Immunochemical Characterization of a Factor IX Inhibitor Following Anamnestic Response

1975 ◽  
Author(s):  
H. R. Roberts ◽  
C. R. Fuller ◽  
H. Worden ◽  
J. Stuart ◽  
H. Reisner ◽  
...  
Keyword(s):  
Light Chain ◽  
Gel Filtration ◽  
Complete Removal ◽  
Igg Subclasses ◽  
Factor Ix ◽  
Biologically Active ◽  
Anamnestic Response ◽  
Polyclonal Igg ◽  
Monospecific Antisera ◽  
A Minor

We previously characterized a human inhibitor for Factor IX in patient P.W.B, with Hemophilia B as an IgG4, λ immunoglobulin of restricted electrophoretic mobility. This restriction to a minor IgG subclass led us to characterize a second Factor IX inhibitor occurring in patient R. J. after an anamnestic response to Factor IX. On preparative zone electrophoresis the inhibitor migrated with a broad zone of mobility in the anodal portion of the γ peak and was restricted to the anodal portion of the IgG containing fractions. Gel filtration on calibrated 1.5 M sepharose columns revealed inhibitor activity in fractions corresponding to a molecular weight of 150,000. The inhibitor was further characterized by the technique of antibody neutralization using monospecific antisera to immunoglobulin classes, subclasses and light chain types in the zone of antibody excess. The inhibitor was completely neutralized by antibody to IgG whereas antisera to IgA, IgM, IgD and IgE had no effect. Neutralization was abolished by absorption of the IgG antiserum with purified IgG. Neutralization with antisera specific for light chains indicated a mixture of light chain types with an estimated ϰ/λ ratio of 6/1. Neutralization with antisera specific for IgG subclasses revealed a mixture of IgG subclasses. The Factor IX inhibitor was thus characterized as a polyclonal IgG immunoglobulin. Sepharose conjugates of R. J. globulin effect complete removal of Factor IX from normal plasma on an immunoabsorbent column and biologically active Factor IX may be eluted with 1600-fold purification.

Feiba And Autoplex: A Comparison Of These Two Activated Prothrombin Complex Concentrates

1981 ◽  
Author(s):  
E Lechler ◽  
B Eggeling ◽  
D Meyer-Börnecke ◽  
H Stoy
Keyword(s):  
Hemophilia A ◽  
Gel Filtration ◽  
Activated Partial Thromboplastin Time ◽  
Factor Ix ◽  
Prothrombin Complex Concentrates ◽  
Minor Degree ◽  
Activated Prothrombin Complex Concentrates ◽  
A Minor

These activated concentrates are used for the treatment of patients with factor VIII inhibitors . Both shorten the activated and non-activated partial thromboplastin time of inhibitor plasma and hemophilia A plasma in vitro. They do not or only to a minor degree improve the prothrombin consumption of hemophilia A plasma in vitro. In gel filtration of AUTOPLEX the activity which shortens the PTT of hemophilia A plasma eluted in a volume higher than that of the nonactivated factors of the prothrombin complex and contains activated factor IX. The activity of FEIBA elutes at a lower filtration volume in a rather broad peak together with the factors of the the non-activated prothrombin complex. BaSO4- adsorbed plasma and purified antithrombin (Behring) abolish the activity of AUTOPLEX more readily than of FEIBA. Both concentrates have only a low amidolytic effect (S 2222) and are not inhibited with SBTI and PMSF. In the crossed two-dimensional immunelectrophoresis with heparin in the agarose of the first dimension and anti-antithrombin (Behring) in the agarose of the second dimension (method of Sas), a mixture of AUTOPLEX and antithrombin results into a two peak precipitation of antithrombin, whereas with FEIBA a broadened intermediate peak develops. In vivo both concentrates do not improve the prothrombin consumption and AUTOPLEX shortens the PTT for at least 90 minutes. In summary, these two concentrates differ considerably.

Characterization of Native Human Thrombopoietin in the Blood of Normal Individuals and of Patients with Haematologic Disorders

1999 ◽  
Vol 82 (07) ◽  
pp. 24-29 ◽  
Author(s):  
Atsushi Matsumoto ◽  
Tomoyuki Tahara ◽  
Haruhiko Morita ◽  
Kensuke Usuki ◽  
Hideya Ohashi ◽  
...  
Keyword(s):  
Size Distribution ◽  
Gel Filtration ◽  
Molecular Size ◽  
Immunoblot Analysis ◽  
Biologically Active ◽  
Amino Acid Residues ◽  
Normal Individuals ◽  
Normal Human ◽  
A Minor ◽  
Two Peaks

SummaryThrombopoietin (TPO) isolated from thrombocytopenic plasma of various animal species has previously been shown to comprise only truncated forms of the molecule, presumably generated by proteolysis. Native TPO has now been partially purified from normal human plasma by immunoaffinity chromatography and was confirmed to be biologically active. Gel filtration in the presence of SDS revealed that TPO eluted in two peaks: a major peak corresponding to the elution position of fully glycosylated recombinant human TPO (rhTPO) consisting of 332 amino acid residues, and a minor peak corresponding to a smaller molecular size. Immunoblot analysis also revealed that most plasma-derived TPO migrated at the same position as fully glycosylated rhTPO, corresponding to a molecular size of ~80 to 100 kDa. Furthermore, the size distribution of circulating TPO in patients with various haematologic disorders did not differ markedly from that of plasma-derived TPO from healthy individuals. These results indicate that the truncation of circulating TPO is not related to disease pa-thophysiology, and that the predominant form of TPO in blood is a biologically active ~80- to 100-kDa species. The size distribution of TPO extracted from normal platelets was similar to that of TPO in plasma; the proportion of truncated TPO was decreased by prior incubation of platelets with hirudin, indicating that the endogenous truncated TPO, at least in platelet extract, was generated by thrombin-mediated cleavage.

Management of Haemophilia A with Antibodies - The Effect of Combined Treatment with Factor VIII, Hydrocortisone and Cyclophosphamide

1985 ◽  
Vol 54 (04) ◽  
pp. 776-779 ◽  
Author(s):  
U Hedner ◽  
L Tengborn
Keyword(s):  
Factor Viii ◽  
Combined Treatment ◽  
Factor Ix ◽  
Treatment Schedule ◽  
High Antibody ◽  
Anamnestic Response ◽  
Short Intervals ◽  
Low Responders ◽  
Antibody Levels ◽  
High Responders

SummaryImmune tolerance has by several methods been induced in haemophiliacs with antibodies. A conversion of “high responders” into “low responders” was previously reported after repeated moderate factor IX doses over periods of 7-10 days in combination with cyclophosphamide and steroids in two patients with haemophilia B and inhibitors. This paper reports similar results in a heamophilia A patient by giving factor VIII, cyclophosphamide, and steroids during relatively short periods of time (7-8 days). The anamnestic response markedly decreased already following the first treatment and never exceeded a level of 1 u/ml (˜ 3 BU/ml) even when boosted with ordinary factor VIII doses for only 3 days. It is concluded that the markedly decreased secondary antibody response is most probably the result of factor VIII given at short intervals (twice a day) for periods of up to about one week when given in combination with cyclophosphamide and steroids. The same effect may be achieved by other methods. The treatment schedule suggested in the present paper is, however, simple and avoids long periods of high antibody levels. Furthermore, the total factor VIII dose used is lower than suggested in most other treatment schedules, which makes the treatment substantially less expensive.

scholarly journals Occurrence of fatty acid epoxide hydrolases in soybean (Glycine max). Purification and characterization of the soluble form

1992 ◽  
Vol 282 (3) ◽  
pp. 711-714 ◽  
Author(s):  
E Blée ◽  
F Schuber
Keyword(s):  
Glycine Max ◽  
Gel Filtration ◽  
Soluble Form ◽  
Soluble Enzyme ◽  
Purification And Characterization ◽  
Epoxide Hydrolases ◽  
Major Activity ◽  
Apparent Homogeneity ◽  
A Minor

Epoxide hydrolases catalysing the hydration of cis-9,10-epoxystearate into threo-9,10-dihydroxystearate have been detected in soybean (Glycine max) seedlings. The major activity was found in the cytosol, a minor fraction being strongly associated with microsomes. The soluble enzyme, which was purified to apparent homogeneity by (NH4)2SO4 fractionation, hydrophobic, DEAE- and gel-filtration chromatographies, has a molecular mass of 64 kDa and a pI of 5.4.

scholarly journals Rat liver alcohol dehydrogenase. Purification and properties

1971 ◽  
Vol 125 (4) ◽  
pp. 1039-1047 ◽  
Author(s):  
M J Arslanian ◽  
E Pascoe ◽  
J G Reinhold
Keyword(s):  
Molecular Weight ◽  
Alcohol Dehydrogenase ◽  
Rat Liver ◽  
Gel Filtration ◽  
Rabbit Antiserum ◽  
Minor Component ◽  
Disc Electrophoresis ◽  
Supernatant Fraction ◽  
Liver Alcohol Dehydrogenase ◽  
A Minor

Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD+ and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn2+ at concentrations above 0.1mm.

Purification and characterization of tuberculin-active components from BCG

1975 ◽  
Vol 21 (12) ◽  
pp. 2019-2027
Author(s):  
M. Laguerre ◽  
R. Turcotte
Keyword(s):  
Physicochemical Properties ◽  
Guinea Pigs ◽  
Gel Filtration ◽  
Biologically Active ◽  
Polyacrylamide Gels ◽  
Active Components ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Antigenic Activity

The tuberculin activity of protoplasmic extracts isolated from living BCG was purified successively by gel filtration on Sephadex G-100 and G-75, and by electrophoresis on 7.5% and on gradient (6–18%) polyacrylamide gels. The tuberculin-active fractions, as determined in BCG-sensitized guinea pigs, were used as the starting material for each of the following fractionation steps.The physicochemical properties and the antigenic activity of the biologically active fractions have shown that a single component, or only a few ones with similar properties, possessed high tuberculin activity. These active components were proteins having relatively high molecular weights (about 72 000) and could behave as antigens.

scholarly journals Heterogeneity of Mycolactones Produced by Clinical Isolates of Mycobacterium ulcerans: Implications for Virulence

2003 ◽  
Vol 71 (2) ◽  
pp. 774-783 ◽  
Author(s):  
Armand Mve-Obiang ◽  
Richard E. Lee ◽  
Françoise Portaels ◽  
P. L. C. Small
Keyword(s):  
Clinical Evidence ◽  
Buruli Ulcer ◽  
Mycobacterium Ulcerans ◽  
Biologically Active ◽  
In Vivo Studies ◽  
Tissue Destruction ◽  
The Core ◽  
A Minor ◽  
Layer Chromatography

ABSTRACT Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe necrotizing skin disease endemic in tropical countries. Clinical evidence suggests that M. ulcerans isolates from Asia, Mexico, and Australia may be less virulent than isolates from Africa. In vivo studies suggest that mycolactone, a polyketide-derived macrolide toxin, plays a major role in the tissue destruction and immune suppression which occur in cases of Buruli ulcer. Mycolactones were extracted from 34 isolates of M. ulcerans representing strains from Africa, Malaysia, Asia, Australia, and Mexico. Thin-layer chromatography, mass spectroscopic analysis, and cytopathic assays of partially purified mycolactones from these isolates revealed that M. ulcerans produces a heterogeneous mixture of mycolactone variants. Mycolactone A/B, the most biologically active mycolactone species, was identified by mass spectroscopy as [M+Na]+ at m/z 765.5 in all cytotoxic isolates except for those from Mexico. Mycolactone C [M+Na]+ at m/z 726.3 was the dominant mycolactone species in eight Australian isolates, and mycolactone D [M+Na]+ m/z 781.2 was characteristic of two Asian strains. Mycolactone species are conserved within specific geographic areas, suggesting that there may be a correlation between mycolactone profile and virulence. In addition, the core lactone, [M+Na]+ m/z 447.4, was identified as a minor species, supporting the hypothesis that mycolactones are synthesized by two polyketide synthases. A cytopathic assay of the core lactone showed that this molecule is sufficient for cytotoxicity, although it is much less potent than the complete mycolactone.

Production of biologically active human factor IX-Fc fusion protein in the milk of transgenic mice

2020 ◽  
Vol 42 (5) ◽  
pp. 717-726
Author(s):  
Hong Yan ◽  
Xiuli Gong ◽  
Miao Xu ◽  
Xinbing Guo ◽  
Yanwen Chen ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Fusion Protein ◽  
Human Factor ◽  
Factor Ix ◽  
Biologically Active ◽  
Human Factor Ix ◽  
Fc Fusion Protein

EXPRESSION IN MAMMALIAN CELLS OF FUSION PROTEINS BETWEEN HUMAN FACTORS IX AND VII

1987 ◽  
Author(s):  
K L Berkner ◽  
S J Busby ◽  
J Gambee ◽  
A Kumar
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Factor Ix ◽  
Biologically Active ◽  
Factor Vii ◽  
Clotting Factor ◽  
Clotting Factors ◽  
Mammalian Expression ◽  
Plasma Factor ◽  
Gla Domain

The vitamin K-dependent plasma proteins demonstrate remarkable similarities in their structures: all have multiple domains in common and extensive homology is observed within many of these domains. In order to investigate the structure-function relationship of these proteins, we have interchanged domains of one protein (factor IX) with that of another (factor VII) and have compared the expression of these fusion proteins with recombinant and native factors IX and VII. Oligonucleotide-directed mutagenesis was used to generate four fusion proteins: factor IX/VII-1, which contains the factor IX leader and gla domain fused to the growth factor and serine protease of factor VII; factor VII/IX-1, a reciprocal fusion protein of factor IX/VII-1; factor IX/VII-2, which contains the factor IX leader adjoined to the mature factor VII protein sequence; and factor VII/IX-2, the reciprocal fusion protein of factor IX/VII-2. The cDNAs encoding all four proteins were cloned into mammalian expression vectors, and to date three of these (factors IX/VII-1, 2 and VII/IX-1) have been transfected into baby hamster kidney (BHK) cells or 293 cells and characterized. Factors IX/VII-1 and VII/IX-1 were both secreted at levels comparable to recombinant factors IX and VII. The factor IX/VII-1 was identical in molecular weight to native or recombinant factor VII (i.e., 53 K). Factor VII/IX-1 was expressed as two proteins with molecular weights around 68 kd, as observed with recombinant factor IX. The factor IX/VII-1 protein has been purified to homogeneity and has been found to possess factor VII biological activity, but at a specific activity approximately 20% that of plasma factor VII. Thus, the gla domain of one clotting factor is capable of directing the activation of another and of generating biologically active protein. In contrast, no activity was observed with the factor IX/VII-2 fusion protein, indicating that there are limits to the interchanges which can generate functional blood clotting factors.

scholarly journals A New Pathway for Arachidonic Acid Metabolism in Human Platelets

1977 ◽  
Author(s):  
S. Rittenhouse-Simmons ◽  
F. A. Russell ◽  
D. Deykin
Keyword(s):  
Arachidonic Acid ◽  
Acid Metabolism ◽  
De Novo ◽  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Kinetic Studies ◽  
Arachidonic Acid Metabolism ◽  
Biologically Active ◽  
Resting Cells ◽  
Active Metabolites

We are reporting a novel pathway of arachidonic acid metabolism in the phosphatides of thrombin-activated platelets. For kinetic studies of arachidonic acid turnover, platelet phosphatides were labeled by incubation of platelet rich plasma with (3H)-arachidonic acid for 15 min. Unincorporated isotope was removed during subsequent gel-filtration. Platelet phosphatides were resolved and quantitated following two-dimensional silica paper chromatography of chloroform/methanol extracts of incubated platelets. Plasmalogen phosphatidylethanolamine (PPE) was examined on paper chromatograms after its breakdown to lysoPPE with HgCl2. In other experiments, gel-filtered platelets were incubated with (14C)-glycerol to monitor de novo phosphatide synthesis. (3H)-Arachidonic acid was released from phosphatidylcholine and phosphatidylinositol of pre-labeled platelets exposed to thrombin and appeared increasingly in PPE in acyl linkage at glycerol-C-2. (3H)-Arachidonic acid was not found in PPE of resting cells. Maximum transfer occurred with 5 U/ml of thrombin and 15 min, of incubation, with t½ of 2½ min., and was Ca+2 dependent. The presence of aspirin, indomethacin, or eicosatetraynoic acid did not prevent the thrombin-activated transfer of (3H)-arachidonic acid to PPE. The stimulated incorporation of (3H)-arachidonic acid into PPE was not accompanied by a stimulation of (14C)-glycerol uptake into this phosphatide. We suggest that perturbation of the platelet may activate a phospholipase A2 leading to turnover of arachidonic acid in PPE, which is rich in this fatty acid. Such turnover may provide substrate for conversion by cyclo-oxygenase and lipoxydase to biologically active metabolites, and therefore, may offer a locus for regulation of prostaglandin synthesis in the human platelet.

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