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M Ezban

Publications and source records attributed to M Ezban.

51 records · Page 3Linked to original sources
PubMed1994

Characterization of chemically crosslinked human factor VIIIa.

The activity of factor VIIIa was enhanced and stabilized by treatment of factor VIII with the crosslinker disuccinimidyl suberate. The activity was > 200-fold higher compared with that of native factor VIIIa and was stable for at least 15 days at 4 degrees C and pH 7.2. The crosslinked factor VIIIa was purified by immunoaffinity chromatography and gel filtration. Electrophoretic analysis revealed high-molecular-mass (approximately 150 kDa) molecules as well as the three bands characteristic of native factor VIIIa. Thus crosslinking appeared to yield molecules stabilized by intra- and/or inter-subunit crosslinks. The material was further fractionated using immobilized von Willebrand factor and the factor VIIIa activity could be ascribed to trimers containing only intra-subunit crosslinks. Moreover, reduction of crosslinked factor VIIIa produced using dithiobis(succinimidylpropionate) suggested that the molecules containing intersubunit crosslinks had not been cleaved by thrombin at arginine 372.

Chromatography, Affinity↗
PubMed1991

Heterogeneity in the tyrosine sulfation of Chinese hamster ovary cell produced recombinant FVIII.

By the use of recombinant technology, several stable Chinese hamster ovary (CHO) cell lines expressing human FVIII were established. Thrombin treatment and SDS-PAGE analysis of the purified recombinant FVIII (rFVIII) revealed a striking difference from plasma-derived FVIII (pFVIII). A 43-kDa fragment of the FVIII heavy chain appears as a double band from rFVIII, while a single band from pFVIII is observed. All other fragments from the two samples appeared similar by SDS-PAGE. The heterogeneity is caused by incomplete tyrosine sulfation of one or more of the three potential tyrosine sulfation sites (Tyr718, Tyr719, Tyr723). To investigate if there is a general limitation and heterogeneity in the tyrosine sulfation of rFVIII, two other potential tyrosine sulfation sites on the FVIII light chain (Tyr1664, Tyr1680) were analyzed. The results show that both sites on the pFVIII light chain and on the rFVIII light chain are completely sulfated. The limitation of CHO cells to tyrosine sulfate rFVIII is therefore only restricted to a few sites. The two sulfated forms of rFVIII can easily be separated by ion-exchange chromatography, indicating the importance of the sulfate groups on the charge and/or conformation of FVIII. Both forms of rFVIII possess identical in vitro coagulation activity, von Willebrand factor binding, and thrombin activation profile. However, the difference in tyrosine sulfation may change other biological properties of FVIII.

Animals↗
PubMed1988

Generation of active coagulation factor VIII from isolated subunits.

Factor VIII-light chain (FVIII-LC) and Factor VIII-heavy chain (FVIII-HC) were isolated separately from human plasma and were without coagulation activity. When FVIII-LC and FVIII-HC preparations were mixed, coagulation activity was generated in the presence of Mn2+, Ca2+, or Co2+. Mn2+ was most effective and with Ca2+ maximal activity was first obtained after 8 days. Bovine Factor X (FX) accelerated recombination and was able to increase the amount of FVIII:C generated up to 10-fold when FVIII-LC/HC were incubated with Ca2+ for 20 h. When recombination was performed in the presence of micromolar concentrations of sulfhydryl reagents, the total amount of FVIII:C generated was increased up to 4-fold and in excess of FVIII-HC it was possible to drive 70% of FVIII-LC into active complex. If FVIII-HC was prepared in the presence of a sulfhydryl reagent it was possible to drive 75% of FVIII-HC into active complex with FVIII-LC in excess. Me2+, which is necessary for recombination, catalyzes disulfide formation, and it is proposed that FVIII subunits have free sulfhydryl groups. The presence of sulfhydryl reagents during purification of FVIII-HC and during recombination retains the individual subunits in a conformation suitable for recombination.

Calcium↗
PubMed1988

Radioimmunoassay for quantitative measurement of factor VIII-heavy chain.

An immunoassay has been developed for the measurement of factor VIII heavy chain (FVIII-HC). IgG from a human inhibitor plasma with specificity for FVIII-HC and FVIII-light chain (FVIII-LC) was coated to microplates with loose wells. After washing, the plates were incubated with sample and after another wash 125I-FVIII-HC was added so that the amount of bound tracer was dependent on the amount of FVIII-HC in sample. When EDTA was included in the assay buffer the assay response was increased 3-fold for normal human plasma. This indicated that the antibody was reactive with a determinant hidden in the FVIII-HC/FVIII-LC complex as EDTA splits the complex. The sensitivity of the assay was 0.004 units/ml (1 unit/ml in normal human plasma pool). Together with a previously published assay for FVIII-LC it has now become possible to measure the relative amounts of FVIII-HC and FVIII-LC in haemophilia A plasma and to study the separate expression of FVIII-HC and FVIII-LC by recombinant DNA technology. Patients with severe haemophilia A had FVIII-HC levels below 0.01 units/ml. However, inhibitor patients in high dose FVIII-treatment showed up to 50 times higher levels of FVIII-HC than FVIII-LC and FVIII:C, indicating the presence of FVIII/anti-FVIII-LC immune complexes. Thus, dependent on assay specificity plasma samples can show very variable content of FVIII:antigen.

Factor VIII↗
PubMed1987

F VIII subunits: purification and antigenic properties.

Factor VIII-Light Chain (FVIII-LC) and FVIII-Heavy Chain (FVIII-HC) were purified from human plasma by the use of immunosorbents containing monoclonal antibodies or human inhibitor antibodies. FVIII-LC was subsequently isolated in essentially pure state by cation exchange chromatography. The preparations obtained contained 50 ng of protein for each unit of FVIII-LC antigen (FVIII-LC:Ag). Affinity purified FVIII-LC and FVIII-HC preparations containing less than 0.3% of the opposite subunit were added in FVIII:C inhibition assay of hemophilia A inhibitor antibodies. FVIII-LC was able to fully block the inhibitor activity in 6 out of 7 hemophilia A plasmas and partially block the inhibitor activity of one plasma. FVIII-HC only blocked FVIII:C inhibiting antibodies form the plasma that was not fully blocked by FVIII-LC. It is suggested that FVIII-LC can be used for immunotherapy of the patients whose FVIII:C inhibiting antibodies are directed towards FVIII-LC. When FVIII-LC was coupled to Sepharose at a concentration of 4800 units of FVIII-LC:Ag per ml Sepharose, 0.2 ml of the immunosorbent was able to bind 900 Bethesda units from 100 ml hemophilia A inhibitor plasma. This opens the possibility to remove FVIII inhibitor antibodies from circulation by extracorporeal immunotherapy with FVIII-LC coupled to Sepharose.

Antibody Affinity↗
PubMed1986

The functional domains of coagulation factor VIII:C.

A lack of factor VIII:C, manifested as a bleeding disorder due to the absence of clot formation, is known as hemophilia A, an X chromosome-linked inherited disease afflicting 1-2 males/10,000. To determine the minimum functional domain(s) essential for factor VIII:C activity, we have expressed the amino-terminal (92-kDa) and carboxyl-terminal (80-kDa) proteolytic cleavage products as individual, secreted polypeptides in monkey cells without the 909-residue central region. We have found that neither terminal domain alone is able to promote coagulation in factor VIII:C-deficient plasma. However, when the 92- and 80-kDa peptides are co-expressed, clotting activity is readily detected. Thus, these two chains alone constitute an active or activatable complex. The central domain is required neither for activity nor for the assembly of an active complex from two chains expressed in trans. These results suggest that a truncated derivative of factor VIII:C may be useful in coagulation therapy.

Amino Acid Sequence↗
PubMed1986

[Adjusted rates of the incidence of mental disorders in 2 populations in general clinical practice].

The present study compares the rates of mental disorder prevalence by means of a direct adjustment method applied in two populations with different socio-demographic characteristics. One, from a private-general hospital with prevalence rates ranging from 25.96% to 40.02% and the other from a health center with rates ranging from 43.1% to 59.1%. In both studies the methodology and the instruments used were similar. Ecological studies have shown that socio-demographic differences exert an important influence on the prevalence. That is the reason for applying a direct adjustment method of rates prevalence to correct the effect of differences from structural population. The results obtained after the rate adjustment reduced considerably such differences.

Adolescent↗
PubMed1985

Specificity of monoclonal antibodies to factor VIII:C.

Three monoclonal antibodies (42 IgG, 47 IgG, 56 IgG) towards factor-VIII:C (VIII:C) have been produced. In ELISA for VIII:C-antigen (VIII:CAg), 47 IgG showed higher affinity for VIII:CAg than 42 IgG and 56 IgG. In solid phase immunoisolation of iodinated VIII:C diluted in EDTA buffer, the three monoclonals, like human VIII:C inhibitors, bound the 77/80 kD-light chain of VIII:C. In the absence of EDTA, 56 IgG bound the heavy chain-light chain complex of VIII:C, while 47 IgG was only able to bind the light chain. When coupled on Sepharose, 56 IgG adsorbed coagulation active VIII:C, while 47 IgG was only able to adsorb coagulation inactive VIII:CAg. In coagulation assay 56 IgG inhibited with 20 BU/mg while 42 IgG and 47 IgG inhibited with 4 BU/mg. A mixture of 42 IgG and 56 IgG showed a synergistic effect and inhibited with 50 BU/mg total IgG. In radioimmunoassay a human VIII:C inhibitor was able to inhibit the VIII:C binding of 42 IgG and 56 IgG but not of 47 IgG. The monoclonals did not inhibit each other. On the contrary, 56 IgG increased the binding of 42 IgG to VIII:C.

Antibodies, Monoclonal↗
PubMed1985

Reactivity of factor VIII inhibitors in a micro ELISA for factor VIII: CAg and in solid phase immunoisolation of VIII: CAg.

Factor VIII coagulant antigen (VIII: CAg) was measured in a sandwich-ELISA. Microplates were used as solid phase and peroxidase conjugated F(ab')2 fragments of IgG isolated from inhibitor plasma was used as label without affinity purification. The capacity of the assay was high and the sensitivity for VIII: CAg was 0.002 U/ml. Using this assay it was possible to measure coagulation inactive VIII: CAg in samples from purification studies. Below 0.05 VIII: CAg U/ml these samples responded in parallel with standard plasma. Seven of 7 inhibitor antibodies tested were able to inhibit binding of peroxidase-conjugate in the VIII: CAg assay, and the inhibitory capacity correlated with coagulation inhibition as measured by the Bethesda method. Using the highest titered antibodies bound to a solid phase, VIII: CAg was isolated and identified in SDS-PAGE as a doublet with a molecular weight of 77-80 kD.

Antigen-Antibody Complex↗
PubMed1980

A new high quality albumin for therapeutic use.

A method of large scale plasma fractionation where PEG precipitation combined with absorption is described. This method permits isolation of most globulins under mild conditions. The final purification of the albumin is achieved by heat treatment at 60 degrees C pH 4.8. The quality of albumin preparations for therapeutic use is discussed from two points of view, purity and nativity. The main problem of purity is the content of high molecular weight aggregates formed by the pasteurization (60 degrees C, 10 h.). The aggregates give a risk for immunological reactions. The nativity of albumin is correlated to the contents of oxidized albumin, and to the metabolism in man. In all the methods discussed, considerable differences are found between albumin preparations produced by different methods.

Hot Temperature↗
PubMed1980

Progress in chemical and in vivo characterization of albumin preparations.

In plasma fractionation industry different methods are used for producing albumin for therapeutic use. We wanted to investigate whether the different ways of fractionating plasma have an effect on the nativity of the albumin molecules. Isoelectric focusing in 6M urea has shown the presence of variable amounts of oxidized albumin in different preparations. The presence of oxidized forms indicates that an initial denaturation has taken place. To evaluate if the alteration in the albumin molecules has any effects in vivo, metabolic studies in rabbits and humans with radioiodinated albumin were undertaken. Our preliminary results show a correlation between the in vivo metabolic studies and the amount of oxidized albumin.

Albumins↗
PubMed1976

DNA-binding proteins in Yoshida ascites tumor fluid.

DNA-binding proteins were isolated from Yoshida ascites tumor fluid by chromatography on DNA-cellulose. This fraction represents 1-2% of the total ascites protein. Most of the DNA-binding proteins will bind to phosphocellulose as well. The proteins migrate by agarose gel electrophoresis at pH 8.6 as alpha and beta globulins. Quantitative immunoelectrophoresis revealed the presence of 12-18 proteins. SDS-polyacrylamide electrophoresis indicated molecular weights ranging from 3-10(4) to 10(6). Seven of the proteins were identified by specific immunoprecipitation as beta1-Eglobulin, beta2-glycoprotein I, fibrinogen split product E (fibrinogen E), coagulation factor XIII (factor XIII), alpha2-macroglobulin, IgG and IgM. Alpha1-antichymotrypsin might also be represented. In nuclear extracts of the tumor cells only factor XIII was present. With the exception of fibrinogen E and P5 all recognized DNA-binding proteins are present in normal rat plasma. With increasing tumor age the concentration of fibrinogen E, factor XIII, P5 and IgM increased both in ascites fluid and in plasma, while the concentration of other DNA-binding-proteins decreased or remained constant. Evidence is presented that the DNA- and phosphocellulose binding ascites protein fraction inhibit tumor cell growth. No inhibition was induced by corresponding protein fractions isolated from normal rat plasma.

Animals↗
PubMedDate not supplied

Antithrombotic effects of recombinant human, active site-blocked factor VIIa in a rabbit model of recurrent arterial thrombosis.

The extrinsic coagulation pathway is activated when circulating factor VII (FVII) gains access to tissue factor (TF) exposed as a consequence of vascular injury. Increasing evidence indicates that this TF-dependent activation of the coagulation plays an important role in the pathophysiology of intravascular thrombus formation. In the present study, we tested the effects of recombinant human, active site-blocked activated FVII (FVIIai) in a rabbit model of carotid artery thrombosis. Cyclic flow variations (CFVs), due to recurrent thrombus formation, were obtained in stenotic rabbit carotid arteries with endothelial injury. Carotid blood flow velocity was measured by a Doppler flow probe. After 30 minutes of CFVs, the animals received FVIIai (100 microg x kg(-1) x min(-1) intracarotid infusion for 10 minutes, n=9). If CFVs were abolished, animals were followed for 30 additional minutes, after which recombinant human activated FVII (FVIIa) was infused into the carotid artery (100 microg x kg(-1) x min(-1) for 10 minutes) to determine whether FVIIai could be displaced from TF by FVIIa, thus restoring CFVs. To establish the duration of action of FVIIai, an additional group of animals received FVIIai at the same dose as above, and after CFVs were inhibited, they were followed until CFVs were restored or for up to 6 hours. To determine whether CFVs could be restored by epinephrine after their abolition with FVIIai, increasing doses of epinephrine were administered to a third group of 6 animals. FVIIai abolished CFVs in 8 of 9 rabbits (P<.01). This effect was reversible, as FVIIa administration restored CFVs in all animals. Prothrombin times and activated partial thromboplastin times did not change significantly throughout the study. One single 10-minute infusion exerted complete antithrombotic effects for at least 6 hours, despite the fact that at this time point, plasma FVIIai levels were well below threshold concentrations. Epinephrine restored CFVs in 3 of 6 animals in which CFVs were inhibited by FVIIai. FVIIai exerts potent antithrombotic effects in this model; these effects were prolonged even after FVIIai was almost completely cleared from the circulation, probably as a result of the tight binding of FVIIai to TF. Thus, FVIIai might represent an antithrombotic substance of potential interest.

Animals↗