The Separation of AHF from Fibrinogen. IV

1966 ◽  
Vol 15 (03/04) ◽  
pp. 338-342
Author(s):  
S van Creveld ◽  
I. A Mochtar ◽  
C. N Pascha
Keyword(s):  
Factor Viii ◽  
Continuous Flow ◽  
Caproic Acid ◽  
Starting Material ◽  
Free Products ◽  
Molar Solution ◽  
Normal Human ◽  
Continous Flow ◽  
Fraction I

SummaryThe anti-hemophilic factor (AHF, factor VIII) has been separated from fibrinogen by means of continous flow electrophoresis. The starting material was Cohn’s fraction I, prepared from normal human resinplasma. These fibrinogen-free products still contained proteins without AHF-activity. Part of these contaminating proteins could be extracted from fraction I by means of a 1 molar solution of epsilon amino caproic acid. The residue, principally consisting of fibrinogen and AHF, was dissolved in a triethylamine buffer and subjected to continuous flow electrophoresis. The resulting AHF-preparations were free from fibrinogen. By immuno-electrophoresis no proteins without AHF-activity could be demonstrated in these preparations.

Studies on the Enzymatic Activity of the Antihemophilic Factor (Factor VIII)

1967 ◽  
Vol 17 (01/02) ◽  
pp. 188-193 ◽  
Author(s):  
S van Creveld ◽  
I. A Mochtar ◽  
C. N Pascha
Keyword(s):  
Enzymatic Activity ◽  
Factor Viii ◽  
Esterase Activity ◽  
Barium Sulphate ◽  
Screening Tests ◽  
Normal Human ◽  
The Stability ◽  
Fraction I ◽  
Antihemophilic Factor ◽  
Esterase Activities

SummaryPreparations of normal human antihemophilic factor (AHF, factor VIII) obtained by continuous free electrophoresis in the “Elphor” apparatus, were investigated for enzymatic activity on various substrates. A number of enzymatic activities could be excluded by screening tests, performed with fraction I of Cohn. In some preparations esterase-activity was demonstrable on the substrates benzoyl-arginyl-ethylester (BAEE) and on glycerol tributyrate. There was, however, no correlation between these esterase-activities and the AHF-activity in the preparations. Moreover, the stability of the esterase at 4° C was much greater than the stability of the AHF-activity. We therefore assume that the AHF has no enzymatic activity on these substrates under the conditions of the tests. Attempts to activate AHF by calcium, magnesium or serum, were unsuccessful. Both esterase-activities were also present in fraction I of Cohn prepared from normal human citrated or resin plasma. The esterase-activity on BAEE is removed from resin plasma by adsorption to barium sulphate. The esterase-activity on glycerol tributyrate is not adsorbed.

Investigations on the Nature of Hemophilia A

1963 ◽  
Vol 09 (01) ◽  
pp. 030-052 ◽  
Author(s):  
Eberhard Mammen
Keyword(s):  
Factor Viii ◽  
Human Plasma ◽  
Barium Sulfate ◽  
Freeze Drying ◽  
Room Temperature ◽  
Hemophilia A ◽  
Normal Human Plasma ◽  
Normal Human ◽  
And Storage ◽  
Prothrombin Activation

SummaryIn this paper an inhibitor is described that is found in hemophilic plasma and serum different from any till now described inhibitor. The inhibitor only inhibits prothrombin activation in the “intrinsic clotting systems”. This inhibitor is probably not present in normal human plasma or serum. It is destroyed by ether and freeze drying, is labile to acid and storage at room temperature. It is stable upon dialysis and has not been adsorbed on barium sulfate, aluminum hydroxide or kaolin. It precipitates at 50% v/v saturation with alcohol. The nature of this inhibitor seems to be a protein or lipoprotein.Factor VIII was isolated from hemophilic plasma. The amount isolated was the same as from normal plasma and the activity properties were not different. Hemophiliacs have normal amounts of factor VIII.

Use of Factor VIII in the Treatment of Hemophilia

1962 ◽  
Vol 07 (02) ◽  
pp. 230-238 ◽  
Author(s):  
A Pavlovsky ◽  
H Peterson ◽  
G Casillas ◽  
C Simonetti ◽  
A Martinez Canaveri ◽  
...  
Keyword(s):  
Factor Viii ◽  
Tannic Acid ◽  
Deae Cellulose ◽  
Fibrinolytic System ◽  
Purification Method ◽  
Fraction I

SummaryThis publication describes the results obtained by treatment of haemophiliacs with factor VIII preparations isolated from Cohn fraction I by use of tannic acid, FI-O-Ta.The authors stress the rapidity of the disappearance of factor VIII after injection. Transfusions are generally well tolerated. One reaction of the pyrogen type has been observed and also a case of activation of the fibrinolytic system.A second purification method by means of chromatography on DEAE-cellulose is described.

The Immunological Properties of Factor VIII

1966 ◽  
Vol 16 (03/04) ◽  
pp. 559-573 ◽  
Author(s):  
L Uszyński
Keyword(s):  
Factor Viii ◽  
Inhibitory Activity ◽  
Hemophilia A ◽  
Severe Form ◽  
Molecular Defect ◽  
Bovine Plasma ◽  
Human Factor Viii ◽  
Antigenic Properties ◽  
Coagulation Tests ◽  
Normal Human

SummaryRabbits immunized against human AHG fibrinogen-free preparations, were shown to produce anti-AHG antibodies. The inhibitory activity of these antibodies was tested by thromboplastin generation test, thrombelastography, and the specific anti-AHG antibodies neutralization test. The latter test permitted quantitative determination of antigenic form of factor VIII. The inhibitory activity of anti-FI-O-Ta serum resulted exclusively from the anti-AHG antibodies which in coagulation tests behaved like circulating anticoagulants directed against factor VIII.The anti-AHG antibodies were neutralizable by normal human serum or plasma even contained only trace of AHG activity after storage. There was no antigenic form of factor VIII in the severely affected patients with hemophilia A, von Willebrand’s disease nor in the normal plasma adsorbed on bentonite. The presented results suggest a molecular defect of factor VIII in patients with hemophilia A. The severe form of this disease depends, probably, on a major impairment of AHG biosynthesis, leading to changes in the antigenic properties of the molecule. The AHG from rabbit, porcine and bovine plasma respectively did not neutralize the anti-AHG antibodies formed in rabbits immunized against human factor VIII preparations.

A Method For Rapid Visualization Of The Size- Distribution Of Factor VIII-Related Protein In Plasma

1981 ◽  
Author(s):  
B A Perret ◽  
R Felix ◽  
M Furlan ◽  
E A Beck
Keyword(s):  
Factor Viii ◽  
Size Distribution ◽  
Protein Pattern ◽  
Related Protein ◽  
Step Method ◽  
Silver Stain ◽  
Molecular Weights ◽  
One Step ◽  
Fresh Plasma ◽  
Normal Human

Factor VUI-related protein circulates in normal human plasma as a series of multimeric forms with apparent molecular weights ranging from 1 to 20×106. So far, combined electrophoretic and immunologic methods permitted demonstration of variable concentration and size- distribution of factor VIII-related protein in von Willebrand’s disease as compared to normal. We now have devised a one-step method for determining the size pattern of this plasma protein. Fresh plasma, containing 1% SDS and 0.8M urea, was layered on top of 2.5% polyacrylamide gels with 2.75% by weight of methylene bisacrylamide/acrylamide. Following extended electrophoresis in 0.2% SDS-0.1M Tris/HCl (pH 7.4), the gels were soaked in 5% formaldehyde and then extensively washed with 10% ethanol. Proteins were visualized employing an ultrasensitive ammoniacal silver stain. This staining revealed a multimeric protein pattern in the upper part of the gel the distribution of which was recorded by densitometry. The protein was identified as factor VIII by two-dimensional immunoelectrophoresis. The method was reproducible and allowed densitometric evaluation within 24 hr.

scholarly journals The factor VIII complex: structure and function

Blood ◽  
1981 ◽  
Vol 58 (1) ◽  
pp. 1-13 ◽  
Author(s):  
LW Hoyer
Keyword(s):  
Factor Viii ◽  
Complex Structure ◽  
Related Protein ◽  
Molecular Defects ◽  
Normal Human ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Antihemophilic Factor ◽  
Immunologic Properties

Abstract Normal human plasma contains a complex of two proteins that are important in hemostasis and coagulation. The factor VIII procoagulant protein (antihemophilic factor) and the factor VIII-related protein (von Willebrand factor) are under separate genetic control, have distinct biochemical and immunologic properties, and have unique and essential physiologic functions. While the nature of their interaction and the details of the biochemical structures remain to be determined, the information now available permits a preliminary understanding of the molecular defects in hemophilia and von Willebrand's diseases.

Crossed Concanavalin A Affinoimmunoelectrophoresis Of Factor VIII - Related Antigen In Whole Plasma

1981 ◽  
Author(s):  
J S Krauss ◽  
M Sheard
Keyword(s):  
Factor Viii ◽  
Concanavalin A ◽  
Human Factor ◽  
Von Willebrand Disease ◽  
Con A ◽  
Factor Viii Related Antigen ◽  
Crossed Immunoelectrophoresis ◽  
Human Factor Viii ◽  
Normal Human ◽  
Von Willebrand

Normal human factor VIII is precipitated by the lectin concanavalin A (con A). In order to study this interaction pooled normal plasma has been subjected to crossed affinoimmunoelectrophoresis with con A in the first dimension and commercial antiserum to factor VIII in the second dimension. Both decreased anodal migration and decreased precipitin height of the factor VIII - related antigen (FVIIIR:ag) confirmed the precipation of FVIIIR:ag by con A.This technique, performed in conjunction with crossed immunoelectrophoresis (CIEP) of FVIIIR:ag, should prove useful in the analysis of von Willebrand disease (vWd) variants whose FVIIIR:ag has decreased carbonhydrate and, therefore, is poorly precipitated by con A.

Rapid Isolation Of Native Urokinase From Normal Human Urine

1981 ◽  
Author(s):  
K Huber ◽  
J Kirchheimer ◽  
B R Binder
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Active Material ◽  
Tween 80 ◽  
Isolation Procedure ◽  
Starting Material ◽  
Rapid Isolation ◽  
Freeze Dried ◽  
Sds Page ◽  
Normal Human

Urokinase (UK) has been isolated to complete homogeneity starting mainly from commercially available prepurified preparations. When native human urine was the starting material, high amounts of urine were necessary because of low yields due to time consuming and complicated procedures. Furthermore, without addition of inhibitors a shift to lower molecular weight (Mr) forms could not be avoided. In order to develop a rapid isolation procedure yielding final UK preparations of complete homogeneity and as unaltered as possible, we avoided concentration procedures at the beginning of the preparation as well as antibody columns because of high activity losses and binding of non active UK antigen, respectively, and employed two affinity chromatography steps.Two liters of native human urine were dialysed and adsorbed to gelatine-Sepharose; UK was completely bound and could be eluted with 0.1M Tris-HCl buffer, 0.7M CaCl2, pH 7.4. Active material was pooled, dialysed, and adsorbed to agmatine-Sepharose. Again, activities applied were totally bound and could be eluted with 0.1M phosphate buffer, 0.4M KC1, pH=7.4. Active material was freeze dried and gel filtrated on Sephadex G-150 in 0.1M Tris-HCl buffer, 1M NaCl, pH=8.0. During the whole isolation procedure Tween-80 (0.1%) was used in the buffer systems. Gelatine (0.02%) was present during dialysis. The procedure resulted in a UK preparation with a Mr=56.000 (SDS-PAGE) of complete homogeneity, and with a yield of l0-20% calculated from the starting material. Specific activity was about 16 pmoles of active enzyme per μg of protein (3H-DFP incorporation). The final product cleaved Glu-plasminogen to plasmin with an apparent KM≃lμM and a kcat≃0.5 s-1.The method described is a simple and efficient procedure resulting in a product with a specific activity comparable to those described for completely purified preparations and in a Mr form identic to that predominantly present in native urine.

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