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.

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.

Human factor VIII procoagulant activity and phospholipid interaction

1981 ◽  
Vol 678 (1) ◽  
pp. 132-136 ◽  
Author(s):  
A LAJMANOVICH ◽  
G HUDRYCLERGEON ◽  
J FREYSSINET ◽  
G MARGUERIE
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Procoagulant Activity ◽  
Human Factor Viii

scholarly journals Structure-Function Studies of Factor VIII/Von Willebrand Protein(s)

1977 ◽  
Author(s):  
Patrick A. McKee
Keyword(s):  
Factor Viii ◽  
Single Molecule ◽  
Gel Filtration ◽  
Protein S ◽  
Fold Increase ◽  
Procoagulant Activity ◽  
Void Volume ◽  
Pas Staining ◽  
Von Willebrand ◽  
Willebrand Factor

Factor VIII (FVIII) procoagulant activity was initially thought to be a glycoprotein with a molecular weight (MW) >1 million and composed of disulfide-1inked ~200,000 MW subunits. A protein with similar properties, except lacking procoagulant activity, is in hemophilic plasma; it was identical to normal FVIII by SDS-gel analyses, isoelectric focusing, and PAS staining. Subsequently it was shown that the FVIII glycoprotein also has von Willebrand factor (vWF) activity, suggesting that both FVIII and vWF activities might be properties of the same molecule. When the FVIII/vWF protein(s) is rechromatographed on 4% agarose and 0.25 M CaCl2, virtually all the protein and vWF activity elute in the void volume, but most of the FVIII procoagulant activity elutes much later. The extent of separation of the two activities depends on the amount of protein applied to the column. Also, exposure of the FVIII/vWF to thrombin before gel filtration strikingly accentuates separation of the two activities. The reduced SDS-gel pattern of the void volume protein peak showed the 200,000 MW subunit while that of the procoagulant peak contained several subunit bands which ranged from ~30,000–100,000 MW. Removal of sialic acid from FVIII/vWF is associated with reduced ristocetin induced platelet aggregation and causes a 50-fold increase in the rate of clearance of protein from the circulation by the hepatocyte. Currently, our data suggest that FVIII procoagulant and vWF activities are properties of a single molecule composed of disulfide-bound identical subunits. Cleavage by thrombin then results in FVIII procoagulant activity. Additional cleavages, to which the molecule appears very sensitive, results in FVIII inactivation. The vWF activity is very stable—even to proteolysis—and it appears to be a function of the carbohydrate side chains of the molecule.

scholarly journals A large region (approximately equal to 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity.

1986 ◽  
Vol 83 (16) ◽  
pp. 5939-5942 ◽  
Author(s):  
J. J. Toole ◽  
D. D. Pittman ◽  
E. C. Orr ◽  
P. Murtha ◽  
L. C. Wasley ◽  
...  
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Procoagulant Activity ◽  
Large Region ◽  
Human 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.

scholarly journals Human Factor VIII Concentrates in Hemophiliac Dogs

1977 ◽  
Author(s):  
Jessica H. Lewis ◽  
Ute Hasiba ◽  
Joel A. Spero
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Rapid Development ◽  
The Third ◽  
Human Factor Viii ◽  
Post Infusion ◽  
Factor Viii Concentrates ◽  
In Vitro Effects

Human factor VIII corrects the clotting defect in dog hemophilic plasma in vitro. The present studies were undertaken to see if this happened in vivo and to look for and document the development of an inhibitor. Four hemophiliac dogs were infused with factor VIII concentrates, the first two on five occasions, the others three times. Factor VIII:C, VIIIR:Ag (defined with antibody to human VIII) and VIIIR:vW were followed at pre, 10 minutes, 2 and 24 hours post infusion. The pre-infusion VIII:C (assayed with human substrate) averaged 0.23 U/ml compared to 6.93 U/ml for normal dogs; VIIIR:Ag was absent in both. VIIIR:vW was low but variable. Following the first injection, all four dogs responded in VIII:C about as calculated. The amounts of VIIIR:Ag and vW were much greater than VIII:C in the concentrates and in the post-first infusion samples from the dogs. On subsequent infusions rises in VIIIR:Ag were not detected and increases in VIII:C and VIIIR:vW were minimal. Precipitating anti-human VIII was found on the third infusion and thereafter. After the first infusion reactions were marked. Vomiting and diarrhea occurred in all, and one dog died in anaphylactic shock about one hour after the third infusion. Lack of response in VIIIR:Ag occurred before anti-VIII could be demonstrated in vitro. This rapid development of an inhibitor suggests that prolonged cross-species VIII therapy will not be successful. The ability of the precipitating anti-VIII elicited in the dogs to destroy VIII:C, VIIIR:Ag and VIIIR:vW is analagous to the in vitro effects of heterologous anti-VIIIs (rabbit and goat).

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 Development and Characterisation of Antibodies to Human Factor VIII in Haemophilic Dogs

1987 ◽  
Vol 57 (03) ◽  
pp. 314-321 ◽  
Author(s):  
Janet D Littlewood ◽  
T W Barrowcliffe
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Kinetic Studies ◽  
Cross Reaction ◽  
High Responder ◽  
Type Ii ◽  
Human Factor Viii ◽  
Factor Viii Concentrates ◽  
Low Responder

SummaryFour haemophilic dogs received infusions of human factor VIII concentrates, and developed inhibitors to human F VIII. These inhibitors cross-reacted with canine F VIII with parallel increases and decreases in titre. Cross-reaction was also found to porcine F VIII but changes in titre did not correlate with anti-human and anti-canine titres. These inhibitors were found to be immunoglobulins, and antibodies were detected against other proteins found in concentrates. Kinetic studies showed that in all four dogs the F VIII inhibitors were Type II antibodies. One of the dogs behaved as a “high-responder”, whilst another was more analogous to a “low-responder” patient. Phospholipid protection experiments in vitro demonstrated that some antibodies could be prevented from inhibiting F VIII, and porcine F VIII was particularly well protected against inhibition.

scholarly journals Immunologic studies on human factor VIII (anti-hemophilic factor A, AHF) components produced by low-ionic-strength dialysis

Blood ◽  
1976 ◽  
Vol 47 (2) ◽  
pp. 253-264 ◽  
Author(s):  
BN Bouma ◽  
JA van Mourik ◽  
S de Graaf ◽  
JM Hordijk-Hos ◽  
JJ Sixma
Keyword(s):  
Ionic Strength ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Human Factor ◽  
Procoagulant Activity ◽  
Factor Activity ◽  
Human Factor Viii ◽  
Low Ionic Strength ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Since dialysis of human factor VIII against buffers of low ionic strength yielded two distinct components, and since the factor VIII fraction isolated from normal plasma showed von Willebrand factor activity as defined by the corrective effect on abnormal platelet retention and ristocetin aggregation in von Willebrand's disease, the present studies were performed to determine if the correcting activities could be attributed to one or both of the components. Dialysis of factor VIII against buffers of low ionic strength led, however, to a decrease in factor VIII procoagulant activity and the reduction of the correcting activities, which suggested that the intact aggregate was required for procoagulant activity and for von Willebrand factor activity. In this respect dialysis of factor VIII at low ionic strength differed from dissociation at high salt concentrations. The two low ionic strength components were identified by the use of a rabbit antiserum against factor VIII, and could be distinguished on the basis of specific antigenic structures. Dialysis of factor VIII at low ionic strength led to a decrease in antigenic determinants closely related to factor VIII function. Specific antibodies to the low ionic strength components inhibited factor VIII activity in normal plasma, but the residual factor VIII was higher than that after inhibition with antibodies against intact factor VIII. Both antibodies interfered with von Willebrand factor activity.

Sign in / Sign up

Export Citation Format

Share Document