Structural analysis of N-linked sugar chains of human blood clotting factor IX

The structures of N-glycans of human blood clotting factor IX were studied. N-Glycans liberated by hydrazinolysis were N-acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine. The pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Each PA-sugar chain was analyzed by two-dimensional sugar mapping combined with glycosidase digestion. The major structures of the N-linked sugar chains of human factor IX were found to be sialotetraantennary and sialotriantennary chains with or without fucose residues. These highly sialylated sugar chains are located on the activation peptide of the protein.     

A rapid method for the isolation of coagulation factor XII from human plasma

Zinc chelate affinity chromatography was used to develop a rapid, three-step procedure to isolate coagulation factor XII from human plasma. The first step was ammonium sulphate fractionction which gave a 2-fold purification and 90% recovery in the 25–50% saturation fraction. The second step was zinc chelate affinity chromatography which gave a 240-fold purification and 67.5% recovery. The third step was zinc chelate affinity chromatography again, but with the application of a pH gradient. The overall recovery of zymogen factor XII was 21.7% and the total purification was 1992-fold. The purified factor XII had an apparent molecular weight of 77 600 as determined by SDS-polyacrylamide gel electrophoresis and a specific activity of 50 units/mg on a clotting assay.     

Injecting skin fibroblasts into muscles to express human clotting factor IX cDNA

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Purification and some characteristics of the coagulation factor IX from human plasma.

Non-activated coagulation factor IX was purified approx. 10,000-fold from human plasma. The final product was electrophoretically homogeneous and comprised a tingle polypeptide chain with a molecular weight of about 70,000 and a pI of 4.3-4.45. The N-terminal amino acid was glycine. The amino acid and the carbohydrate contents were analysed and a monospecific antiserum to the factor was raised in rabbits.     

Calcium and thiol reactivity of human plasma clotting factor XIII

1. The reaction of iodoacetate, 2-chloromercuri-4-nitrophenol and 5,5′-dithiobis-(2-nitrobenzoate) with thrombin-cleaved Factor XIII (i.e. Factor XIII_a) was accompanied by enzyme inhibition. 2. The reaction with iodoacetate and 5,5′-dithiobis-(2-nitrobenzoate) was absolutely dependent on Ca²⁺, and the rate of reaction increased with the Ca²⁺ concentration up to very high, non-physiological concentrations. 3. 2-Chloromercuri-4-nitrophenol reacted with Factor XIII_a in the absence of Ca²⁺, but at a much slower rate. 4. Stopped-flow methods were used to quantify the reaction with 5,5′-dithiobis-(2-nitro-benzoate) because of the Ca²⁺-dependent dissociation of Factor XIII_a (a′₂b₂) and subsequent aggregation of the a′ chains into turbid precipitates. 5. The 3-carboxy-4-nitrothio-phenolate released was consistent with the reaction of 2 thiol groups/molecule of Factor XIII_a. The isolated b chains of Factor XIII did not react with either of the chromophoric reagents. This indicated that the a′ chains of Factor XIII_a were responsible for the thiol reactivity of the enzyme. 6. The Ca²⁺ dependence of the enzyme inhibition by these thiol reagents was very dependent on protein concentration. This is discussed in relation to the Ca²⁺-induced dissociation of Factor XIII_a. 7. The acceptor substrate, casein, decreased the Ca²⁺ concentration required for enzyme inhibition by both the mercurial and the aromatic disulphide compounds. Dansylcadaverine did not affect Ca²⁺ dependence of inhibition.     

Application of semi-industrial monolithic columns for downstream processing of clotting factor IX

It has been shown in a previous study that monolithic columns can be used for downstream processing of different concentrates of clotting factor IX [K. Branovi? et al., J. Chromatogr. A 903 (2000) 21]. This paper demonstrates that such supports are useful tools also at an early stage of the purification process of factor IX from human plasma. Starting with the eluate after solid-phase extraction with DEAE-Sephadex, the use of monolithic columns has allowed much better purification than that achieved with conventional anion-exchange supports. The period of time required for separation is also much reduced. In up-scaling experiments, separations are carried out with 8, 80 and 500 ml columns. A volume of 1830 ml of DEAE-Sephadex eluate, containing a total of 27.6 g of protein and 48500 IU of factor IX is applied to the 500 ml monolithic column. This corresponds to a separation on a pilot scale. The results of this separation after up-scaling are comparable to those obtained with the 8 ml column on a laboratory scale.     

An RNA aptamer that discriminates bovine factor IX from human factor IX

An RNA aptamer has been selected by SELEX against bovine factor IX using an RNA pool containing 74-nucleotides randomized region. Selected RNA aptamer (Clone 5) could discriminate bovine factor IX effectively from human factor IX. Interestingly, the nucleotide regions 73-78 and 80-83 of the selected aptamer were determined to be important for bovine factor IX-binding using phosphate interference. Based on phosphate interference and binding studies the minimal motif for aptamer with discriminating ability is found with the nucleotide regions from 65 to 106. The discriminating ability of this mini aptamer is calculated as more than 1,000 fold. The equilibrium dissociation constant (K(d)) for the above complex was 10 nM as determined by surface plasmon resonance. Based on the available structural informations, probable binding site of aptamer on the target was predicted.     

The isolation of factor V from bovine plasma

1. The activity per unit of chlorophyll of certain carboxylases, and of other enzymes involved in photosynthesis, was determined in leaf extracts of the tropical grasses, sugar-cane, maize and sorghum, and compared with the activities for wheat, oat and silver-beet. Maximum rates of photosynthetic carbon dioxide uptake were also measured for comparison with enzyme activities. 2. Phosphopyruvate carboxylase activity was about 60 times greater in the tropical grasses than in wheat, oat and silver-beet and was severalfold higher than the rates of photosynthetic carbon dioxide uptake. Most of the enzyme was located in the chloroplast fraction of cell extracts. 3. Phosphopyruvate carboxylase was apparently the major photosynthetic carbon dioxide-fixing enzyme in the tropical grasses, although malic enzyme may contribute to a lesser extent. 4. Tropical grasses contained less than one-tenth of the ribulose diphosphate carboxylase activity present in wheat, oat and silver-beet. For the tropical grasses this activity, determined with a saturating concentration of bicarbonate, was approx. 10% of the rate of photosynthesis. 5. The fraction-1 protein content of leaf extracts paralleled the ribulose diphosphate carboxylase activity. 6. In contrast, the activity of several other enzymes of the Calvin cycle was similar in the different species examined.     

Removal and inactivation of viruses during the manufacture of a high-purity antihemophilic factor IX from human plasma

The purpose of this study was to evaluate the efficacy and mechanisms of the solvent/detergent (S/D) treatment, DEAE-toyopearl 650M anion-exchange column chromatography, heparin-sepharose 6FF affinity column chromatography, and Viresolve NFP filtration steps employed in the manufacture of high-purity antihemophilic factor IX (Green-Nine VF) from human plasma, with regard to removal and/or inactivation of blood-borne viruses. A variety of experimental model viruses for human pathogenic viruses, including human immunodeficiency virus (HIV), bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), hepatitis A virus (HAV), murine encephalomyocarditis virus (EMCV), and porcine parvovirus (PPV), were all selected for this study. Samples from relevant stages of the production process were spiked with each virus and subjected to scale-down processes mimicking the manufacture of high-purity factor IX. Samples were collected at each step, immediately titrated using a 50% tissue culture infectious dose (TCID₅₀), and virus reduction factors were evaluated. S/D treatment using the organic solvent, tri (n-butyl) phosphate (TNBP), and the detergent, Tween 80, was a robust and effective step in inactivation of enveloped viruses. Titers of HIV, BHV, and BVDV were reduced from the initial titer of 6.06, 7.72, and 6.92 log₁₀ TCID₅₀, respectively, reaching undetectable levels within 1 min of S/D treatment. DEAE-toyopearl 650M anion-exchange column chromatography was found to be a moderately effective step in the removal of HAV, EMCV, and PPV with log reduction factors of 1.12, 2.67, and 1.38, respectively. Heparin-sepharose 6FF affinity column chromatography was also moderately effective for partitioning BHV, BVDV, HAV, EMCV, and PPV with log reduction factors of 1.55, 1.35, 1.08, 1.19, and 1.61, respectively. The Viresolve NFP filtration step was a robust and effective step in removing all viruses tested, since HIV, BHV, BVDV, HAV, EMCV, and PPV were completely removed during the filtration step with log reduction factors of ≥ 5.51, ≥ 5.76, ≥ 5.18, ≥ 5.34, ≥ 6.13, and ≥ 4.28, respectively. Cumulative log reduction factors of HIV, BHV, BVDV, HAV, EMCV, and PPV were ≥ 10.52, ≥ 12.07, ≥ 10.49, ≥ 7.54, ≥ 9.99, and ≥ 7.24, respectively. These results indicate that the production process for GreenNine VF has a sufficient virus reduction capacity for achievement of a high margin of virus safety.     

Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma

Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull‐down, and OptiPrep^TM density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep^TM density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active.     

Calcium and the assays of human plasma clotting Factor XIII

1. A continuous fluorimetric assay for blood-clotting Factor XIII based on the incorporation of dansylcadaverine into casein was investigated. 2. Hammarsten casein was fractionated to yield the beta-casein, which was dephosphorylated and acetylated to give a substrate which itself did not bind calcium and which was not cross-linked by the activated Factor. 3. The modified beta-casein was used as substrate for a continuous fluorimetric assay and as substrate for the incorporation of radioactive glycine ethylester. 4. A linked fluorimetric assay for the zymogen is described. 5. The K(m) for calcium was redetermined and at 0.2mm was in the physiological range and much lower than the values reported by others using substrates which interact with calcium. 6. The K(m) for casein is about 14mum.     

The synthesis of sulfated dextran beads for isolation of human plasma coagulation factors II,IX, and X

We have prepared a new matrix for the chromatography of coagulation factors using bead-polymerized dextran (Sephadex) sulfated by anhydrous reaction with chlorosulfonic acid. The material is a useful adsorbent for isolation of human plasma coagulation Factors II, IX, and X. Good recoveries (70, 40, and 50% for Factors II, IX, and X, respectively) of proteins homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and >99% free of contaminating activities can be obtained from human plasma in 2 days. This new chromatographic material greatly simplifies the isolation of these coagulation factors.     

Blood clotting factor IX. Loss of activity after cleavage of sialic acid residues

Enzymatic cleavage of sialic acid from human blood clotting factor IX results in a loss of factor IX clotting activity. The loss of clotting activity and the rate of release of sialic acid follow the same time courses. Control experiments have ruled out several explanations for the loss of factor IX activity: proteolytic degradation, inhibitory effects of free sialic acid, and non-specific inhibition of the clotting assays. Furthermore, no inhibition was seen when similar enzymatic cleavage was carried out on factor X and factor VIII. Therefore, we suggest that the loss of factor IX activity is the direct result of cleavage of sialic acid from the protein. Most of the inhibition appeared to be an effect on the activity of factor IXa itself, and thus far, little or no effect has been shown on the activation of factor IX to IXa. The structural basis for this unusual effect of sialic acid on protein function currently is being investigated.     

Blood clotting factor IX Kashihara: amino acid substitution of valine-182 by phenylalanine

Hemophilia B Kashihara is a severe hemorrhagic disorder in which the factor IX antigen is present in normal amounts but factor IX biological activity is markedly reduced. In addition, purified factor IX Kashihara is not activated by purified factor XIa in the presence of calcium ions. Amino acid sequence analysis of one of the tryptic peptides isolated from factor IX Kashihara indicated that Val-182 (equivalent to Val-17 in the chymotrypsin numbering system) had been replaced by Phe. No substitution was found in the members of the catalytic triad His-221, Asp-269, and Ser-365 of factor IX Kashihara. The Val-to-Phe replacement found in factor IX Kashihara appears to sterically hinder the cleavage of Arg 180-Val 181 by factor XIa required for the activation of this zymogen.