Identification and characterization of novel lysine-independent apolipoprotein(a)-binding sites in fibrin(ogen) alphaC-domains

Accumulation of lipoprotein(a) (Lp(a)) in atherosclerotic plaques is mediated through interaction of fibrin-(ogen) deposits with the apolipoprotein(a) (apo(a)) moiety of Lp(a). It was suggested that because apo(a) competes with plasminogen for binding to fibrin, causing inhibition of fibrinolysis, it could also promote atherothrombosis. Because the fibrin(ogen) alphaC-domains bind plasminogen and tissue-type plasminogen activator with high affinity in a Lys-dependent manner, we hypothesized that they could also bind apo(a). To test this hypothesis, we studied the interaction between the recombinant apo(a) A10 isoform and the recombinant alphaC-fragment (Aalpha-(221-610)) corresponding to the alphaC-domain by enzyme-linked immunosorbent assay and surface plasmon resonance. Both methods revealed a high affinity interaction (Kd = 19-21 nm) between the immobilized alphaC-fragment and apo(a), indicating that the former contains an apo(a)-binding site. This affinity was comparable to that of apo(a) for fibrin. At the same time, no interaction was observed between soluble fibrinogen and immobilized apo(a), suggesting that, in the former, this and other apo(a)-binding sites are cryptic. Further experiments with truncated recombinant variants of the alphaC-fragment allowed localization of the apo(a)-binding site to the Aalpha-(392-610) region. The presence of epsilon-aminocaproic acid only slightly inhibited binding of apo(a) to the alphaC-fragment, indicating the Lys-independent nature of their interaction. In agreement, the influence of plasminogen or tissue-type plasminogen activator on binding of apo(a) to the alphaC-fragment was minimal. These results indicate that the alphaC-domains contain novel high affinity apo(a)-binding sites that may provide a Lys-independent mechanism for bringing Lp(a) to places of fibrin deposition such as injured vessels or atherosclerotic lesions.     

Probing interactions between the coagulants thrombin, Factor XIII, and fibrin(ogen)

Thrombin cleaves fibrinopeptides A and B from fibrinogen leading to the formation of a fibrin network that is later covalently crosslinked by Factor XIII (FXIII). Thrombin helps activate FXIII by catalyzing hydrolysis of the FXIII activation peptides (AP). In the current work, the role of exosites in the ternary thrombin-FXIII-fibrin(ogen) complex was further explored. Hydrolysis studies indicate that thrombin predominantly utilizes its active site region to bind extended Factor XIII AP (FXIII AP 33-64 and 28-56) leaving the anion-binding exosites for fibrin(ogen) binding. The presence of fibrin-I leads to improvements in the K(m) for hydrolysis of FXIII AP (28-41), whereas peptides based on the cardioprotective FXIII V34L sequence exhibit less reliance on this cofactor. Surface plasmon resonance measurements reveal that d-Phe-Pro-Arg-chloromethylketone-thrombin binds to fibrinogen faster than to FXIII a(2) and dissociates from fibrinogen more slowly than from FXIII a(2). This system of thrombin exosite interactions with differing affinities promotes efficient clot formation.     

Mechanism of fibrin(ogen) forced unfolding

Fibrinogen, upon enzymatic conversion to monomeric fibrin, provides the building blocks for fibrin polymer, the scaffold of blood clots and thrombi. Little has been known about the force-induced unfolding of fibrin(ogen), even though it is the foundation for the mechanical and rheological properties of fibrin, which are essential for hemostasis. We determined mechanisms and mapped the free energy landscape of the elongation of fibrin(ogen) monomers and oligomers through combined experimental and theoretical studies of the nanomechanical properties of fibrin(ogen), using atomic force microscopy-based single-molecule unfolding and simulations in the experimentally relevant timescale. We have found that mechanical unraveling of fibrin(ogen) is determined by the combined molecular transitions that couple stepwise unfolding of the γ chain nodules and reversible extension-contraction of the α-helical coiled-coil connectors. These findings provide important characteristics of the fibrin(ogen) nanomechanics necessary to understand the molecular origins of fibrin viscoelasticity at the fiber and whole clot levels.     

Force spectroscopy of the fibrin(ogen)-fibrinogen interaction

Fibrin aggregation is of vital importance in many physiological and pathological processes, such as blood coagulation, wound healing, and thrombosis. In the present study, we investigated the forces involved in the initial steps of the fibrinogen fibrin aggregation by force spectroscopy using the atomic force microscope. Our data confirm the existence of strong specific interactions between fibrin and fibrin(ogen), with unbinding forces ranging from 290 to 375 pN and a logarithmic dependence on the loading rate between 0.8 and 23 nN/s.     

Structural organization of the fibrin(ogen) alpha C-domain

We hypothesized that the alpha C-domain of human fibrinogen (residues hA alpha 221-610) and of other species consists of a compact COOH-terminal region (hA alpha 392-610) and a flexible NH(2)-terminal connector region (hA alpha 221-391) which may contain some regular structure [Weisel and Medved (2001) Ann. N.Y. Acad. Sci. 936, 312-327]. To test this hypothesis, we expressed in E. coli recombinant fragments corresponding to the full-length human alpha C-domain and its NH(2)- and COOH-terminal regions as well as their bovine counterparts, bA alpha 224-568, bA alpha 224-373, and bA alpha 374-568(538), respectively, and tested their folding status by fluorescence spectroscopy, circular dichroism (CD), and differential scanning calorimetry (DSC). All three methods revealed heat-induced unfolding transitions in the full-length bA alpha 224-568 and its two COOH-terminal fragments, indicating that the COOH-terminal portion of the bovine alpha C-domain is folded into a compact cooperative structure. Similar results were obtained by CD and DSC with the full-length and the COOH-terminal h392-610 human fragments. The NH(2)-terminal fragments of both species, b224-373 and h221-392, did not exhibit any sign of a compact structure. However, their heat capacity functions, CD spectra, and temperature dependence of ellipticity at 222 nm were typical for peptides in the extended helical poly(L-proline) type II conformation (PPII), suggesting that they contain this type of regular structure. This is consistent with the presence of proline-rich tandem repeats in the sequence of both bovine and human connector regions. These results indicate that both bovine and human fibrinogen alpha C-domains consist of a compact globular cooperative unit attached to the bulk of the molecule by an extended NH(2)-terminal connector region with a PPII conformation.     

纤维蛋白（原）与动脉粥样硬化关系的研究进展

动脉粥样硬化是常见的血管病变,众多研究表明纤维蛋白(原)是动脉粥样硬化的独立危险因素.纤维蛋白(原)能够调节炎性细胞黏附和迁移,使血液处于高凝状态,刺激血管平滑肌细胞增殖和迁移.本文综述纤维蛋白(原)和动脉粥样硬化发病机制之间的关系.     

Novel aspects of fibrin(ogen) fragments during inflammation

Coagulation is fundamental for the confinement of infection and/or the inflammatory response to a limited area. Under pathological inflammatory conditions such as arthritis, multiple sclerosis or sepsis, an uncontrolled activation of the coagulation system contributes to inflammation, microvascular failure and organ dysfunction. Coagulation is initiated by the activation of thrombin, which, in turn, triggers fibrin formation by the release of fibrinopeptides. Fibrin is cleaved by plasmin, resulting in clot lysis and an accompanied generation of fibrin fragments such as D and E fragments. Various coagulation factors, including fibrinogen and/or fibrin [fibrin(ogen)] and also fibrin degradation products, modulate the inflammatory response by affecting leukocyte migration and cytokine production. Fibrin fragments are mostly proinflammatory, however, Bβ15-42 in particular possesses potential antiinflammatory effects. Bβ15-42 inhibits Rho-kinase activation by dissociating Fyn from Rho and, hence prevents stress-induced loss of endothelial barrier function and also leukocyte migration. This article summarizes the state-of-the-art in inflammatory modulation by fibrin(ogen) and fibrin fragments. However, further research is required to gain better understanding of the entire role fibrin fragments play during inflammation and, possibly, disease development.     

New strategies in the determination of fibrin and fibrin(ogen) derivatives by monoclonal antibodies

Until recently only tests with a limited specificity were available for the assessment of the products of activated coagulation and/or fibrinolysis. Those assays were based on polyclonal antibodies, which crossreact with fibrinogen, and as a consequence they were performed on serum samples i.e. after removal of fibrinogen by clotting. Serum preparation, however, is a notorious source of artefactually high or low levels of fibrin(ogen) degradation products, and is not suitable for the determination of coagulation products. Recently, highly specific monoclonal antibodies (MoAb's) have been developed, the majority of which do not crossreact with fibrinogen. This has enabled new strategies to be developed, i.e. assays using these MoAb's on plasma samples. Furthermore, the new assays can discriminate between (individual) fibrin and fibrinogen degradation products, and coagulation products can be assessed in the same plasma samples.     

New colored and fluorescent amine substrates for activated fibrin stabilizing factor (Factor XIIIa) and for transglutaminase

Two fluorescent (FITC and 6-chloro-2-methoxyacridine) and an intensely colored (dabsyl) derivative of cadaverine were synthesized, following earlier work from this laboratory with dansyl-cadaverine, in order to enlarge the scope of possibilities for labeling some gamma-glutamine sites in proteins. Enzyme affinities of the amine substrates for human Factor XIIIa and for guinea pig liver transglutaminase were measured. The utility of dabsylcadaverine was further demonstrated by activity staining of these enzymes, following electrophoresis in agarose, and by measuring the Factor XIII zymogen of human plasma colorimetrically.     

Mapping of a minimal apolipoprotein(a) interaction motif conserved in fibrin(ogen) beta - and gamma -chains

Lipoprotein(a) (Lp(a)) is a major independent risk factor for atherothrombotic disease in humans. The physiological function(s) of Lp(a) as well as the precise mechanism(s) by which high plasma levels of Lp(a) increase risk are unknown. Binding of apolipoprotein(a) (apo(a)) to fibrin(ogen) and other components of the blood clotting cascade has been demonstrated in vitro, but the domains in fibrin(ogen) critical for interaction are undefined. We used apo(a) kringle IV subtypes to screen a human liver cDNA library by the yeast GAL4 two-hybrid interaction trap system. Among positive clones that emerged from the screen, clones were identified as fibrinogen beta- and gamma-chains. Peptide-based pull-down experiments confirmed that the emerging peptide motif, conserved in the carboxyl-terminal globular domains of the fibrinogen beta and gamma modules specifically interacts with apo(a)/Lp(a) in human plasma as well as in cell culture supernatants of HepG2 and Chinese hamster ovary cells, ectopically expressing apo(a)/Lp(a). The influence of lysine in the fibrinogen peptides and of lysine binding sites in apo(a) for the interaction was evaluated by binding experiments with apo(a) mutants and a mutated fibrin(ogen) peptid. This confirmed the lysine binding sites in kringle IV type 10 of apo(a) as the major fibrin(ogen) binding site but also demonstrated lysine-independent interactions.     

Anion-binding exosite of human alpha-thrombin and fibrin(ogen) recognition

Activation of prothrombin to alpha-thrombin generates not only the catalytic site and associated regions but also an independent site (an exosite) which binds anionic substances, such as Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)]. Like human alpha-thrombin with high fibrinogen clotting activity (peak elution at I = 0.40 +/- 0.01 M, pH 7.4, approximately 23 degrees C), catalytically inactivated forms (e.g., i-Pr2P-alpha- and D-Phe-Pro-Arg-CH2-alpha-thrombins) were eluted with only slightly lower salt concentrations (I = 0.36-0.39 M), while gamma-thrombin with very low clotting activity was eluted with much lower concentrations (I = 0.29 M) and the hirudin complex of alpha-thrombin was not retained by the resin. In a similar manner, hirudin complexes of alpha-, i-Pr2P-alpha-, and gamma-thrombin were not retained by nonpolymerized fibrin-agarose resin. Moreover, the ionic strengths for the elution from the CG-50 resin of seven thrombin forms were directly correlated with those from the fibrin resin (y = 0.15 + 0.96x, r = 0.95). In other experiments, the 17 through 27 synthetic peptide of the human fibrinogen A alpha chain was not an inhibitor of alpha-thrombin, while the NH2-terminal disulfide knot (NDSK) fragment was a simple competitive inhibitor of alpha-thrombin with a Ki approximately 3 microM (0.15 M NaCl, pH 7.3, approximately 23 degrees C). These data suggest that alpha-thrombin recognizes fibrin(ogen) by a negatively charged surface, noncontiguous with the A alpha cleavage site but found within the NDSK fragment. Such interaction involving an anion-binding exosite may explain the exceptional specificity of alpha-thrombin for the A alpha cleavage in fibrinogen and alpha-thrombin incorporation into fibrin clots.     

GST-Fbe can recognize beta-chains of fibrin(ogen) on explanted materials

Staphylococcus epidermidis, a coagulase-negative staphylococcus (CoNS), is one of the leading pathogens of nosocomial infections, particularly associated with foreign body infections. Adherence of S. epidermidis to fibrinogen deposited on the surfaces of implants is important for the development of foreign body infections. A gene (fbe) encoding a fibrinogen-binding protein from S. epidermidis (Fbe) was identified by shotgun phage display. A portion of fbe was cloned into a GST-fusion vector. Affinity to glutathione-Sepharose by the GST-tag and affinity to fibrinogen-Sepharose by the Fbe part were applied to purify the recombinant Fbe. The purity and efficacy of the methods used in protein purification was compared. Furthermore, the potential physiological role of Fbe was studied by the interaction between GST-Fbe and components extracted from explanted materials in vitro.     

A recombinant subtilisin with keratinolytic and fibrin(ogen)olytic activity

KerS14 is a keratinase with great potential in tannery, since it degrades keratin without damaging collagen, a feature suitable for various industrial uses. The enzyme was previously characterized and described as a serino endopeptidase belonging to the subtilisin group. However, KerS14 low thermal stability impairs its biotechnological potential. The present work presents several attempts to improve KerS14 thermal stability. KerS14 ORF was cloned into pET-5a vector with a His-tag at C-terminal, and four different mutants enzymes (G61C, S98C, P239R and G61C-S98C) were produced by site-direct mutagenesis. The recombinant enzyme and four mutants were expressed, purified and characterized regarding their thermal stability, optimum temperature and pH. The presence of a His-tag was shown to increase the KerS14 thermal stability, and to decrease the thermal stability of mutant enzymes. In addition, the recombinant enzyme has a remarkable fibrin(ogen)olytic activity. This indicates that the enzyme has a potential for application in cardiovascular diseases, besides its use in tanning as a dehairing agent.     

Interaction of fibrin(ogen) with the endothelial cell receptor VE-cadherin: mapping of the receptor-binding site in the NH2-terminal portions of the fibrin beta chains

Interaction of fibrin with endothelial cells stimulates capillary tube formation thus promoting angiogenesis. This interaction occurs via endothelial cell receptor VE-cadherin and fibrin beta chain 15-42 regions [Bach, T. L., et al. (1998) J. Biol. Chem. 272, 30719-30728]. To clarify the mechanism of this interaction, we expressed in Escherichia coli a number of recombinant fibrin(ogen) fragments containing the beta15-42 region or the VE-cad(1-2) and VE-cad(1-4) fragments encompassing two and four extracellular NH2-terminal domains of VE-cadherin, respectively, and tested interaction between them by surface plasmon resonance and ELISA. Neither the recombinant Bbeta1-57 or Bbeta1-64 fragments, nor beta15-57 or beta15-64 prepared from the latter fragments by thrombin treatment to remove fibrinopeptides B, bound the recombinant VE-cadherin fragments. At the same time, a dimeric recombinant thrombin-treated (beta15-66)2 fragment, which had been disulfide-linked via Cys65 to mimic the dimeric arrangement of the beta chains in fibrin, bound VE-cad(1-4) well, but not VE-cad(1-2); no binding was observed with the untreated (Bbeta1-66)2 dimer. We next mutated several residues in the dimer, His16, Arg17, Pro18, and Asp20, and tested the interaction of the thrombin-treated mutants with VE-cad(1-4) by ligand blotting and surface plasmon resonance. No binding was observed with the H16A and R17Q single mutants and the H16P, P18V double mutant while the P18A and D20N single mutants bound VE-cad(1-4) with the same affinity as the thrombin-treated wild-type dimer. These results indicate that the VE-cadherin binding site in fibrin includes NH2-terminal regions of both fibrin beta-chains, that His16 and Arg17 are critical for the binding, and that the third and/or fourth extracellular domains of VE-cadherin are required for the binding to occur.     

Differential splicing of thymosin beta 4 mRNA

A cDNA clone was isolated from a mouse pre-B cell line, the sequence of which has a very high homology with rat and human thymosin beta 4 genes. However, the mouse clone has an insertion of 98 bp relative to the published rat and human sequences upstream of the coding region. By isolation of a second set of clones from a different cDNA library and by cloning a PCR amplified region of mouse genomic DNA it was confirmed that the insertion is not a cloning artifact. Furthermore, it was shown by RNase protection assays with RNA from the pre-B cell line that two sizes of thymosin beta 4 mRNA exist, a long form containing the 98 nucleotide insertion, and a short form that corresponds to the known rat and human mRNA. The short form is about 50 times more abundant than the long form. Analysis of genomic DNA by sequencing and Southern blotting revealed that both forms are encoded by a single gene in the mouse. The two forms of mRNA arise by differential RNA splicing; the long mRNA contains three separate exons, whereas the short mRNA is missing exon 2. The long mRNA is present in two different pre-B cell lines, spleen and thymus, but could not be detected in brain, liver, and kidney. It is possible that the longer mRNA, which encodes a hydrophobic NH2-extension of six additional amino acids, plays a role in lymphocyte function or development. In contrast to the mouse which has a single thymosin beta 4 gene, rat and human have multiple homologs. Most or all of these also contain sequences that cross-hybridize with the newly discovered exon 2. A polymorphic thymosin beta 4 gene has been found in human DNA.     

Biotechnological production of acetylated thymosin beta4

Thymosin beta4 (43 aa) is a highly conserved acidic peptide which regulates actin polymerization in mammalian cells by sequestering globular actin. Thymosin beta4 is undergoing clinical trials as a drug for the treatment of venous stasis ulcers, corneal wounds and injuries, as well as acute myocardial infarction. Currently, thymosin beta4 is produced with solid-phase chemical synthesis. Biotechnological synthesis of this peptide presents difficulties because N-terminal amino acid residue of thymosin beta4 is acetylated. In this study we propose a method for producing the recombinant precursor of thymosin beta4 and its subsequent targeted chemical acetylation. Desacetylthymosin beta4 was synthesized as a part of a hybrid protein with thioredoxin and a specific TEV (tobacco etch virus) protease cleavage site. The following scheme was developed for the purification of desacetylthymosin beta4: (i) the biosynthesis of a soluble hybrid protein (HP) in Escherichia coli; (ii) isolation of the HP by ion exchange chromatography; (iii) cleavage of the HP with TEVprotease; (iv) purification of desacetylthymosin beta4 by ultra-filtration. N-terminal acetylation of desacetylthymosin beta4 was performed with acetic anhydride under acidic conditions (pH 3). The reaction yield was 55%. Thymosin beta4 was then purified by reverse-phase high performance liquid chromatography. The proposed synthetic approach to recombinant thymosin beta4 is suitable for scale-up and can provide for the medical use of highly purified preparation with a yield of 20 mg from 1 L of culture.     

Polymerisation of chemically cross-linked actin:thymosin beta(4) complex to filamentous actin: alteration in helical parameters and visualisation of thymosin beta(4) binding on F-actin.

The beta-thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin beta(4) with F-actin. Thymosin beta(4) at 200 microM was chemically cross-linked to F-actin. In the presence of phalloidin, the chemically cross-linked actin:thymosin beta(4) complex was incorporated into F-actin. These mixed filaments were of normal appearance when inspected by conventional transmission electron microscopy after negative staining. We purified the chemically cross-linked actin:thymosin beta(4) complex, which polymerised only when phalloidin and the gelsolin:2-actin complex were present simultaneously. Using scanning transmission electron microscopy, the mass-per-length of control and actin:thymosin beta(4) filaments was found to be 16.0(+/-0.8) kDa/nm and 18.0(+/-0.9) kDa/nm, respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis of the helical parameters revealed an increase of the crossover spacing of the two right-handed long-pitch helical strands from 36.0 to 40.5 nm. Difference map analysis of 3-D helical reconstruction of control and actin:thymosin beta(4) filaments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible with published data of the atomic structure of thymosin beta(4). The deduced binding sites of thymosin beta(4) to actin were in agreement with those identified previously. However, parts of the difference map might represent subtle conformational changes of both proteins occurring upon complex formation.     

Lack of effect of thrombin on fibrin(ogen)-endothelial cell interaction

In the present study we investigate the fibrin(ogen)-endothelial cell binding and the effect of thrombin on the endothelial cells in relation to fibrin(ogen) binding capacity. Endothelial cell fibrinogen binding was concentration and time-dependent, reaching saturation at 1.4 M of added ligand. At equilibrium, the number of fibrinogen molecules bound per endothelial cell in the monolayer was 5.8±0.7×10⁶. When endothelial cells were activated by different concentrations of thrombin (0–0.1 NIH units ml^–1), no increase in fibrinogen binding capacity was observed at all the thrombin concentration tested. Whereas disruption of endothelial cell monolayers was observed at thrombin concentrations higher than 0.05 NIH units ml^–1, no increase in the amount of fibrinogen bound was observed. Therefore, resting and thrombin-activated endothelial cells show the same fibrinogen binding capacity.The adhesion of endothelial cells in suspension on immobilized fibrinogen or fibrin was studied to ascertain whether the behavior of fibrin is similar to that of fibrinogen. The extent of endothelial cell attachment to immobilized fibrinogen and fibrin was similar (4275±130 cells cm^–2 for fibrinogen and 4350±235 cells cm^–2 for fibrin) and represent approximately 40% of the added endothelial cells. However, endothelial cell adhesion to immobilized fibrin was significantly faster than endothelial cell adhesion to immobilized fibrinogen. The maximum binding rate was 66±9 and 46±8 cells cm^–2 min^–1 for fibrin and fibrinogen, respectively. Therefore, the fibrinopeptides released by thrombin from fibrinogen induce qualitative changes which enhance the fibrin interaction with the endothelial cells.