Interaction between fibrinogen and insulin-like growth factor-binding protein-1 in human plasma under physiological conditions

Fibrinogen is a plasma glycoprotein and one of the principle participants in blood coagulation. It interacts with many proteins during formation of a blood clot, including insulin-like growth factors (IGFs) and their binding proteins (IGFBP). Fibrinogen complexes were found as minor fractions in fibrinogen preparations independently of the coagulation process, and their presence influences the kinetics of polymerization. The idea of this work was to investigate whether fibrinogen in human plasma interacts with IGFBPs independently of the tissue injury or coagulation process. The results have shown that fibrinogen forms complexes with IGFBP-1 under physiological conditions. Several experimental approaches have confirmed that complexes are co-isolated with fibrinogen from plasma, they are relatively stable, and they appear as a general feature of human plasma. Several other experiments excluded the possibility that alpha-2 macroglobulin/IGFBP-1 complexes or IGFBP-1 oligomers contributed to IGFBP-1 immunoreactivity. The role of fibrinogen/IGFBP-1 complexes is still unknown. Further investigation in individuals expressing both impaired glucose control and coagulopathy could contribute to identification and understanding of their possible physiological role.     

Isolation of Bothrops jararaca Snake Antithrombin from the Supernatant of Fibrinogen Purification

A novel method of antithrombin (AT) purification from Bothrops jararaca snake plasma was developed to obtain this protein using a waste supernatant from B. jararaca fibrinogen purification. The AT purification was achieved by affinity chromatography on HiTrap Heparin HP. The results showed an efficient purification process yielding pure AT (purity 65-fold and specific activity 368.91). In conclusion, we showed a feasible purification method of AT from B. jararaca plasma using a discarded material. This feature is important, considering the limitation of material, such as snake plasma, and could also be useful to obtain pure plasma proteins from other animals, including human plasma.     

Bothrops jararaca fibrinogen and its resistance to hydrolysis evoked by snake venoms

Fibrinogen is an essential protein involved in several steps of hemostasis, being associated with the final steps of the blood coagulation mechanism. Herein, we describe the purification and characterization of a reptile fibrinogen, obtained from Bothrops jararaca plasma. Native B. jararaca fibrinogen showed a molecular mass of 372 kDa, and the reduced and alkylated fibrinogen molecule showed three chains of 71, 60 and 55 kDa, which are similar to the molecular masses of human and bovine Aα, Bβ and γ fibrinogen chains. Remarkably, B. jararaca fibrinogen was clotted by bovine thrombin, but B. jararaca, Crotalus durissus terrificus and Lachesis muta rhombeata venoms could not induce its clotting or hydrolysis. Thus, despite the similarities between B. jararaca and mammalian fibrinogens, the former shows distinctive features, which protect B. jararaca snakes from accidental envenomation.     

The role of the carbohydrate moiety in the biologic properties of fibrinogen

The carbohydrate moiety of some glycoproteins influences their secretion and functional properties. We have examined the importance of the oligosaccharide chains of fibrinogen in this regard. Fibrinogen was labeled de novo by the addition to rabbit hepatocyte monolayer cultures of either 3H-amino-acids or [2-3H] mannose, in the presence or absence of tunicamycin, a potent inhibitor of glycosylation. Inhibition of glycosylation, which ranged from 75 to 80%, was determined by incorporation of [2-3H]mannose as quantitated by gel filtration. Synthesis and secretion of fibrinogen were quantitated by 3H-amino-acid incorporation, using anti-fibrinogen immunoaffinity column chromatography of medium and cell homogenates. Tunicamycin did not appreciably inhibit fibrinogen synthesis, as compared to a 30-40% inhibition of overall protein synthesis, determined by incorporation of 3H-amino-acids into trichloroacetic acid-precipitable material. There was no evidence that secretion of fibrinogen was impaired. Fibrinogen from medium was copurified by adding cold plasma fibrinogen as carrier. Nonglycosylated fibrinogen was found to be functional as demonstrated by incorporation of radioactivity into clots of the copurified material at a rate identical to that of glycosylated fibrinogen. When clotted in the presence of Ca2+ and Factor XIII, cross-linking of glycosylated and nonglycosylated fibrin was demonstrable on fluorography of sodium dodecyl sulfate-polyacrylamide gels, showing disappearance of gamma-chain and appearance of gamma-gamma-dimers.     

Ozone-induced damage of fibrinogen molecules: identification of oxidation sites by high-resolution mass spectrometry

Fibrinogen is highly susceptible to oxidation compared to other plasma proteins. Fibrinogen oxidation damages its structure and affects the protein function. Ozone-induced oxidative modifications of the fibrinogen Aα, Bβ, and γ polypeptide chains upon addition of various amounts of the oxidiser were studied by mass spectrometry. Amino acid residues located on all three chains and main structural parts of the protein were revealed to be involved in oxidation. The αC-connector was shown to be most vulnerable to oxidation as compared to other structural parts while the E region turned out to be the most protected area of the protein. For the first time, it was established that numerous amino acid residues responsible for the conversion of fibrinogen to fibrin remain unaffected upon fibrinogen oxidation. The data obtained in this study indicate that none of the identified residues, which are considered crucial for the binding of both hole “a” and hole “b” to knob “A” and knob “B”, respectively, as well as those responsible for the thrombin binding to fibrinogen E region, have been subjected to chemical alterations under moderate oxidation. The data on fibrinogen oxidation acquired in the current study enable one to assume that some of the structural fibrinogen parts and easily oxidisable residues could be endowed with antioxidant properties. New findings presented here could be essential for the detection of adaptive molecular mechanisms capable of mitigating the detrimental action of reactive oxygen species (ROS) on the functioning of oxidatively damaged fibrinogen. Data are available via ProteomeXchange with identifier PXD012046.

• Highlights

• Various oxidative modifications were detected in fibrinogen by mass spectrometry

• αC-connector has been shown to be most susceptible to oxidation

• E region proved to be least vulnerable to the action of the oxidising agent

• Some of the Met residues in the fibrinogen structure could operate as ROS scavengers

     

Recombinant human fibrinogen expressed in the yeast Pichia pastoris was assembled and biologically active

Fibrinogen is a large plasma glycoprotein with a molecular mass of 340 kDa that plays a critical role in the final stage of blood coagulation. Human plasma fibrinogen is a dimeric molecule comprising two sets of three different polypeptides (Aα, 66 kDa; Bβ, 55 kDa; γ, 48 kDa). To express recombinant human fibrinogen in the methylotrophic yeast Pichia pastoris, we constructed an expression vector containing three individual fibrinogen chain cDNAs under the control of the mutated AOX2 (mAOX2) promoter. First, P. pastoris GTS115 was transformed with the vector, but the expressed recombinant fibrinogen suffered severe degradation by yeast-derived proteases under conventional nutrient culture conditions. Fibrinogen degradation was prevented by using the protease A-deficient strain SMD1168 as a host strain and regulating the pH of the culture to between 5.5 and 7.0. Western blot analysis revealed that the Aα, Bβ and γ chains of recombinant fibrinogen were assembled and secreted as a complete molecule. The Bβ chain of the recombinant fibrinogen was N-glycosylated but the Aα chain, as in plasma fibrinogen, was not. The γ chains however were heterologous, one being N-glycosylated and the other not. The recombinant fibrinogen was capable of forming a thrombin-induced clot in the presence of factor XIIIa and both the glycosylated and the non-glycosylated γ chains were involved in the formation of cross-linking fibrin. The present study indicates that the recombinant fibrinogen expressed in P. pastoris, although different from plasma fibrinogen in post-translational modification, is correctly assembled and biologically active.     

Pro-inflammatory effect of fibrinogen and FDP on vascular smooth muscle cells by IL-6, TNF-α and iNOS

AimsAtherosclerosis is a chronic inflammatory response of the arterial wall to multiple endothelial injuries. As one of the inflammatory markers, fibrinogen has been implicated in pathogenesis of atherosclerosis. But, it is not completely understood whether atherogenesis of fibrinogen is related to its pro-inflammatory effect on vascular smooth muscle cells (VSMCs). The purpose of the present study was to observe effects of fibrinogen and fibrin degradation products (FDP) on interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and inducible nitric oxide synthase (iNOS) generation in rat VSMCs.Main methodsRat VSMCs were cultured, and fibrinogen and FDP were used as stimulants for IL-6, TNF-α, and iNOS. IL-6 and TNF-α level in the supernatant were measured by ELISA, mRNA expression of IL-6, TNF-α, and iNOS were assayed with RT-PCR, and protein expression of iNOS was detected with western blot and immunocytochemistry.Key findingsFibrinogen and FDP both significantly stimulated mRNA and protein expressions of IL-6, TNF-α and iNOS in VSMCs in time- and concentration-dependent ways. The pro-inflammatory potency of FDP is higher than fibrinogen, which seems to mean that smaller fragments of the protein have greater pro-inflammatory activity. Fibrinogen and FDP promote more protein expressions of IL-6 and TNF-α compared to iNOS, suggesting that fibrinogen and FDP produce a pro-inflammatory effect on VSMCs mainly by IL-6 and TNF-α.SignificanceThese findings are helpful to better understand pro-inflammatory effect of fibrinogen on VSMCs involved in atherogenesis, and imply a therapeutic strategy targeting hyperfibrinogenemia in atherosclerosis.     

Semi-Automated Hydrophobic Interaction Chromatography Column Scouting Used in the Two-Step Purification of Recombinant Green Fluorescent Protein

Background

Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported.

Methods and Results

Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A₃₉₅) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation.

Conclusions

Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in development other of HIC-compatible protein purification schemes.     

Overexpression,Refolding, Purification of Erbin PDZ Domain from Escherichia coli and Preparation of its Polyclonal Antibody

Abstract

The Erbin was recently identified. The antibody against Erbin has not been commercially available. As a new member of peripheral protein LAP family and novel type of adaptor protein, its functions and binding partners are not completely known. In the present study, cDNA encoding PDZ domain of Erbin was inserted in a prokaryotic expression vector. His-tagged recombinant protein was overproduced in E. coli and purified by Ni-NTA column chromatography. About 14.4 mg of the purified protein was obtained from 500 mL of cell culture. The purity of the recombinant protein was higher than 90%. The polyclonal antibody against this protein was raised. The antibody can recognize both denatured and natural Erbin protein. It will be used to further identify the new binding partners of Erbin and study its unknown functions.     

Purification of glucoamylase by acarbose (BAY g-5421) affinity chromatography

Aspergillus niger and Rhizopus sp. glucoamylases were purified on an affinity chromatography column from commercially available, impure enzyme preparations. Up to 2 mg of glucoamylase protein was bound without leakage to a 1-ml affinity gel column (0.7 X 2.5 cm) possessing a covalently linked acarbose ligand (1 mg acarbose/g wet gel), and the bound enzyme was specifically released by irrigation of the column with a solution of maltose. A complete cycle of purification was accomplished in about 8 h. Glucoamylases were recovered, in more than 80% yield, free of alpha-amylase activity and possessing specific activities comparable to those of preparations obtained by time-consuming, multistep procedures involving several ion-exchange and hydrophobic column fractionations. Thus, acarbose affinity chromatography provides a general method for the rapid and efficient purification of the glucoamylases, and seems to be ideally suited for scale-up for the commercial purification of these enzymes.     

Functional impact of oxidative posttranslational modifications on fibrinogen and fibrin clots

Fibrinogen is a circulating multifunctional plasma protein vital for hemostasis. Activation of the coagulation cascade converts soluble fibrinogen to insoluble polymerized fibrin, which, along with platelets, forms the hemostatic clot. However, inappropriate formation of fibrin clots may result in arterial and venous thrombotic disorders that may progress to life-threatening adverse events. Often thrombotic disorders are associated with inflammation and the production of oxidants. Fibrinogen represents a potential target for oxidants, and several oxidative posttranslational modifications that influence fibrinogen structure and function have been associated with disease pathogenesis. Here, we review various oxidative modifications of fibrinogen and the consequences of these modifications on protein structure and the ability to form fibrin and how the resulting alterations affect fibrin architecture and viscoelastic and biochemical properties that may contribute to disease.     

Reactive carbonyl compounds (RCCs) cause aggregation and dysfunction of fibrinogen

Fibrinogen is a key protein involved in coagulation and its deposition on blood vessel walls plays an important role in the pathology of atherosclerosis. Although the causes of fibrinogen (fibrin) deposition have been studied in depth, little is known about the relationship between fibrinogen deposition and reactive carbonyl compounds (RCCs), compounds which are produced and released into the blood and react with plasma protein especially under conditions of oxidative stress and inflammation. Here, we investigated the effect of glycolaldehyde on the activity and deposit ion of fibrinogen compared with the common RCCs acrolein, methylglyoxal, glyoxal and malondialdehyde. At the same concentration (1 mmol/L), glycolaldehyde and acrolein had a stronger suppressive effect on fibrinogen activation than the other three RCCs. Fibrinogen aggregated when it was respectively incubated with glycolaldehyde and the other RCCs, as demonstrated by SDS-PAGE, electron microscopy and intrinsic fluorescence intensity measurements. Staining with Congo Red showed that glycolaldehyde- and acroleinfibrinogen distinctly formed amyloid-like aggregations. Furthermore, the five RCCs, particularly glycolaldehyde and acrolein, delayed human plasma coagulation. Only glycolaldehyde showed a markedly suppressive effect on fibrinogenesis, none did the other four RCCs when their physiological blood concentrations were employyed, respectively. Taken together, it is glycolaldehyde that suppresses fibrinogenesis and induces protein aggregation most effectively, suggesting a putative pathological process for fibrinogen (fibrin) deposition in the blood.     

A solid-phase radioimmunoassay for fibrinogen.

A solid-phase radioimmunoassay for fibrinogen has been developed utilizing [¹⁴C]-methylated fibrinogen as standard antigen and fibrinogen-specific antibodies covalently linked to Sepharose. Fibrinogen was [¹⁴C]-methylated by reductive alkylation using [¹⁴C] formaldehyde and sodium borohydride. The methylated fibrinogen was unaltered in clotting ability and antigenicity.The assay, an isotope dilution assay, is quantitative for picomole amounts of fibrinogen. It is specific for fibrinogen in homologous plasma and in the presence of a variety of other proteins.     

Purification of factor X by hydrophobic interaction chromatography

Human factor X has been purified to homogeneity by hydrophobic interaction chromatography on phenyl-sepharose. The coagulation protein did not interact with the resin in the presence of 2–3 M NaCl whereas contaminants were retained. This single purification step, in conjunction with classical purification strategies, is a powerful tool in generating high purity factor X and is based on resins which are readily available.     

Purification of Human α2-Antiplasmin with Chicken IgY Specific to Its Carboxy-Terminal Peptide

ABSTRACT

α₂-antiplasmin, a plasma glycoprotein of the serpin superfamily, is the primary physiological inhibitor of plasmin, the key enzyme in fibrin degradation. Previous purification methods utilize lengthy multistep protocols with low yields or use monoclonal antibodies that are expensive or difficult to make. With a relatively small investment, a chicken was immunized with keyhole limpet hemocyanin-conjugated to α₂-antiplasmin C-terminal 26 residue synthetic peptide and the peptide-specific antibody (IgY) was isolated from the egg yolks of hens using the peptide affinity column. Based on the interaction between this IgY and α₂-antiplasmin, pure α₂-antiplasmin was isolated from human plasma in two steps: (a) citrated plasma was precipitated with 15% PEG-8000 to remove the bulk of plasma proteins while retaining the majority of α₂-antiplasmin activity; and (b) the α₂-antiplasmin was affinity-purified from the supernatant using the IgY column. Yields were typically 48% and the purity and authenticity of the α₂-antiplasmin were verified by gel electrophoresis, Western Blot analysis, N-terminal sequence, and amino acid analysis.     

Increased thrombin-induced polymerization of fibrinogen associated with high protein carbonyl levels in plasma from patients post myocardial infarction

Increased levels of protein carbonyls have been measured in plasma of patients following a myocardial infarction (Mocatta et al. J. Am. Coll. Cardiol. 49:1993–2000; 2007). In this study, we have examined representative plasma samples from this group of patients. We show that carbonyls are formed preferentially on fibrinogen and that there is a strong correlation between fibrinogen and total plasma protein carbonyls. Functional properties of fibrinogen isolated from post myocardial plasmas were investigated by measuring thrombin-catalyzed polymerization. Fibrinogen from plasma with upper quartile protein carbonyls (mean 0.16 nmol/mg protein) polymerized ～1.4 times more rapidly and gave 1.4-fold higher maximum turbidity (12 per group, P < 0.001) than fibrinogen from lower quartile carbonyl plasma (mean 0.007 nmol/mg), which behaved similarly to control plasma. Significant differences were also apparent when related to the carbonyl content of the fibrinogen itself. These changes in the high carbonyl plasma reflect a faster rate of lateral aggregation of small oligomers to form fibrin polymers that comprise thicker, more loosely woven fibers. In vivo this could be translated into a tendency to clot faster and form more fragile clots. High protein carbonyls in fibrinogen were not associated with an increased content of nitrotyrosine or chlorotyrosine. Nitrotyrosine levels were significantly lower in fibrinogen than total plasma protein, with no difference between patients and controls. Chlorotyrosine levels in fibrinogen (but not total protein) were significantly higher for the post myocardial patients. Our data suggest that high fibrinogen protein carbonyls in heart disease could be a prothrombotic risk factor.     

Purification of cytochrome c peroxidase for monitoring H2O2 production

One of the most precise methods of determining hydrogen peroxide (H₂O₂) formation by biological systems is based on measuring the rate of enzyme-substrate complex formation between H₂O₂ and cytochrome c peroxidase (CCP). The main problem with this method is that CCP is not commercially available and has to be prepared in the laboratory. We have modified some currently available methods for purifying a highly active preparation of CCP in about 4 d. It includes a batch extraction of protein using DEAE-sepharose followed by concentration either by lyophilization or by passing the extract through a small DEAE-sepharose column instead of by ultrafiltration. The concentrated preparation is passed through a Sephadex G-75 column and the final CCP crystallized against water. The final preparations had a purity index (PI, ratio of absorbance at 408 nm/280 nm, equivalent to heme/protein ratio) above 1.2. These changes make the overall procedure very simple, preserving enzyme activity and spectral properties. In addition, we point out that special care has to be taken to eliminate cytochrome c from crude CCP extracts. Cytochrome c not only introduces an artifact when determining PI, but is also may act as a hydrogen donor for CCP when monitoring H₂O₂ formation, thus decreasing the sensitivity of this method.     

Purification of antibody against Ara h 2 by a homemade immunoaffinity chromatography column

Antibodies are used extensively in numerous applications both in vivo and in vitro. To purify anti-Ara h 2 polyclonal antibody, a homemade immunoaffinity chromatography (IAC) column method was established. The properties of homemade column were compared with those of the mAb affinity protein G (MPG) agarose high flow, a commercially available column successfully used in capturing polyclonal antibodies. During antibody purification from rabbits’ antiserum against Ara h 2, the column capacity, recovery, and purification factor were characterized for IAC and MPG. The homemade IAC could separate the corresponding antibody with higher specificity and lower cost but with lower recovery and column capacity than those of MPG. Thus, the homemade IAC is a specific, inexpensive, and suitable method that can be used for various laboratory purifications.     

ETRAP (efficient trapping and purification) of target protein polyclonal antibodies from GST-protein immune sera

Recombinant GST (glutathione transferase) proteins are widely used as immunogens to generate polyclonal antibodies. Advantages of using GST proteins include: commercially available cloning vectors, vast literature for protein expression in Escherichia coli, the ease of protein purification, immunogen can be used as an ELISA standard and GST can be removed in some systems. However, there are disadvantages: GST oligomerization, inclusion body formation and target protein insolubility after GST removal. Perhaps the most detrimental is the significant generation of anti-GST antibodies by the host animal. A two-column procedure using a glutathione-GST column and a glutathione-(GST-protein) column can yield affinity-purified anti-(GST-protein) polyclonal antibody. Several passes over the first column are often required, though, to completely extract the anti-GST antibodies from the immune sera. We reasoned that knowledge of the target protein linear epitope(s) would allow construction of a peptide affinity resin for a single-pass 'one and done' purification termed ETRAP (efficient trapping and purification). In the present paper, we describe our efforts and present data on rabbits and sheep immunized with GST proteins having target protein molecular masses of ~8, 21 and 33?kDa. The titre and purity of the target antibodies using the ETRAP protocol were comparable to the more laborious multi-column purifications but with a considerable saving in time.     

Malabarase,a serine protease with anticoagulant activity from Trimeresurus malabaricus venom

In the present study we describe the purification and characterization of Malabarase, a serine protease from Trimeresurus malabaricus venom. Purification was achieved by gel-permeation chromatography on Sephadex G-75 followed by ion-exchange chromatography on CM Sephadex C-25. Homogeneity of Malabarase was confirmed by RP-HPLC. Malabarase is a monomer that migrated as a single protein band on SDS-PAGE under both reducing and non-reducing conditions. The molecular mass of Malabarase was determined to be 23.4 kDa using MALDI-TOF mass spectrometry. Malabarase is the first serine protease purified from T. malabaricus venom and is selective for fibrinogen. Malabarase hydrolyzes Aα and Bβ but not γ-chains of fibrinogen similar to the metalloproteases, Malabarin and Trimarin, isolated from the same venom. However, the action of Malabarase on plasma coagulation is opposite than those of Malabarin, Trimarin and the whole venom. Malabarase significantly prolonged plasma coagulation time from 152–341 s; whereas Malabarin, Trimarin, and whole venom, greatly reduce plasma clotting time from 152 to 12, 48, and 14 s, respectively. Malabarase did not show hemorrhagic or myotoxic activity. In contrast, Malabarin, Trimarin and whole venom are highly hemorrhagic and myotoxic. These observations support the specificity of Malabarase towards fibrinogen and its non-toxic nature. In conclusion, Malabarase is a fibrinogen-specific, anti-coagulant, and non-toxic serine protease. Its selective action and non-toxic nature might make it useful for treating thrombotic disorders.