Self-assembly of soluble unlinked and cross-linked fibrin oligomers

Self-assembly of soluble unlinked and cross-linked fibrin oligomers formed from desA-fibrin monomer under the influence of factor XIIIa was studied in the presence of non-denaturing urea concentrations. By methods of elastic and dynamic light scattering combined with analytical ultracentrifugation, desA-fibrin oligomers formed in both the presence and absence of the factor XIIIa were shown to be ensembles consisting of soluble rod-like double-stranded protofibrils with diverse weight and size. Unlinked and cross-linked soluble double-stranded protofibrils can reach the length of 350–450 nm. The structure of soluble covalently-linked protofibrils is stabilized by isopeptide γ-dimers. Electrophoretic data indicate a complete absence of isopeptide bonds between α-chains of desA-fibrin molecules. The molecular mechanism of formation of soluble rod-like fibrin structures and specific features of its covalent stabilization under the influence of factor XIIIa are discussed.     

Genes from the medicinal leech (Hirudo medicinalis) coding for unusual enzymes that specifically cleave endo-ɛ(γ-Glu)-Lys isopeptide bonds and help to dissolve blood clots

We previously detected in salivary gland secretions of the medicinal leech (Hirudo medicinalis) a novel enzymatic activity, endo-?(γ-Glu)-Lys isopeptidase, which cleaves isopeptide bonds formed by transglutaminase (Factor XIIIa) between glutamine γ-carboxamide and the ?-amino group of lysine. Such isopeptide bonds, either within or between protein polypeptide chains are formed in many biological processes. However, before we started our work no enzymes were known to be capable of specifically splitting isopeptide bonds in proteins. The isopeptidase activity we detected was specific for isopeptide bonds. The enzyme was termed destabilase. Here we report the first purification of destabilase, part of its amino acid sequence, isolation and sequencing of two related cDNAs derived from the gene family that encodes destabilase proteins, and the detection of isopeptidase activity encoded by one of these cDNAs cloned in a baculovirus expression vector. The deduced mature protein products of these cDNAs contain 115 and 116 amino acid residues, including 14 highly conserved Cys residues, and are formed from precursors containing specific leader peptides. No homologous sequences were found in public databases.     

The Mechanism of Cross-Linking of Fibrinogen and Its Early Structural Homolog—XFragment

We studied the mechanism of the cross-linking of fibrinogen, as well as its closest structural homolog Xfragment, under the influence of a fibronectin-stabilizing factor (factor XIIIa). The data on elastic and dynamic light scattering indicate the formation of single-stranded polymers without any structural rigidity that acquire a ramified and compact structure upon reaching critical mass. The values of coefficients of translational diffusion, mean-mass molecular weight, averaged scattering factor, and the accumulation of -dimers indicate that preincubating of fibrinogen and fragment Xsolutions significantly accelerates the enzymatic formation of a covalently bound macromolecular protein complex. We propose that enzymatic cross-linking proceeds only with the gradual accumulation of structurally imperfect molecules of fibrinogen and fragment Xthat are prone to intermolecular D–Dend-to-end contacts.     

Staphylococcus aureus fibronectin-binding protein serves as a substrate for coagulation factor XIIIa: evidence for factor XIIIa-catalyzed covalent cross-linking to fibronectin and fibrin

In this study, we have investigated the interactions of a Staphylococcal recombinant fibronectin-binding protein A (rFnbA) with fibronectin, fibrinogen, and fibrin. Using analytical size-exclusion chromatography, we evaluated the stoichiometry of reversible binding of FnbA to fibronectin and demonstrated that, in solution, it can accommodate at least two molecules of fibronectin. Results of ELISA experiments demonstrated that rFnbA binds with equally high affinity to both immobilized fibrinogen and fibrin. When included into a thrombin-induced fibrin polymerization reaction, rFnbA strongly inhibited fibrin assembly in a dose-dependent manner. In this study, we have shown that rFnbA can act as a substrate for coagulation factor XIIIa. Factor XIIIa catalyzes the incorporation of amine donor (dansylacadaverine) and amine acceptor (peptide patterned on the N-terminal sequence of fibronectin) synthetic probes into rFnbA, suggesting that it serves as a bifunctional substrate containing reactive glutamine and lysine residues. We have demonstrated that the reversible complex formed by rFnbA and fibronectin or rFnbA and fibrin is covalently stabilized by the transglutaminase action of factor XIIIa. Incubation of rFnbA in the presence of either of its ligands and factor XIIIa results in the introduction of intermolecular epsilon-(gamma-glutamyl)lysine isopeptide bond(s) and the formation of high molecular mass heteropolymers. These findings suggest a novel mechanism by which pathogenic Staphylococcus aureus may utilize the transglutaminase activity of factor XIIIa for attachment to soluble proteins, cell surfaces, and matrixes.     

Post-translational modification of glutamine and lysine residues of HIV-1 aspartyl protease by transglutaminase increases its catalytic activity

The human immunodeficiency virus type 1 aspartyl protease (HIV-1 PR) is a homodimeric aspartyl endopeptidase that is required for virus replication. HIV-1 PR was shown to act invitro as acyl-donor and -acceptor for both guinea pig liver transglutaminase (TG, EC 2.3.2.13) and human Factor XIIIa. These preliminary evidences suggested that the HIV-1 PR contains at least three TG-reactive glutaminyl and one lysyl residues. We report here that the incubation of HIV-1 PR with TG increases its catalytic activity. This increase is dependent upon the time of incubation, the concentration of TG and the presence of Ca²⁺. Identification of ε-(γ-glutamyl)lysine in the proteolytic digest of the TG-modified HIV-1 PR suggested intramolecular covalent cross-linking of this protease which may promote a non-covalent dimerization and subsequent activation of this enzyme via a conformational change. This hypothesis is supported by the observation that the TG-catalyzed activation of HIV-1 PR was completely abolished by spermidine (SPD) which acts as a competitive inhibitor of ε-(γ-glutamyl)lysine formation. Indeed, in the presence of 1 mM SPD the formation of the isopeptide was decreased of about 80%. The main products of the TG-catalyzed modification of HIV-1 PR in the presence of SPD were N₁-mono(γ-glutamyl)SPD and N₈-mono(γ-glutamyl)SPD. Negligible amount of N₁,N₈-bis(γ-glutamyl)SPD were found. The significance of these results is discussed with respect to the activation of the protease by post-translational modification and design of potential inhibitors.     

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.     

epsilon-(gamma-Glutamyl)lysine isopeptide bonds in normal and virus transformed human fibroblasts.

Normal human lung cells (WI-38) possessed 20–40 times more ε-(γ-glutamyl)lysine isopeptide bonds than Simian virus transformed counterparts, WI-38 VA13A and WI-38 VA13-2RA. Normal cells arrested in an essentially nonmitotic state had more isopeptide bonds than proliferating cells. Isopeptide content paralleled the transglutaminase activity of these cells. The results suggest that isopeptide crosslinks contribute to a cellular architecture conducive to a nonproliferating state.     

Gly-Pro-Arg-Pro modifies the glutamine residues in the alpha- and gamma-chains of fibrinogen: inhibition of transglutaminase cross-linking

During blood clotting Factor XIIIa, a transglutaminase, catalyzes the formation of covalent bonds between the epsilon-amino group of lysine and the gamma-carboxamide group of peptide-bound glutamine residues between fibrin molecules. We report that glycyl-L-prolyl-L-arginyl-L-proline (GPRP), a tetrapeptide that binds to the fibrin polymerization sites (D-domain) in fibrin(ogen), inhibits transglutaminase cross-linking by modifying the glutamine residues in the alpha- and gamma-chains of fibrinogen. Purified platelet Factor XIIIa, and tissue transglutaminase from adult bovine aortic endothelial cells were used for the cross-linking studies. Gly-Pro (GP) and Gly-Pro-Gly-Gly (GPGG), peptides which do not bind to fibrinogen, had no effect on transglutaminase cross-linking. GPRP inhibited platelet Factor XIIIa-catalyzed cross-linking between the gamma-chains of the following fibrin(ogen) derivatives: fibrin monomers, fibrinogen and polymerized fibrin fibers. GPRP functioned as a reversible, noncompetitive inhibitor of Factor XIIIa-catalyzed incorporation of [3H]putrescine and [14C]methylamine into fibrinogen and Fragment D1. GPRP did not inhibit 125I-Factor XIIIa binding to polymerized fibrin, demonstrating that the Factor XIIIa binding sites on fibrin were not modified. GPRP also had no effect on Factor XIIIa cross-linking of [3H]putrescine to casein. This demonstrates that GPRP specifically modified the glutamine cross-linking sites in fibrinogen, and had no effect on either Factor XIIIa or the lysine residues in fibrinogen. GPRP also inhibited [14C]putrescine incorporation into the alpha- and gamma-chains of fibrinogen without inhibiting beta-chain incorporation, suggesting that the intermolecular cross-linking sites were selectively affected. Furthermore, GPRP inhibited tissue transglutaminase-catalyzed incorporation of [3H]putrescine into both fibrinogen and Fragment D1, without modifying [3H]putrescine incorporation into casein. GPRP also inhibited intermolecular alpha-alpha-chain cross-linking catalyzed by tissue transglutaminase. This demonstrates that the glutamine residues in the alpha-chains involved in intermolecular cross-linking are modified by GPRP. This is the first demonstration that a molecule binding to the fibrin polymerization sites on the D-domain of fibrinogen modifies the glutamine cross-linking sites on the alpha- and gamma-chains of fibrinogen.     

Oxidized modification of fragments <Emphasis Type="Italic">D</Emphasis> and <Emphasis Type="Italic">E</Emphasis> from fibrinogen induced by ozone

Ozone-induced free-radical oxidation of fragments D and E from fibrinogen has been studied. The methods of elastic and dynamic light scattering in combination with electrophoresis of unreduced samples have shown the acceleration of enzymatic covalent crosslinking of molecules of oxidation-modified fragment D under the action of factor XIIIa. UV and IR spectroscopy shows that free-radical oxidation of amino acid residues of polypeptide chains catalyzed by ozone affects the cyclic and amino groups, giving rise to generation of mainly oxygen-containing products. Comparison of the IR spectra obtained for the oxidation-modified D and E fragments revealed more significant transformation of functional groups for the D fragment. EPR spectroscopy showed that the rotational correlation time of spin labels bound to the ozonized proteins decreased in comparison with the non-ozonized proteins. The rotation correlation time of the radicals covalently bound to the ozonized D and E fragments suggests that D fragment of fibrinogen is more sensitive to free-radical oxidation followed by local structural changes. Possible causes of different degrees of oxidation for fragments D and E are discussed.     

Interactions of human fibrinogens with factor XIII: roles of calcium and the gamma' peptide

Plasma factor XIII is the zymogen of the transglutaminase factor XIIIa. This enzyme catalyzes the formation of isopeptide cross-links between fibrin molecules in nascent blood clots that greatly increase the mechanical stability of clots and their resistance to thrombolytic enzymes. We have characterized the solution interactions of factor XIII with two variants of fibrinogen, the soluble precursor of fibrin. Both the predominant fibrinogen gamma(A)/gamma(A) and the major variant gamma(A)/gamma' form complexes with a 2 fibrinogen:1 factor XIII ratio. The absence of detectable concentrations of 1:1 complexes in equilibrium mixtures containing free factor XIII and 2:1 complexes suggests that this interaction is cooperative. Factor XIII binds fibrinogen gamma(A)/gamma' approximately 20-fold more tightly than fibrinogen gamma(A)/gamma(A), and the interaction with fibrinogen gamma(A)/gamma' (but not fibrinogen gamma(A)/gamma(A)) is accompanied by a significant release of Ca(2+). Taken together, these results suggest that the strikingly anionic gamma' C-terminal sequence contains features that are important for factor XIII binding. Consistent with this notion, a synthetic 20-residue polypeptide containing the gamma' sequence was found to associate with factor XIII in a 2:1 molar ratio and act as an efficient competitor for fibrinogen gamma(A)/gamma' binding.     

The comparative ability of plasma and tissue transglutaminases to use collagen as a substrate

Heat denatured type I and type III calf skin collagen were found to be substrates for guinea pig liver transglutaminase (R-glutaminyl-peptide:amine gamma-glutamyl-yltransferase, EC 2.3.2.13) but not for active plasma factor XIII (factor XIIIa). Liver transglutaminase was shown to catalyse incorporation of 14C-putrescine into subunits of denatured collagen of both types, cross-linking of the latter into high molecular weight polymers and their co-cross-linking to fibrin and fibrinogen. Factor XIIIa is inactive in these respects. None of these reactions was catalysed by liver transglutaminase and plasma factor XIIIa when nondenatured collagens both soluble or in the forms of reconstituted fibrils served as substrates. Some cross-linking of cleavage products of collagen type I (obtained by treatment with collagenase from human neutrophiles) was induced by liver transglutaminase and factor XIIIa. The results indicate that although appropriate glutamine and lysine residues for a epsilon-(gamma-glutamine) lysine cross-linked formation are present in collagen, the native conformation of collagen prevents the action of liver transglutaminase and factor XIIIa.     

Isopeptide bond in collagen- and fibrinogen-binding MSCRAMMs

The internal isopeptide bonds are amide bonds formed autocatalytically between the side chains of Lys and Asn/Asp residues and have been discovered recently. These bonds are well conserved in Gram-positive bacterial pilin proteins and are also observed over a wide range of Gram-positive bacterial surface proteins. The presence of these bonds confers the pilus subunits with remarkable properties in terms of thermal stability and resistance to proteases. Like pili, microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) are also surface proteins found only in Gram-positive bacteria. They specifically interact with the extracellular matrix (ECM) molecules like collagen, fibrinogen, fibronectin, laminin, etc. Many biophysical and biochemical studies have been carried out to characterize the isopeptide bonds in pili proteins from Gram-positive bacteria, but no attempts have been made to study the isopeptide bonds in MSCRAMMs. This short review aims to study the significance of the isopeptide bonds in relation to their function, by analyzing the crystal structures of collagen- and fibrinogen-binding MSCRAMMs. In this analysis, interestingly, we observed that the putative isopeptide bonds are restricted to the collagen-binding MSCRAMMs. Based on analogy with bacterial pilus subunits, we hypothesize that the collagen-binding MSCRAMMs possessing putative isopeptide bonds exhibit similar structural properties, which could help the bacteria in colonizing the host and provide resistance against host–defense mechanisms.     

Effect of fibronectin on fibrinogen conversion into fibrin

The effects of fibronectin on fibrinogen clotting induced by thrombin or reptilase and on fibrin monomer polymerization in a pure system in the absence of factor XIIIa were studied. It was shown that within a broad range of concentrations and molar ratios of the mixed proteins, fibronectin does not alter significantly the fibrinogen clotting time either under thrombin or under reptilase action. The effect of fibronectin on the fibrin self-assembly consists in a slight acceleration of this process, whose degree is directly dependent on the fibronectin/fibrin monomer molar ratio as well as on the absolute fibrin monomer content at a constant molar ratio. The stimulating effect of fibronectin is amplified by Ca2+. The experimental results suggest that fibronectin can noncovalently bind the fibrin monomer and/or intermediate polymers in the non-enzymatic phase of fibrinogen conversion to fibrin.     

Factor XIII-induced crosslinking in solutions of fibrinogen and fibronectin

In solutions containing fibrinogen and fibronectin, factor XIIIa catalyzes the formation of two types of crosslinked polymers: hybrid oligomers consisting of equimolar amounts of fibrinogen and fibronectin, and fibrinogen oligomers. The two types of oligomers are produced in amounts proportional to the starting concentration of fibronectin and fibrinogen in the reaction mixture. Increasing the fibronectin concentration relative to the fibrinogen concentration results in the production of more hybrid and less fibrinogen type oligomers. The lowest molecular weight hybrid oligomer, a dimer, is formed by ligation of one molecule of fibrinogen and fibronectin. The A alpha-chain of fibrinogen and one fibronectin subunit participate in the crosslinking. Larger size hybrid oligomers form by the joining of two hybrid dimers to each other via gamma-chain dimerization in the fibronectin moiety of the dimers. In fibrinogen oligomer formation, fibrinogen molecules are ligated by gamma-chain dimerization in a step-wise fashion producing fibrinogen dimers, trimers, tetramers, etc. without A alpha-chain crosslinking. The hybrid type and the fibrinogen type of oligomer grow in size and eventually become crosslinked to each other yielding large molecular weight complexes that interact to form a gel network.     

Enzymatic and kinetic properties of blood coagulation factor XIIIa and guinea pig liver transglutaminase utilizing (6-[N-(4-aminobutyl)-N-ethylamino]-2,3-dihydrophthalazine-1,4-dione,as a novel,specific and sensitive chemiluminescent substrate

A novel and sensitive chemiluminescent assay is described to quantitate the acyl transfer activities of blood coagulation factor XIIIa or liver transglutaminase using aminobutyl-N-ethyl-isoluminol as acyl acceptor and N,N′-dimethylcasein, human plasma fibrinogen or fibronectin as acyl donors. The method involved covalently linking aminobutyl-N-ethyl-isoluminol through its free amino group with the γ-carboxamide of protein-bound glutamine resulting in an isopeptide bond; a reaction catalysed by both transglutaminase and factor XIIIa. The protein-bound aminobutyl-N-ethyl-isoluminol was separated from non-conjugated amine by precipitation with trichloroacetic acid. The protein–amine conjugate was dissolved in 500 mmol/L NaOH, oxidized using 15 mmol/L ammonium persulphate and light emission quantitated using a luminometer. Optimal conditions were established to detect factor XIIIa and transglutaminase activities with the chemiluminescent assay. Specificity was demonstrated by lack of activity in the presence of ethylenediamine tetra-acetic acid or unactivated factor XIII, or boiled enzymes, and by competitive inhibition with putrescine and 5′-(biotinamido) pentylamine. The enzymatic and kinetic properties of factor XIIIa and transglutaminase in utilizing aminobutyl-N-ethyl-isoluminol as an acyl acceptor substrate were comprehensively documented. The reaction could be carried out in either a purified system or a complex plasma or cell lysates milieu. The assay is sensitive, specific, and eliminates a need for radioactive reagents. The assay could be used to photolabel reactive glutamines in substrates. The assay could also be adapted to a variety of solid- and solution-phase formats and is amenable to X-ray film and/or light photography imaging. © 1998 John Wiley & Sons, Ltd.     

Intramolecular isopeptide but not internal thioester bonds confer proteolytic and significant thermal stability to the S. pyogenes pilus adhesin Spy0125

Streptococcus pyogenes and other Gram‐positive bacterial pathogens present long macromolecular filaments known as pili on their surface that mediate adhesion and colonization. These pili are covalent polymers, assembled by sortases. Typically, they comprise a putative adhesin at their tip, a backbone subunit present in multiple copies and a basal subunit that is covalently anchored to the peptidoglycan layer of the cell surface. The crystal structures of pilin subunits revealed the presence of unusual covalent linkages in these proteins, including intramolecular isopeptide and internal thioester bonds. The intramolecular isopeptide bonds in backbone pilins are important for protein stability. Here, using both the wild‐type protein and a set of mutants, we assessed the proteolytic and thermal stability of the S. pyogenes pilus tip adhesin Spy0125, in the presence and absence of its intramolecular isopeptide and internal thioester bonds. We also determined a crystal structure of the internal thioester bond variant Spy0125^Cys426Ala. We find that mutations in the intramolecular isopeptide bonds compromise the stability of Spy0125. Using limited proteolysis and thermal denaturation assays, we could separate the contribution of each intramolecular isopeptide bond to Spy0125 stability. In contrast, mutation in the internal thioester bond had a lesser effect on protein stability and the crystal structure is essentially identical to wild type. This work suggests that the internal thioester in Spy0125, although having a minor contributory role, is not required for protein stability and must have a different primary function, most likely mediating a covalent interaction with host cell ligands. Proteins 2014; 82:517–527. © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

The Influence of the End Products of Plasmin-Mediated Hydrolysis of Fibrinogen and Fibrin (E F and E f Fragments) on Fibrinogen Cross-linking

We studied the influence of the end products of plasmin-mediated hydrolysis of fibrinogen and nonstabilized fibrin (E _F and E _f fragments) on covalent cross-linking of fibrinogen molecules catalyzed by a fibrin-stabilizing factor (factor XIIIa). The data on elastic and dynamic light scattering reveal no difference in the spatial structure of covalently linked fibrinogen molecules in the presence of the hydrolysis end products E _F and E _f. In contrast to the inactive fragment E _F, fragment E _f significantly accelerates the enzymatic reaction. This is also confirmed by electrophoresis of the reduced samples indicating a relatively fast accumulation of -dimers and A-polymers as compared to the control samples. Possible molecular mechanisms of this effect are discussed.     

Role of subunit crosslinking in fibrin solubility.

¹²⁵I-labelled fibrinogen was clotted by thrombin in the presence of activated Factor XIII and the rates of formation of γ dimers and α polymers were measured. These changes in fibrin subunits were correlated with the solubility of fibrin in 1% monochloroacetic acid. In the presence of the factor XIIIa inhibitor, glycine methyl ester, fibrin solubility was found to depend on the level of α polymers formed. A preferential inhibition of α polymer formation rather than γ dimer was observed in the presence of glycine methyl ester.