Studies on Human Factor VIII and its Antibodies Using Radiolabelling and Affinity Chromatography

1981 ◽  
Vol 45 (03) ◽  
pp. 267-271
Author(s):  
M L Kavanagh ◽  
C N Wood ◽  
J F Davidson
Keyword(s):  
Factor Viii ◽  
Affinity Chromatography ◽  
Human Factor ◽  
Gel Filtration ◽  
Chromatography Method ◽  
Human Antibodies ◽  
Immunoglobulin Preparations ◽  
Human Factor Viii ◽  
Subsequent Application ◽  
Covalently Linked

SummaryAn immuno-affinity chromatography method was used to isolate human factor VIII and its antibodies and the mechanism of the affinity system was investigated using iodine labelling.Rabbit antibodies to human factor VIII were insolubilised onto CNBr — activated Sepharose 2B which was used for the preparation of affinity columns. Both VIII:C and VIIIR:Ag were adsorbed onto such columns from factor VIII preparations. The subsequent application of immunoglobulin preparations containing human antibodies to factor VIII resulted in the adsorption of these antibodies onto the columns. Adsorbed material was eluted from the affinity columns with 0.2 M glycine - HCl, pH 2.3.When 125I-labelled factor VIII and 131I-labelled human antibodies to factor VIII were used in this affinity system, the eluted material could be separated into three fractions by gel filtration on Bio-Gel A 1.5 m. Fraction 1 occurred at the void volume position, fraction 3 at a position corresponding to the elution position of IgG and fraction 2 at an intermediate position. 131I-labelled material was present in all three peaks. 125I-labelled material was present mainly in peak 1, with a little in peak 2. The results support the view that VIIIR: Ag, which binds heterologous antibodies, is non-covalently linked to a smaller subunit, VIII.C, which binds homologous antibodies.

The Immunological Characterization of Human Antibodies to Factor VIII Isolated by Immuno-Affinity Chromatography

1981 ◽  
Vol 45 (01) ◽  
pp. 060-064 ◽  
Author(s):  
M L Kavanagh ◽  
C N Wood ◽  
J F Davidson
Keyword(s):  
Factor Viii ◽  
Affinity Chromatography ◽  
Gel Filtration ◽  
Immunological Characterization ◽  
Human Antibodies ◽  
Heavy Chains ◽  
Light Chain Type ◽  
Antibody Preparations ◽  
Chain Type

SummaryNine human antibodies to factor VIII were isolated from haemophilic plasmas by affinity chromatography and gel filtration and six were subsequently subjected to immunological characterization. Three partially purified preparations were similarly characterized. Eight of the antibodies were characterized as being exclusively IgG and one preparation was found to contain IgM. Seven of the antibodies contained only a single light chain type, four being of type lambda and three of type kappa. Two antibody preparations contained both kappa and lambda light chains. In four of the preparations, only a single heavy chain sub-class could be demonstrated, three of IgG3 and one of IgG4. Of the remainder, three were a mixture of IgG3 and IgG4 sub-classes and one contained both IgG2 and IgG4. IgG sub-classification could not be achieved with the IgM-containing preparation. These results demonstrate a restricted heterogeneity of light and heavy chains in human antibodies to factor VIII.

scholarly journals Studies of the human factor VIII/von Willebrand's factor protein. II. Identification and characterization of the von Willebrand protein

Blood ◽  
1975 ◽  
Vol 46 (3) ◽  
pp. 417-430
Author(s):  
HR Gralnick ◽  
BS Coller
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Related Protein ◽  
Human Factor Viii ◽  
Von Willebrand’S Factor ◽  
Von Willebrand ◽  
Identification And Characterization

The purified factor VIII-related protein we have previously characterized from normal cryoprecipitate possesses both procoagulant activity and vWf activity. We have attempted to isolate and characterize this protein from three patients with severe vWd. This protein is absent or markedly diminished in amount in these vWd patients, as judged by gel filtration, polyacrylamide-gel electrophoresis, and immunoprecipitation assays. Likewise, the procoagulant and vWf activities are deficient. As vWf activity is one of the major biologic functions of either the normal or hemophilic factor VIII-related protein, the purified protein should be designated the f VIII/vWf protein.

Rabbit Antibodies Against the Procoagulant Activity (VIII:C) of Human Factor VIII

1981 ◽  
Vol 46 (04) ◽  
pp. 699-705 ◽  
Author(s):  
T H Tran ◽  
G A Marbet ◽  
F Duckert
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Gel Filtration ◽  
Procoagulant Activity ◽  
Solid Matrix ◽  
Factor Viii Activity ◽  
Physiological Conditions ◽  
Human Factor Viii ◽  
Plasma Factor ◽  
Factor Viii Antigen

SummaryThe procoagulant activity VIII:C was separated from factor VIII antigen (VIIIR:Ag) by gel filtration in the presence of 0.25 mol/l calcium chloride. Antibodies (anti-VIII:C) were obtained by immunization of rabbits with VIII:C. The last step of the purification procedure of antibodies consists of an adsorption on VIIIR:Ag-Sepharose 2 BCL as immunoadsorbent to remove contaminating traces of antibodies against VIIIR:Ag. The anti- VIII:C titer remains unchanged during this adsorption (29 Bethesda units per mg). In solution, anti-VIII:C neutralies factor VIII activity (in plasma, cryoprecipitate or in purified form) and the fragment VIII:C without reacting with VIIIR:Ag. Once immobilized on a solid matrix, i.e.2% agarose, it loses over 95% of its inhibitory capacity. The immobilized anti-VIIIR:Ag binds stoichiometrically the antigen and the activity of plasma factor VIII. These results together suggest that factor VIII is composed of 2 different entities, but undissociated under physiological conditions. Immunophysical analyses as a function of pH and temperature of anti-VIII:C and its complex with factor VIII show properties similar to those of homologous antibodies. The antigen determinants of VIII:C (VIII:CAg) are destroyed at low pHs or high temperatures, and VIII:C can no more form a complex with anti-VIII:C. Purified anti-VIII:C is also used in a two-stage assay to detect VIII:CAg or cross-reacting material in some severe haemophiliacs.

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.

Concentration-dependent dissociation of factor VIII in 1 M NaCl

1976 ◽  
Vol 230 (2) ◽  
pp. 434-440 ◽  
Author(s):  
Sussman ◽  
W Rosner ◽  
HJ Weiss
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Gel Filtration ◽  
Procoagulant Activity ◽  
Void Volume ◽  
Gradual Transition ◽  
Human Factor Viii ◽  
Factor Viii Concentrates ◽  
High Concentrations ◽  
Dilution Curves

Plasma, cryoprecipitate, Hemofil, and human factor VIII concentrate were dissolved in 1.0 M NaCl and chromatographed on Bio-Gel A-5m. With high concentrations of factor VIII the activity eluted as a symmetrical peak in the void volume; with a low factor VIII concentration the procoagulant activity was retarded. Dilution curves were performed for several human factor VIII concentrates. When the concentration of factor VIII was decreased, elution patterns showed a gradual transition from a peak in the void volume to a peak with a Ve/Vo of 1.7. Cryoprecipitate exhibited a similar behavior in 1.0 M NaCl, but the percent dissociation was greater than expected at high concentrations of factor VIII. When gel filtration was performed with 0.25 M CaCl2, significant dissociation occurred at all concentrations of factor VIII tested. The behavior of factor VIII in 1.0 M NaCl closely fit a theoretically derived curve for the dissociation of a protein from its binder. We conclude that the dissociation of factor VIII in 1 M NaCl is dependent on the concentration and purification of the procoagulant protein.

scholarly journals Human factor VIII: a calcium-linked protein complex

Blood ◽  
1983 ◽  
Vol 62 (5) ◽  
pp. 1006-1015 ◽  
Author(s):  
ME Mikaelsson ◽  
N Forsman ◽  
UM Oswaldsson
Keyword(s):  
Factor Viii ◽  
Protein Complex ◽  
Human Factor ◽  
Protein Complexes ◽  
Gel Filtration ◽  
Spectrophotometric Analysis ◽  
Human Factor Viii ◽  
Coagulant Activity ◽  
Heparin Plasma ◽  
Contrast Factor

Abstract The possible role of Ca2+ as an essential constituent part of the human factor VIII complex has been investigated by stability studies, metal determinations, and gel filtration experiments. In citrated plasma, the factor VIII coagulant activity (VIII:C) deteriorated during storage in a biphasic manner. Collection of blood in heparin, instead of chelating anticoagulants, or neutralization of citrate by addition of CaCl2 to heparinized citrate phosphate dextrose (CPD) plasma rendered VIII:C noticeably stable. At physiologic levels of ionized calcium, VIII:C was almost completely stable during incubation of plasma for 6 hr at 37 degrees C. The influence of other divalent ions was also studied. Highly purified factor VIII complex was subjected to atomic absorption spectrophotometric analysis and found to contain about 1.0 mole calcium per 220,000 daltons. This intrinsic calcium could be readily removed by EDTA. When heparin plasma and CPD plasma were chromatographed on Sepharose CL-6B at 37 degrees C, all the factor-VIII-related activities eluted together as large protein complexes. In contrast, factor VIII coagulant antigen (VIII:CAg) and factor-VIII-related antigen (VIIIR:Ag) were completely dissociated upon exposure to EDTA. From these observations it is concluded that human factor VIII circulates in normal plasma as a calcium-linked protein complex.

scholarly journals Human factor VIII: a calcium-linked protein complex

Blood ◽  
1983 ◽  
Vol 62 (5) ◽  
pp. 1006-1015
Author(s):  
ME Mikaelsson ◽  
N Forsman ◽  
UM Oswaldsson
Keyword(s):  
Factor Viii ◽  
Protein Complex ◽  
Human Factor ◽  
Protein Complexes ◽  
Gel Filtration ◽  
Spectrophotometric Analysis ◽  
Human Factor Viii ◽  
Coagulant Activity ◽  
Heparin Plasma ◽  
Contrast Factor

The possible role of Ca2+ as an essential constituent part of the human factor VIII complex has been investigated by stability studies, metal determinations, and gel filtration experiments. In citrated plasma, the factor VIII coagulant activity (VIII:C) deteriorated during storage in a biphasic manner. Collection of blood in heparin, instead of chelating anticoagulants, or neutralization of citrate by addition of CaCl2 to heparinized citrate phosphate dextrose (CPD) plasma rendered VIII:C noticeably stable. At physiologic levels of ionized calcium, VIII:C was almost completely stable during incubation of plasma for 6 hr at 37 degrees C. The influence of other divalent ions was also studied. Highly purified factor VIII complex was subjected to atomic absorption spectrophotometric analysis and found to contain about 1.0 mole calcium per 220,000 daltons. This intrinsic calcium could be readily removed by EDTA. When heparin plasma and CPD plasma were chromatographed on Sepharose CL-6B at 37 degrees C, all the factor-VIII-related activities eluted together as large protein complexes. In contrast, factor VIII coagulant antigen (VIII:CAg) and factor-VIII-related antigen (VIIIR:Ag) were completely dissociated upon exposure to EDTA. From these observations it is concluded that human factor VIII circulates in normal plasma as a calcium-linked protein complex.

scholarly journals Studies of the human factor VIII/von Willebrand's factor protein. II. Identification and characterization of the von Willebrand protein

Blood ◽  
1975 ◽  
Vol 46 (3) ◽  
pp. 417-430 ◽  
Author(s):  
HR Gralnick ◽  
BS Coller
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Related Protein ◽  
Human Factor Viii ◽  
Von Willebrand’S Factor ◽  
Von Willebrand ◽  
Identification And Characterization

Abstract The purified factor VIII-related protein we have previously characterized from normal cryoprecipitate possesses both procoagulant activity and vWf activity. We have attempted to isolate and characterize this protein from three patients with severe vWd. This protein is absent or markedly diminished in amount in these vWd patients, as judged by gel filtration, polyacrylamide-gel electrophoresis, and immunoprecipitation assays. Likewise, the procoagulant and vWf activities are deficient. As vWf activity is one of the major biologic functions of either the normal or hemophilic factor VIII-related protein, the purified protein should be designated the f VIII/vWf protein.

scholarly journals Immunodepletion of human plasma factor VIII

Blood ◽  
1980 ◽  
Vol 56 (4) ◽  
pp. 604-607
Author(s):  
F Ofosu ◽  
K Cassidy ◽  
MA Blajchman ◽  
J Hirsh
Keyword(s):  
Factor Viii ◽  
Affinity Chromatography ◽  
Human Plasma ◽  
Human Factor ◽  
Human Factor Viii ◽  
Plasma Factor

Affinity chromatography of human cryosupernatants on anti-human factor VIII-Sepharose yielded a plasma devoid of detectable factor VIIIC, VIIIR:Ag, and VIIIR:WF activities. This plasma was indistinguishable from severe congenital hemophilic plasma when used as substrate in factor VIII coagulant assays.

scholarly journals Effects of Plasmin on Human Factor VIII (AHF)

Blood ◽  
1973 ◽  
Vol 41 (1) ◽  
pp. 105-111 ◽  
Author(s):  
R. Pasquini ◽  
E. J. Hershgold
Keyword(s):  
Factor Viii ◽  
Gel Electrophoresis ◽  
Human Factor ◽  
Gel Filtration ◽  
Antigenic Determinants ◽  
Hemorrhagic Diathesis ◽  
Activity Assay ◽  
Factor Viii Activity ◽  
Immunologic Reactivity ◽  
Human Factor Viii

Abstract Highly purified, fibrinogen-free human factor VIII was incubated with plasmin, and the liberated split products of the factor VIII were analyzed by gel filtration, acrylamide gel electrophoresis, bioassay, and for immunologic reactivity. At least three fragments retaining different antigenic determinants are released from the factor VIII after prolonged digestion and at least three new fragments are seen in acrylamide gel electrophoresis. The split products were not anticoagulant in the factor VIII activity assay. In fact, the breakdown products in the hydrolysate increased the factor VIII activity of normal plasma mixed with it. Therefore, it is not likely that the factor VIII split products formed in fibrinolytic states contribute actively to the hemorrhagic diathesis.

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