scholarly journals Apparent molecular weight of purified human factor VIII procoagulant protein compared with purified and plasma factor VIII procoagulant protein antigen

Blood ◽  
1983 ◽  
Vol 62 (5) ◽  
pp. 1114-1117
Author(s):  
MJ Weinstein ◽  
CA Fulcher ◽  
LE Chute ◽  
TS Zimmerman
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Apparent Molecular Weight ◽  
Discontinuous System ◽  
Major Band ◽  
Molecular Weights ◽  
Human Factor Viii ◽  
Plasma Factor ◽  
Factor Viii Concentrates ◽  
Electrophoretic Techniques

We have compared apparent molecular weights of purified factor VIII procoagulant protein (VIII:C) and VIII:C antigen (VIII:CAg) by two different NaDodSO4 gel electrophoretic techniques. In a discontinuous NaDodSO4–7.5% polyacrylamide system, reduced and unreduced VIII:C, purified from commercial factor VIII concentrates by a monoclonal antibody immunoadsorption technique, showed a major doublet at mol wt 0.79 and 0.8 X 10(5) and less intense bands extending up to 1.9 X 10(5). In NaDodSO4–4% polyacrylamide/0.5% agarose gels (NaDodSO4–4% PAAGE), purified VIII:C had a major band of mol wt 1.0 X 10(5), with minor bands of mol wt 0.96, 1.1, 1.4, 1.6, 1.8, 2.2, and 2.4 X 10(5). In NaDodSO4–4% PAAGE of 125I-anti-VIII:C-Fab-VIII:CAg complexes, the major and minor forms of VIII:CAg in purified VIII:C had the same molecular weight as above when calculated by subtracting the molecular weight of 125I-Fab from 125I-Fab-VIII:CAg. In both plasma and factor VIII concentrate, a band of mol wt 2.4 X 10(5) predominated, and minor VIII:CAg forms of mol wt 2.6, 1.8, 1.2 and 1.0 X 10(5) were also visible. We conclude that the molecular weight of plasma VIII:CAg forms agree with those derived from protein stains of purified VIII:C in the NaDodSO4–4% PAAGE system, but that consistently lower molecular weight values are obtained for purified VIII:C in the discontinuous system. Both native and either disaggregated or proteolyzed VIII:C species are present in the purified VIII:C preparation.

scholarly journals Apparent molecular weight of purified human factor VIII procoagulant protein compared with purified and plasma factor VIII procoagulant protein antigen

Blood ◽  
1983 ◽  
Vol 62 (5) ◽  
pp. 1114-1117 ◽  
Author(s):  
MJ Weinstein ◽  
CA Fulcher ◽  
LE Chute ◽  
TS Zimmerman
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Apparent Molecular Weight ◽  
Discontinuous System ◽  
Major Band ◽  
Molecular Weights ◽  
Human Factor Viii ◽  
Plasma Factor ◽  
Factor Viii Concentrates ◽  
Electrophoretic Techniques

Abstract We have compared apparent molecular weights of purified factor VIII procoagulant protein (VIII:C) and VIII:C antigen (VIII:CAg) by two different NaDodSO4 gel electrophoretic techniques. In a discontinuous NaDodSO4–7.5% polyacrylamide system, reduced and unreduced VIII:C, purified from commercial factor VIII concentrates by a monoclonal antibody immunoadsorption technique, showed a major doublet at mol wt 0.79 and 0.8 X 10(5) and less intense bands extending up to 1.9 X 10(5). In NaDodSO4–4% polyacrylamide/0.5% agarose gels (NaDodSO4–4% PAAGE), purified VIII:C had a major band of mol wt 1.0 X 10(5), with minor bands of mol wt 0.96, 1.1, 1.4, 1.6, 1.8, 2.2, and 2.4 X 10(5). In NaDodSO4–4% PAAGE of 125I-anti-VIII:C-Fab-VIII:CAg complexes, the major and minor forms of VIII:CAg in purified VIII:C had the same molecular weight as above when calculated by subtracting the molecular weight of 125I-Fab from 125I-Fab-VIII:CAg. In both plasma and factor VIII concentrate, a band of mol wt 2.4 X 10(5) predominated, and minor VIII:CAg forms of mol wt 2.6, 1.8, 1.2 and 1.0 X 10(5) were also visible. We conclude that the molecular weight of plasma VIII:CAg forms agree with those derived from protein stains of purified VIII:C in the NaDodSO4–4% PAAGE system, but that consistently lower molecular weight values are obtained for purified VIII:C in the discontinuous system. Both native and either disaggregated or proteolyzed VIII:C species are present in the purified VIII:C preparation.

Affinity Chromatography of Human Factor VIII Using Human and Rabbit Antibodies to Factor VIII

1979 ◽  
Vol 42 (04) ◽  
pp. 1306-1315 ◽  
Author(s):  
Janet L Lane ◽  
H Ekert ◽  
A Vafiadis
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Affinity Chromatography ◽  
Protein Concentration ◽  
Gel Filtration ◽  
Subunit Structure ◽  
Molecular Weights ◽  
Human Factor Viii ◽  
Sds Page ◽  
Normal Human

SummaryFactor VIII, purified by gel filtration on Sepharose 2B, has an 8 band multiple subunit structure, with molecular weights ranging from 30,000 to 230,000, on reduction and SDS-PAGE at a protein concentration of 400 μg/gel. Affinity chromatography of this factor VIII preparation with insolubilized haemophilic antibody to factor VIII showed that 45-81% VIII:C and 0-33% VIILRag were attached to the column. Elution of the column with 0.25 M CaCl2 did not show VIII:C or VIILRag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed that 95 % of the protein bound by haemophilic antibody had a molecular weight similar to the low molecular weight subunits of the reduced factor VIII.In control experiments with normal Human IgG, 3% of VIII:C and 5% of VIILRag were attached to the column. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the protein, showed 2 faint bands with molecular weight consistent with heavy and light chains of IgG.Similar experiments with antibody to factor VIII showed that 67-83% of VIILC and 61-76% of VIII:Rag were attached to the column. Elution of the column with 0.25 M CaCl2 showed 10% of the applied VIII:C, but no VIII:Rag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed an 8 band subunit structure similar to the reduced factor VIII.

scholarly journals Studies on the Structure and Subunit Composition of Human Antihaemophilic Factor

1977 ◽  
Author(s):  
J. J. Gorman
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Peptide Mapping ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Protein S ◽  
Molecular Forms ◽  
Major Band ◽  
Human Factor Viii ◽  
Von Willebrand

Human antihaemophilic factor has been purified by hydroxylapatite chromatography following precipitation from plasma and gel filtration on Sepharose 6B.Application to hydroxyiapatite was in 0.02 M tris HCl (pH 7.35) – 0.14 M NaCl and after washing with 5mM phosphate (pH 6.8) – 0.1 M NaCI the antihaemophilic factor was eluted with 0. 1M phosphate (pH 6.8) – 0.1M NaCl. Factor VIII coagulant activity, factor VIII related antigen and von Willebrand factor activity eluted simultaneously.The protein(s) had a molecular weight in excess of 500,000 and multiple subunits as shown by electrophoresis in 5% acrylamide gels containing sodium dodecyl sulphate;without reduction the protein failed to enter these gels but following reduction multiple bands were observed, the major band had a molecular weight around 200,000.Thin layer peptide mapping demonstrated structural inter-relationship between the 200,000 dalton protein and three of the smaller species, however, two other unrelated smaller species were evident.It is apparent from these findings that human factor VIII may exist as multiple molecular forms due to heterogeneity of one subunit (MW around 200,000) and the molecular structure may include other smaller non-identical subunits. The structure-function relationships of these subunits remains to be elucidated.

Subunit Structure of Human Factor VIII

1975 ◽  
Author(s):  
J. A. van Mourik ◽  
W. T. LaBruyère ◽  
I. A. Mochtar
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Factor Viii ◽  
Apparent Molecular Weight ◽  
Subunit Structure ◽  
Single Chain ◽  
Covalent Bonds ◽  
Single Polypeptide Chain ◽  
Human Factor Viii ◽  
Low Ionic Strength

Several investigations have shown that factor VIII is a high molecular weight aggregate and that both non-covalent and disulphide bonds contribute to the stabilization of the macromolecular factor VIII aggregate. As shown previously (1) disruption of non-covalent bonds can be accomplished at relatively low ionic strength and neutral pH and results via a series of homologous oligomers (having constant charge/mass ratios) in 2 immunologically non-related subunits. Thus, the generally accepted concept that factor VIII is constructed of identical subunits seems incorrect. For several reasons we assume that the observed fragmentation at low ionic strength is not due to proteolytic breakdown. However, this view is not favoured by the observation that tryptic and plasmic digestion of factor VIII (in aggregated form) results in gel electrophoresis patterns comparable with those obtained of factor VIII after low ionic strength dissociation. Further, evidence will be presented that the assumption that reduction of factor VIII under denaturating conditions results in a single polypeptide chain is also no longer tenable. As far as the reduction of aggregated factor VIII is concerned our observations agree with data from the literature, that is, reduction of factor VIII results in 1 major subunit with an apparent molecular weight of approximately 270.000. However, when factor VIII is first dissociated at low ionic strength followed by reduction, smaller fragments appear in the electrophoresis pattern. Thus, it seems most likely that the apparent single chain subunit is in fact, constructed of smaller non-covalently linked fragments.(1) J. A. van Mourik et al. Thrombosis Research, 4, 155, 1974.

PARADOXICAL INCREASE IN HUMAN FACTOR VIII AFTER INFUSION OF PORCINE FACTOR VIII CONCENTRATE IN A PATIENT WITH ACQUIRED VARIANT VON WILLEBRAND'S DISEASE

1987 ◽  
Author(s):  
J Ball ◽  
R G Malía ◽  
M Greaves ◽  
F E Preston
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Human Factor ◽  
De Novo ◽  
Similar Time ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Human Factor Viii ◽  
Plasma Factor ◽  
Abnormal Pattern

A patient with acquired variant von Willebrand's disease was given an infusion of 2000 units of high purity porcine factor VIII (Hyate). Quantitative factor VIII parameters were assessed following infusion and human factor VIII multimers were analysed by radioimmunoelectrophoresis and autoradiography. We have previously described the patient to have acquired von Willebrand's disease due to a circulating inhibitor to the factor VIII complex (B. J. Haematol-, 54,233,1983). Prior to infusion plasma from the patient contained factor VIIIC, RRCo, and vWFAg at less than 10 u/dl- Plasma factor VIII multimers showed an abnormal pattern with no high molecular weight bands present despite a normal triplet structure in the low molecular weight forms. After the infusion of porcine factor VIII concentrate a large increase in the levels of plasma VIIIC was detected with a disappearance half-life of 3.5 hours. A specific non-crossreacting immunoradiometric assay (IRMA) showed that plasma levels of porcine vWFAg did not rise significantly after the infusion. Despite this, human vWFAg levels were notably elevated at 1 hour (40 u/dl by Laurell) and 2 hours (30 u/dl by IRMA) post infusion. Similarly, ristocetin induced platelet aggregation and plasma RRCo levels showed significant elevations , 2 hours after the infusion. Factor VIII multimers assessed on plasma samples taken over a similar time period revealed the transient appearance of a normal compliment of human factor VIII multimeric forms 2 hours after the infusion of porcine factor VIII concentrate. This study indicates that the abnormal pattern of factor VIII multimeric bands present in inhibitor-related variant acquired von Willebrand's disease can be transiently normalised by infused porcine factor VIII concentrate. Whether this represents antibody displacement or de novo synthesis is yet to be determined.

Studies on the Characterization of Factor VIII and a Co-factor VIII

1974 ◽  
Vol 31 (02) ◽  
pp. 328-338
Author(s):  
M. M. P Paulssen ◽  
H. L. M. A Vandenbussche-Scheffers ◽  
P. B Spaan ◽  
T de Jong ◽  
M. C Planje
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
The Body ◽  
Haemophilia A ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Polysaccharide Content ◽  
Plasma Factor ◽  
Two Factors

SummaryFactor VIII occurs in the body in two different forms. In lymph factor VIII is bound to chylomicra. In plasma, factor VIII is bound to a protein.After delipidation of chylomicra we obtained a glycoprotein with a high polysaccharide content and a molecular weight of approx. 160,000.In plasma, factor VIII is attached to a protein which is present in normal concentrations in plasma of patients with haemophilia A and in serum (co-factor VIII).This factor is deficient in both the plasma and the serum of patients with von Willebrand’s disease.The binding between factor VIII and co-factor VIII is reversible.Some properties of these two factors are described.

The Fractionation Properties of Human Factor VIII (Antihaemophilic Factor)

1960 ◽  
Vol 04 (02) ◽  
pp. 253-260 ◽  
Author(s):  
Franco Gobbi
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Maximum Yield ◽  
Factor Vii ◽  
Precipitation Method ◽  
Salting Out ◽  
Human Factor Viii ◽  
Plasma Factor ◽  
Two Factors ◽  
Thromboplastic Activity

SummaryThe fractionation properties of human Factor VIII (antihaemophilic factor, AHF, antihaemophilic globulin) have been studied using a plasma of congenital afibrinogenaemia as a starting material.From a fibrinogen-free plasma, Factor VIII does not precipitate with ethanol at a final concentration of 8%; on the contrary the maximum yield is reached at an ethanol concentration of 25%.With a precipitation method carried out by a one to ten dilution of plasma with distilled water and acidification by N/10 hydrochloric acid to a pFI 5.2, Factor VIII does not precipitate with the euglobulin fraction; when normal plasma is used, such a precipitation is almost complete.With the salting-out fractionation method by ammonium sulphate, Factor VIII precipitates at a concentration between 25 and 33% of saturation either from fibrinogen-free and from normal human plasma.A non-specific thromboplastic activity appears in the fractions prepared by every method. This activity, which is probably due to the activation of seric accelerators, is easily removed by Al(OH)s adsorption. Thus, in order to insure the specificity of Factor VIII assays, the preliminary adsorption of the fractions is indispensable before testing their antihaemophilic activity.Fibrinogen and Factor VIII have different and definite precipitation patterns. When these two factors are associated the fractionation properties of AHF appear quite modified, showing a close similarity to those of fibrinogen. This fact can explain the technical difficulties encountered in the attempt to purify the antihaemophilic factor, and the lack of reproducible procedures for removing fibrinogen without affecting Factor VII.

Molecular Weights of Factor VIII (AHF) and Factor IX (PTC) by Electron Irradiation

1962 ◽  
Vol 08 (02) ◽  
pp. 270-275 ◽  
Author(s):  
David L Aronson ◽  
John W Preiss ◽  
Michael W Mosesson
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Electron Irradiation ◽  
Factor Ix ◽  
Molecular Weights

SummaryThe molecular weights of AHF (factor VIII) and of PTC (factor IX) have been estimated by their sensitivity to inactivation by 7 kilovolt electrons. The molecular weight of AHF was found to be 180 000 by this method and that of PTC was found to be 110 000.

scholarly journals Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle.

1977 ◽  
Vol 72 (1) ◽  
pp. 194-208 ◽  
Author(s):  
L D Hodge ◽  
P Mancini ◽  
F M Davis ◽  
P Heywood
Keyword(s):  
Cell Cycle ◽  
Molecular Weight ◽  
Nuclear Matrix ◽  
Apparent Molecular Weight ◽  
Molecular Weight Range ◽  
Weight Range ◽  
Molecular Weights ◽  
Liver Nuclei ◽  
Hela S3 ◽  
Biochemical Analyses

A subnuclear fraction has been isolated from HeLa S3 nuclei after treatment with high salt buffer, deoxyribonuclease, and dithiothreitol. This fraction retains the approximate size and shape of nuclei and resembles the nuclear matrix recently isolated from rat liver nuclei. Ultrastructural and biochemical analyses indicate that this structure consists of nonmembranous elements as well as some membranous elements. Its chemical composition is 87% protein, 12% phospholipid, 1% DNA, and 0.1% RNA by weight. The protein constituents are resolved in SDS-polyacrylamide slab gels into 30-35 distinguishable bands in the apparent molecular weight range of 14,000 - 200,000 with major peptides at 14,000 - 18,000 and 45,000 - 75,000. Analysis of newly synthesized polypeptides by cylindrical gel electrophoresis reveals another cluster in the 90,000-130,000 molecular weight range. Infection with adenovirus results in an altered polypeptide profile. Additional polypeptides with apparent molecular weights of 21,000, 23,000, and 92,000 become major components by 22 h after infection. Concomitantly, some peptides in the 45,000-75,000 mol wt range become less prominent. In synchronized cells the relative staining capacity of the six bands in the 45,000-75,000 mol wt range changes during the cell cycle. Synthesis of at least some matrix polypeptides occures in all phases of the cell cycle, although there is decreased synthesis in late S/G2. In the absence of protein synthesis after cell division, at least some polypeptides in the 45,000-75,000 mol wt range survive nuclear dispersal and subsequent reformation during mitosis. The possible significance of this subnuclear structure with regard to structure-function relationships within the nucleus during virus replication and during the life cycle of the cell is discussed.

scholarly journals Further Experience on the Effect of High Pre-Donation Factor VIII Levels in Blood Donors on the Factor VIII Content of Small-Pool Fractions

1977 ◽  
Author(s):  
H. Beeser ◽  
H. Eqli
Keyword(s):  
Factor Viii ◽  
High Plasma ◽  
Activity Levels ◽  
Factor Viii Activity ◽  
Factor Viii Level ◽  
Plasma Factor ◽  
Viii Level ◽  
Factor Viii Concentrates ◽  
Small Pool ◽  
Concentrate Preparation

Because of the well known wide normal range of the factor VIII activity between 60 to 170% I man, selecting of donors with high activity levels would be of advantaae for the preparation of factor VIII concentrates. This is especially true for preparing small-pool fractions, as for technical reasons the final product cannot be controlled for its factor VIII content. In preliminary investigations, we reported on elsewhere, high factor VIII activity in donors estimated before a donation had been rarely reproducible before a second donation after 8-12 weeks. So as a preliminary result of finding a donor’s factor VIII level varying from donation to donation selecting of plasmas with high factor VIII content for concentrate preparation could only be establishedby re-estimating the activity before each donation. Proceeding in this way would be much too troublesome. To get more reliable information whether a healthy subject’s high factor VIII plasma level is distinctly varying or rather constant we assayed the plasma of 200 donors with factor VIII activity > 120% two times more before donation. The results confirmed our preliminary findings, especially the fact that a high plasma factor VIII activity in experienced donors was rarely reproducible when re-estimated before a second and third donation. As a consequence selecting of donors with high factor VIII procoaqulant activity for preparing small-pool factor VIII concentrates is impracticable.

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