3 of the A alpha chain appears to increase the rate of release of the corresponding phosphorylated peptide A from fibrinogen, due to enhanced binding of thrombin (lower value of the Michaelis-Menten constant KM). However, phosphorylation does not affect the rate of release of the unphosphorylated A or B peptides. Increase in the length of the gamma chain (at the C-terminus) does not affect the rate of release of any of the fibrinopeptides. The rate of release of the A peptide from fragment E (which is devoid of the B peptide) is similar to that for the complete fibrinogen molecule. These results are in agreement with an earlier conclusion [Martinelli, R. A., & Scheraga, H. A. (1980) Biochemistry 19, 2343] that the A alpha and B beta chains behave independently in their competition for thrombin; i.e., the hydrolyzable Arg-Gly bonds of the A alpha and B beta chains are both accessible to thrombin.     

Characterization of the kinetic pathway for liberation of fibrinopeptides during assembly of fibrin

The time dependence of the release of fibrinopeptides from fibrinogen was studied as a function of the concentration of fibrinogen, thrombin, and Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The release of fibrinopeptides during fibrin assembly was shown to be a highly ordered process. Rate constants for individual steps in the formation of fibrin were evaluated at pH 7.4, 37 degrees C, gamma/2 = 0.15. The initial event, thrombin-catalyzed proteolysis at Arg-A alpha 16 to release fibrinopeptide A (kcat/Km = 1.09 X 10(7) M-1s-1) was followed by association of the resulting fibrin I monomers. Association of fibrin I was found to be a reversible process with rate constants of 1 X 10(6) M-1s-1 and 0.064 s-1 for association and dissociation, respectively. Assuming random polymerization of fibrin I monomer, the equilibrium constant for fibrin I association (1.56 X 10(7) M-1) indicates that greater than 80% of the fibrin I protofibrils should contain more than 10 monomeric units at 37 degrees C, pH 7.4, when the fibrin I concentration is 1.0 mg/ml. Association of fibrin I monomers was shown to result in a 6.5-fold increase in the susceptibility of Arg-B beta 14 to thrombin-mediated proteolysis. The 6.5-fold increase in the observed specificity constant from 6.5 X 10(5) M-1s-1 to 4.2 X 10(6) M-1s-1 upon association of fibrin I monomers and the rate constant for fibrin association indicates that most of the fibrinopeptide B is released after association of fibrin I monomers. The interaction between a pair of polymerization sites in fibrin I dimer was found to be weaker than the interaction of fibrin I with Gly-Pro-Arg-Pro and weaker than the interaction of fibrin I with fibrinogen.     

Direct evidence for specific interactions of the fibrinogen alphaC-domains with the central E region and with each other

The carboxyl-terminal regions of the fibrinogen Aalpha chains (alphaC regions) form compact alphaC-domains tethered to the bulk of the molecule with flexible alphaC-connectors. It was hypothesized that in fibrinogen two alphaC-domains interact intramolecularly with each other and with the central E region preferentially through its N-termini of Bbeta chains and that removal of fibrinopeptides A and B upon fibrin assembly results in dissociation of the alphaC regions and their switch to intermolecular interactions. To test this hypothesis, we studied the interactions of the recombinant alphaC region (Aalpha221-610 fragment) and its subfragments, alphaC-connector (Aalpha221-391) and alphaC-domain (Aalpha392-610), between each other and with the recombinant (Bbeta1-66)2 and (beta15-66)2 fragments and NDSK corresponding to the fibrin(ogen) central E region, using laser tweezers-based force spectroscopy. The alphaC-domain, but not the alphaC-connector, bound to NDSK, which contains fibrinopeptides A and B, and less frequently to desA-NDSK and (Bbeta1-66)2 containing only fibrinopeptides B; it was poorly reactive with desAB-NDSK and (beta15-66)2 both lacking fibrinopeptide B. The interactions of the alphaC-domains with each other and with the alphaC-connector were also observed, although they were weaker and heterogeneous in strength. These results provide the first direct evidence for the interaction between the alphaC-domains and the central E region through fibrinopeptide B, in agreement with the hypothesis given above, and indicate that fibrinopeptide A is also involved. They also confirm the hypothesized homomeric interactions between the alphaC-domains and display their interaction with the alphaC-connectors, which may contribute to covalent cross-linking of alpha polymers in fibrin.     

Distribution of receptor sites on large glycoprotein molecules by electron microscopy. Application to epiglycanin.

Electron microscopy was used to map the loci of immunochemically active sites on individual glycoprotein molecules. The positions of specific galactose residues and asparagine-linked carbohydrate chains containing specific mannose residues in epiglycanin, a glycoprotein of extended conformation from the surface of TA3 mouse mammary tumour cells, were observed in complexes with Ricinus communis toxin and concanavalin A respectively. The maximum number of Ricinus communis toxin molecules attached to a single epiglycanin molecule was 23, and the average number was 16. Only one concanavalin A molecule was observed attached to any epiglycanin molecule, and this at one end of the molecule, suggesting the presence of only one receptor for this lectin. By means of this new approach for mapping specific residues, evidence has been obtained that suggests microheterogeneity in epiglycanin with respect to the locations of carbohydrate chains containing receptors for Ricinus communis toxin.     

Examining thrombin hydrolysis of the factor XIII activation peptide segment leads to a proposal for explaining the cardioprotective effects observed with the factor XIII V34L mutation

In the blood coagulation cascade, thrombin cleaves fibrinopeptides A and B from fibrinogen revealing sites for fibrin polymerization that lead to insoluble clot formation. Factor XIII stabilizes this clot by catalyzing the formation of intermolecular cross-links in the fibrin network. Thrombin activates the Factor XIII a(2) dimer by cleaving the Factor XIII activation peptide segment at the Arg(37)-Gly(38) peptide bond. Using a high performance liquid chromatography assay, the kinetic constants K(m), k(cat), and k(cat)/K(m) were determined for thrombin hydrolysis of fibrinogen Aalpha-(7-20), Factor XIII activation peptide-(28-41), and Factor XIII activation peptide-(28-41) with a Val(34) to Leu substitution. This Val to Leu mutation has been correlated with protection from myocardial infarction. In the absence of fibrin, the Factor XIII activation peptide-(28-41) exhibits a 10-fold lower k(cat)/K(m) value than fibrinogen Aalpha-(7-20). With the Factor XIII V34L mutation, decreases in K(m) and increases in k(cat) produce a 6-fold increase in k(cat)/K(m) relative to the wild-type Factor XIII sequence. A review of the x-ray crystal structures of known substrates and inhibitors of thrombin leads to a hypothesis that the new Leu generates a peptide with more extensive interactions with the surface of thrombin. As a result, the Factor XIII V34L is proposed to be susceptible to wasteful conversion of zymogen to activated enzyme. Premature depletion may provide cardioprotective effects.     

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.     

Fibrin assembly after fibrinopeptide A release in model systems and human plasma studied with magnetic birefringence.

Magnetically induced birefringence was used to monitor fibrin polymerization after the release of the small negatively charged A fibrinopeptides from human fibrinogen by the action of the snake-venom-derived enzymes reptilase and ancrod. A range of conditions was investigated. Fibrin polymerization in solutions of purified fibrinogen shows a distinct break near the gelation point. On addition of Ca2+ or albumin the lag period is shortened, fibre thickness is increased and the break in assembly almost vanishes, probably because both of these additives promote lateral aggregation. There are minor differences in the kinetics, depending on the venom enzyme used. The kinetics of fibrin assembly in model systems containing either Ca2+ or albumin and in human plasma with a largely dormant coagulation cascade are very similar. Therefore in the latter condition there is no significant alteration in the assembly process due to interaction between fibrin or the venom enzymes and any of the plasma proteins. When the cascade is activated, the polymerization progress curves have a character that resembles a combination of the reactions observed when the venom enzymes and endogenously generated thrombin separately induce coagulation, except for a region near gelation where, paradoxically, polymerization appears to be slower on activation. The low-angle neutron-diffraction patterns from oriented gels made with thrombin or reptilase are identical. Therefore at low resolution the packing of the monomers within fibres is the same when fibrinopeptide A only or both fibrinopeptides A and B are removed.     

Binding of human fibroblast interferon to concanavalin A-agarose. Involvement of carbohydrate recognition and hydrophobic interaction.

Human fibroblast interferon binds to a concanavalin A-agarose (Con A-Sepharose) equilibrated with methyl alpha-D-mannopyranoside, or levan; in contrast, it is only partially retarded on a similar column equilibrated with ethylene glycol. Interferon does not bind, however, to a lectin column equilibrated with both methyl alpha-D-mannopyranoside and ethylene glycol. Thus, a hydrophobic interaction between fibroblast interferon and the immobilized lectin seems to account for a large portion of the binding forces involved. Other hydrophobic solutes, such as dioxane, 1, 2-propanediol, and tetraethylammonium chloride, were found equally or more efficient than ethylene glycol in displacing interferon from the lectin column. The elution pattern of interferon from a concanavalin A-agarose (Con A-Sepharose) column, at a constant ehtylene glycol concentration and with an increasing mannoside concentration, reveals the existence of four distinct interferon components. The selective adsorption to and elution from a concanavalin A-agarose (Con A-Sepharose) column resulted in about a 3000-fold purification of human fibroblast interferon and complete recovery of activity. The specific activity of the partially purified interferon preparation is about 5 X 10(7) units per mg of protein. The chromatographic behavior of human leukocyte interferon is remarkable in that it does not bind to concanavalin A-agarose at all indicating the absence of carbohydrate moieties recognizable by the lectin, or if present, their masked status. When concanavalin A was coupled to an agarose matrix (cyanogen bromide activated) at pH 8.0 and 6.0 human fibroblast interferon bound to both lectin-agarose adsorbents and could be recovered with methyl alpha-D-mannopyranoside. Concanavalin A, immobilized directly on agarose matrix at pH 8.0 and 6.0, thus displays only carbohydrate recognition toward interferon. By contrast, unless a hydrophobic solute was included in the solvent containing methyl mannoside, human fibroblast interferon could not be recovered from concanavalin A-agarose coupled at pH 9.0. When concanavalin A was immobilized via molecular arms, in tetrameric as well as dimeric forms, the binding of interferon again occurred exclusively through carbohydrate recognition. Thus, the hydrophobic interaction can be eliminated by appropriate immobilization of the lectin, and then adsorbed glycoproteins, as exemplified here by interferon, can be recovered readily with methyl mannoside alone.     

The proteolytic action of Arvin on human fibrinogen

1. Human fibrinogen was subjected to proteolysis by enzyme preparations (clinical Arvin and IRC-50 Arvin) from the venom of Agkistrodon rhodostoma. 2. IRC-50 Arvin releases three peptides from fibrinogen, and these were identified as fibrinopeptides AP, AY and A. 3. The less purified ;clinical' Arvin releases, in addition to fibrinopeptides AP, AY and A, small amounts of two heptapeptides derived from fibrinopeptides AP and A, probably because it contains another enzyme as well as Arvin. 4. No fibrinopeptide B is released by either Arvin preparation. 5. Thus, although Arvin is known to differ from ;reptilase' from Bothrops jararaca in that it does not activate the enzyme that cross-links fibrin (fibrin-stabilizing factor), it is identical with reptilase with respect to the peptides that it liberates from fibrinogen.     

Lamprey fibrinopeptide B is A glycopeptide

One of the peptides released from lamprey fibrinogen during its transformation into fibrin has been found to contain covalently bound carbohydrate. The peptide, which also contains tyrosine O-sulfate, corresponds to the mammalian fibrinopeptide B and is the amino-terminal segment of the lamprey fibrinogen β-chain. As noted previously, this peptide is the only one released when lamprey fibrinogen is coltted by mammalian thrombin. Of the more than fifty sets of fibrinopeptides characterized from various species, this is the first one found to contain carbohydrate.     

Localization of the binding site on fibrin for the secondary binding site of thrombin

Affinity chromatography of active site inhibited thrombin on immobilized fragments derived from the central (desAB-NDSK) and terminal (D1) globular domains of fibrinogen revealed that the site responsible for the binding of thrombin at its secondary fibrin binding site is located in the central domain. Chromatography of various domains of the central nodule (desAB-NDSK, fibrinogen E, and fibrin E) having nonidentical amino acid sequences showed that all of these fragments are capable of binding to PMSF-thrombin-Sepharose, suggesting that the thrombin binding site resides within the peptide regions common to all of these fragments: alpha(Gly17-Met51), beta(Val55-Met118), and gamma(Tyr1-Lys53). Competitive affinity chromatography of the same binding domains revealed that there is no detectable difference in their binding constants to PMSF-thrombin-Sepharose, indicating that the alpha(Lys52-Lys78), beta(Gly15-Lys54)/(Tyr119-Lys122), and gamma(Thr54-Met78) peptide segments do not contribute significantly to the binding of thrombin. Chromatography of the isolated chains of fibrinogen E showed that the alpha(Gly17-Lys78) peptide region itself contains a strong binding site for PMSF-thrombin-Sepharose. The location of the binding site suggests that the secondary site interaction may play an important role in determining the cleavage specificity of thrombin on fibrinogen and can affect the rate of release of the fibrinopeptides. Affinity chromatography of fragments prepared from polymerized fibrin showed that cross-linked DD (D x D) itself does not bind to thrombin, whereas the D x DE complex remained attached to the column, suggesting that the binding site on fragment E for thrombin is distinct from its binding site for D x D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of native chicken fibrinogen at 2.7 A resolution

The crystal structure of native chicken fibrinogen (320 kDa) complexed with two synthetic peptides has been determined at a resolution of 2.7 A. The structure provides the first atomic-resolution view of the polypeptide chain arrangement in the central domain where the two halves of the molecule are joined, as well as of a putative thrombin-binding site. The amino-terminal segments of the alpha and beta chains, including fibrinopeptides A and B, are not visible in electron density maps, however, and must be highly disordered. The alphaC domain is also very disordered. A residue by residue analysis of the coiled coils with regard to temperature factor shows a strong correlation between mobility and plasmin attack sites. It is concluded that structural flexibility is an inherent feature of fibrinogen that plays a key role in both its conversion to fibrin and its subsequent destruction by plasmin.     

Interaction of a peptide inhibitor with two forms of monomeric fibrin differing in the degree of activation

The study is devoted to the interaction of peptide inhibitor of fibrin self-assemblage with two forms of fibrin monomer: deprived of fibrinopeptides A and preserving fibrinopeptides B (desAA-monomer) and fully activated (desAABB-monomer). It is shown that peptide inhibitor hinders the coagulative conversion of fibrinogen by thrombin, limiting the enzymic and nonenzymic stages of the process. The indispensible condition for the formation of inhibitor-monomeric fibrin associates is a preliminary modification of fibrinogen by thrombin.     

The chemical characterization of favin, a lectin isolated from Vicia faba.

We have determined the subunit structure of the glucose- and mannose-binding lectin favin, from Vicia faba. The molecule is composed of two nonidentical polypeptide chains held together by noncovalent interactions. We have determined the complete amino acid sequence of the smaller alpha chain (Mr = 5,571) and shown that it is homologous to the alpha chain of the lectins from lentil and pea and to residues 72 to 120 of concanavalin A (Con A). The larger beta chain (Mr = 20,000) contains carbohydrate and is homologous to the beta chain of lentil, pea, soybean, peanut, and red kidney bean lectins and is homologous to a portion of the Con A molecule beginning at residue 122. Favin also contains a minor component, beta' (Mr = 18,700), that closely resembles the beta chain but lacks carbohydrate and may, on the basis of apparent molecular weight, lack some part of the COOH-terminal region of the polypeptide chain. Although favin is similar to Con A, it, like the lentil and pea lectins, appears to lack residues corresponding to positions 1 to 71 of Con A. Because these residues contribute significantly to the carbohydrate binding site of Con A, the lack of this region in the otherwise homologous lectin favin suggests that the carbohydrate binding site of favin differs from that of Con A or that the region represented by residues 1 to 71 of Con A is located in a different portion (i.e. in the beta chain) of the favin molecule.