scholarly journals Immunological Evidence that Human Factor VIII is Composed of Two Linked Moieties

1977 ◽  
Author(s):  
J. Koutts ◽  
J.-M. Lavergne ◽  
D. Meyer
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Solid Phase ◽  
Molecular Size ◽  
Detectable Effect ◽  
Prolonged Incubation ◽  
Human Factor Viii ◽  
Single Protein ◽  
Antigen Antibody ◽  
Ristocetin Cofactor

Whether the three measurable parameters of factor VIII (procoagulant activity, VIII:C; ristocetin cofactor activity, VIIIR:WF; and factor VIII related antigen, VIIIR:AG) reside on a single protein remains disputed. A solid phase immunoadsorption system, in which homologous antibodies to VIII:C arising in haemophiliacs were insolubilized onto Sepharose, was used to examine the action of such antibodies and the inter-relationship between VIII:C, VIIIR:WF and VIIIR:AG. Homologous antibodies were shown to bind specifically VIII:C and to induce a spontaneous separation of VIII:C from VIIIR:WF/VIIIR:AG. The bond between VIII:C and the homologous antibodies bound to Sepharose appeared to be very stable and could not be broken with the usual antigen-antibody dissociating agents. Following prolonged incubation with antibody-sepharose, concentrated VIIIR:WF/VIIIR:AG (20 u/ml), completely devoid of VIII:C and inhibitor-neutralizing activity, was obtained. The loss of VIII:C had no detectable effect on the molecular size, antigenicity or electrophoretic mobility of the original molecule. The concentrated VIIIR:WF/VIIIR:AG was used to absorb heterologous antisera raised against factor VIII. Specific heterologous antisera to VIII:C, no longer neutralizing VIIIR:WF nor precipitating with VIIIR:AG, were obtained. Immunization of rabbits with VIIIR:WF/VIIIR:AG resulted in antisera which potently neutralized VIIIR:WF and precipitated with VIIIR:AG but also weakly neutralized VIII:C. These antibodies, like 4 other heterologous antibodies to Factor VIII studied, did not neutralize VIII:C which had been dissociated from VIIIR:WF/VIIIR:AG.The results indicate that VIII:C and VIIIR:WF/VIIIR:AG are two different, but linked entities.

scholarly journals The Effects of Injection of Human Factor VIII Antibody Into Rabbits

Blood ◽  
1973 ◽  
Vol 42 (4) ◽  
pp. 509-521 ◽  
Author(s):  
S. M-C. Shen ◽  
D. I. Feinstein ◽  
S. I. Rapaport
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Rabbit Model ◽  
Kinetic Studies ◽  
Factor Vii ◽  
Factor V ◽  
Initial Values ◽  
Human Factor Viii ◽  
Hemolytic Complement Activity ◽  
Antigen Antibody

Abstract Rabbits were injected with an immunoglobulin fraction of human serum containing a factor VIII antibody. Factor VIII levels fell abruptly, persisted below 10% of a rabbit plasma standard for 12 hr, and returned to normal by 120-168 hr. The factor VIII antigen-antibody reaction did not result in Intravascular clotting as evaluated by kinetic studies with 125I-fibrinogen. However, small falls in factor V and factor VII levels were observed over a 6-hr period after the injection. Platelets fell to about one-half of initial values within 15 min, rose to 80% of initial levels over 2 hr, and subsequently declined to 65%-70% of initial levels. WBC levels fell to below 20% of initial values 2 hr after the injection but returned to about 75% of initial values by 6 hr. Total hemolytic complement activity was unaffected. Animals made granulocytopenic with nitrogen mustard and animals with hereditary C'6 deficiency behaved similarly to normal animals. One may conclude that the injection of human factor VIII antibody into rabbits produces a rabbit model with impaired intrinsic coagulation suitable for studies of the mechanism of endotoxin-induced intravascular clotting.

Antisera To Human Factor VIII Raised In Balb/C Mice. The Production Of Monoclonal Antibodies To Factor VIII

1981 ◽  
Author(s):  
D E Joshua ◽  
T Exner ◽  
H Kronenberg ◽  
K A Rickard
Keyword(s):  
Monoclonal Antibodies ◽  
Factor Viii ◽  
Human Factor ◽  
Anticoagulant Activity ◽  
Parental Line ◽  
Plasma Samples ◽  
Radioimmune Assay ◽  
Human Factor Viii ◽  
Factor Viii Antigen ◽  
Ristocetin Cofactor

The aim of this series of experiments is to raise antisera to human Factor VIII in Balb/C mice, study the properties of these antisera, and use the spleen cells from such hyperimmune mice to raise a panel of monoclonal antibodies directed against the varying antigenic determinants of the human factor VIII complex. Antibody activity against factor VIII antigen was determined by a microtitre solid phase radioimmune assay. This involved exposing test antisera to human factor VIII-coated PVC wells, and detection of bound mouse antibody by subsequent exposure to 125I labelled rabbit antimouse immunoglobulin. Anticoagulant assays and ristocetin cofactor inhibition assays were based on standard methods adapted to microtitre plates. Balb/C mice were immunized with gel filtered factor VIII complex and boosted immediately prior to fusion. Fusion was performed with the parental line P3-NSI/l-Ag-l in polyethylene glycol (PEG). Compared to rabbits Balb/C mice produced considerably higher anticoagulant activity. Pooled rabbit anti-factor VIII coagulant activity was 1.2 Oxford units/ml whereas the anticoagulant activity of 6 mice ranged between 30-185 Oxford units/ml. Incubation of mouse antisera with human plasma samples and subsequent assay of residual mouse anti-VIII antigen demonstrated that normal and haemophiliac plasma markedly inhibited binding of the antisera whereas plasma samples from patients with Von Willebrand's disease did not. This system has been optimized as a microtitre radioimmune assay for human factor VIII antigen. There is good correlation with the Laurell “rocket” technique (r=0.86; n=14). A number of monoclonal antibodies which bind to the factor VIII complex have been produced. They have no anticoagulant activity nor activity against ristocetin cofactor. They recognize antigens present in plasma fractions after DEAE Sephadex A-50 separation which differ from those fractions recognized by the heterologous Balb/C antisera.

Von Willebrand Activity of Low Molecular Weight Human Factor VIII Increases by Binding to Gold Granules

1981 ◽  
Vol 45 (03) ◽  
pp. 242-246 ◽  
Author(s):  
Miha Furlan ◽  
Beat A Perret ◽  
Eugene A Beck
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Human Factor ◽  
Low Molecular Weight ◽  
Human Factor Viii ◽  
Large Factor ◽  
Cofactor Activity ◽  
Von Willebrand ◽  
Ristocetin Cofactor ◽  
Ristocetin Cofactor Activity

SummaryHuman factor VIII/von Willebrand protein is a population of multimers which vary in size but contain apparently identical subunits. Large-molecular-weight forms possess higher ristocetin cofactor/von Willebrand activity than the native smaller oligomers. Disulfide reduction of large factor VIII multimers results in progressively decreasing molecular size and a loss of ristocetin cofactor activity. Small molecular forms of factor VIII were adsorbed onto gold granules (average diameter 20-30 nm) and thereby increased their ristocetin cofactor activity. The amount of adsorbed material and the extent of activation were dependent on the pH of the colloid suspension. The maximum recovery of von Willebrand activity was observed at pH 4.75. Aggregation of fixed human platelets by factor VIII-coated gold particles was dependent on ristocetin concentration and was not competitively inhibited by unbound low-molecular-weight factor VIII. These results suggest that the subunits of the native small factor VIII species possess potential binding affinity for platelet receptors, which is manifested following formation of large factor VIII polymers. We conclude that an optimal size of remarkably high molecular weight is required for efficient aggregation of platelets by factor VIII as occurs during the primary phase of hemostasis.

scholarly journals Isolation of Specific Homologous Antibodies to Factor VIII by Immunoadsorption

1977 ◽  
Author(s):  
J.-M. Lavergne ◽  
D. Meyer ◽  
J. Koutts ◽  
N. Ardaillou ◽  
J. P. Girma ◽  
...  
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Solid Phase ◽  
High Titer ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Radioactivity ◽  
The Third ◽  
Human Factor Viii ◽  
Subsequent Elution ◽  
Willebrand Factor

Current immunological studies of Factor VIII use heterologous antibodies which predominantly measure Willebrand Factor (WF) and give little information on Factor VIII procoagulant activity (VIII:C). Purification of homologous antibodies specific for VIII:C has been hampered by the fact that they do not form immune precipitates. We have attempted to isolate such antibodies by solid phase immunoadsorption and subsequent elution. Human Factor VIII was specifically bound to goat anti-human Factor VIII IgG previously immobilized onto Sepharose 6 B beads. IgG isolated from a Haemophiliac with a high titer anti-VIII:C antibody (700 Oxford U/ml) was labeled with 125-I and reacted for 72 hours with these beads. The column was then washed with 0.1 M glycine, 0.5 M NaCl, pH 10 buffer to remove non specifically adsorbed material. Specifically adsorbed material was then eluted with 2.5 M MgCl2, pH 7.3, and the peak of radioactivity was filtered on Biogel A-5 m. Both anti-VIII:C and radioactivity were recovered in three distinct peaks. The third peak, corresponding to IgG, contained anti-VIII:C activity with a 10 fold purification as estimated by specific radioactivity. The second peak, eluting just in front of the IgG, had half the specific radioactivity of the third peak. The first peak, corresponding to the Vo, contained Factor VIII related antigen and very little anti-VIII:C activity. The pattern by SDS-polyacrylamide gel electrophoresis is compatible with the existence of Factor VIII (WF-VIII:C)-anti-VIII:C complexes in the first peak; VIII:C-anti-VIII:C complexes in the second, and free anti-VIII:C IgG in the third one. Thus the method leads to the formation of stable VIII:C-anti-VIII:C complexes, allowing the purification of specific human anti-VIII:C antibodies.

scholarly journals Inhibition of Human Platelet Aggregation by Plasmin Digests of Human Factor VIII

Blood ◽  
1973 ◽  
Vol 42 (5) ◽  
pp. 749-751 ◽  
Author(s):  
M. B. Donati ◽  
G. de Gaetano ◽  
J. Vermylen ◽  
M. Verstraete
Keyword(s):  
Platelet Aggregation ◽  
Factor Viii ◽  
Human Factor ◽  
Human Platelet ◽  
Hemorrhagic Diathesis ◽  
Thrombin Time ◽  
Prolonged Incubation ◽  
Human Platelet Aggregation ◽  
Human Factor Viii

Abstract Human factor VIII, upon prolonged incubation at 37°C with plasmin, inhibits platelet aggregation induced by ADP, adrenaline, or collagen. The human factor VIII digests are not anticoagulant in the thrombin time or partial thromboplastin time. Factor VIII split products may interfere in vivo with platelet function and thus could contribute in some way to the hemorrhagic diathesis of fibrinolytic states.

scholarly journals Antibodies to Factor VIII. I. Variations in Stability of Antigen-Antibody Complexes in Hemophilia A

Blood ◽  
1973 ◽  
Vol 42 (3) ◽  
pp. 437-444 ◽  
Author(s):  
J.-P. Allain ◽  
D. Frommel
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Direct Consequence ◽  
Antigenic Stimulation ◽  
Factor Viii Activity ◽  
Human Factor Viii ◽  
Antigen Antibody ◽  
Stimulation Of

Abstract Human factor VIII-anti-factor VIII complexes were formed in vitro in slight antigen excess, using plasma of hemophiliacs who were found to have antibodies neutralizing AHF activity. These complexes, stable at +37°C and pH 7.4, were submitted to classical procedures known to favor dissociation of antibody from antigen. The methods used to obtain dissociation, incubations at +56°C and at pH 4.2, inactivated both unbound factor VIII and that released as a consequence of dissociation. The extent of dissociation was measured by the recovery of anti-factor VIII activity. Increasing resistance of complexes towards dissociation was observed in the plasma of the patients whose titer of inhibitor was increasing after recent transfusions. These observations suggested the emergence, as a direct consequence of renewed antigenic stimulation, of a population of different antibodies characterized by higher combining strength.

scholarly journals The Apparent Separation of Factor VIII Related Antigen and Coagulant Activity from von Willebrand Factor Activity by an Immunoadsorbent

1977 ◽  
Author(s):  
H. A. Cooper ◽  
D. Lee ◽  
M. A. Lamb ◽  
R. H. Wagner
Keyword(s):  
Factor Viii ◽  
Human Plasma ◽  
Human Factor ◽  
Solid Phase ◽  
Residual Activity ◽  
Normal Human Plasma ◽  
Human Factor Viii ◽  
Coagulant Activity ◽  
Plasma Fractions ◽  
Normal Human

An antibody was raised in rabbits to the small active fragment of human factor VIII. The antigen was obtained by Ca2+ dissociation of a human factor VIII preparation made from a multidonor pool of plasma. After two adsorptions with 0.1 volume of normal human plasma, the antibody neutralized the F. VIII coagulant activity of normal human plasma, but did not precipitate with any plasma or plasma fractions nor did it neutralize vWF activity as measured by ristocetin aggregation of fixed washed platelets. A solid phase immunochemical reagent was prepared by CNBr binding of the partially purified rabbit antibody to 1% agarose beads. Non-immune beads were similarly prepared with IgG fractions from a normal non-immunized rabbit. Using a batch technique the beads were studied for their ability to remove F. VIII coagulant, F. VIII Ag, and vWF activity from normal human plasma. Assay of the supernatant plasma after 2 hrs, 22°, from 10 replicate experiments gave the following results for residual activity, as per cent of non-immune bead control:F. VIII (37.5±4), F. VIII-Ag (30.8±9.7), and vWF (72.1±16). The experiment was repeated with 6 replicate samples with higher ratio of beads to plasma with essentially similar results. This unexpected separation of F. VIII-Ag from vWF activity prompted further investigation into how these activities are related to the molecular structure of F. VIII and vWF.

A Simplified Immunoradioactive Assay For Human Factor VIII Coagulation Antigen

1981 ◽  
Author(s):  
K B Thomas ◽  
M A Howard ◽  
J Koutts ◽  
B G Firkin
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Solid Phase ◽  
Biological Activities ◽  
Test Sample ◽  
High Titre ◽  
Standard Curve ◽  
Normal Individuals ◽  
Human Factor Viii ◽  
Von Willebrand

Normal human factor VIII (FVIII) is a high molecular weight glycoprotein expressing two measurable biological activities, FVIII procoagulant activity (VIII:C) and von Willebrand factor activity (vWf). The corresponding antigenic moieties are referred to respectively as FVIII coagulation antigen (VIII:CAg) and FVIII related antigen (VIII: RAg). Since VIII:C is extremely labile measurement of VIII: CAg is of importance for pre-natal diagnosis of haemophilia where VIII:C is reduced and von Willebrand’s disease (vWd) where all measurable parameters of FVIII are reduced.Current immunoradiometric assays for VIII:CAg depend on both the availability of high titre human antibodies directed against VIII:C and the possibility of preparing highly specific 125I-IgG from human antiserumWe have developed a solid phase immunoradiometric assay for VIII:CAg. FVIII in a test sample binds to human anti- VIII:C IgG immobilised on the surface of a polystyrene tube. The bound FVIII is detected using rabbit 125I-anti human FVIII-IgG. The standard curve was linear for dilutions of normal pooled plasma (NPP) of 1/5 to 1/500. The lower limit of the assay was 0.002U/ml of NPP. Using this assay the concentration of VIII:CAg correlated well with the VIII:C levels in the plasma from normal individuals (r=0.84, n=15). Homozygote vWd patients had undetectable levels of VIII:CAg, VIII:C, VIII:RAg or vWf activities. Patients with haemophilia A with less than O.OlU/ml VIII:C could be divided into two groups on the basis of the VIII:CAg assay, CRM and CRMf. Markedly reduced levels of VIII:CAg were detected in serum.The advantage of this assay over the previously available assays for VIII:CAg are that smaller volumes of high titre human anti VIII IgG are required and the time consuming step of preparing 125I-human anti VIII-IgG is eliminated. The rabbit 125I-IgG specific for human VIII is the same as used in routine immunoradiometric assays for FVIII:RAg

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.

Sign in / Sign up

Export Citation Format

Share Document