The Development Of Monoclonal Antibodies To Factor VIII Related Antigen (VIIIRAG)

1981 ◽  
Author(s):  
J E Thomas ◽  
R A Furlong ◽  
J C Giddings ◽  
I R Peake ◽  
A L Bloom
Keyword(s):  
Factor Viii ◽  
Serum Antibody ◽  
Selective Medium ◽  
Dilution Method ◽  
Gel Chromatography ◽  
Spleen Cells ◽  
Human Factor Viii ◽  
Mouse Immunoglobulin ◽  
Serum Antibody Titre ◽  
Ristocetin Cofactor

A human VIIIRAg rich fraction was prepared from human factor VIII concentrate by gel chromatography on Sepharose CL6B to give 40 u/ml VIIIRAg and 0.06 u/ml factor VIII clotting antigen (VIIICAg). Balb/c mice were immunised with this fraction (1-2 u VIIIRAg per mouse) and polyclonal antibody to VIIIRAg was detected in the serum of the mice by conventional immunodiffusion and by a specially developed immunoradlometric test (IRMT). This test utilised polystyrene tubes coated with polyclonal (sheep) antibody to VIIIRAg which were then incubated with a source of human VIIIRAg (pooled normal plasma). After incubation with mouse anti human VIIIRAg the bound mouse immunoglobulin was detected by binding of 125I labelled rabbit anti mouse IgG. This was prepared from commercial rabbit anti mouse IgG by immunoadsorption and elution from immobilised mouse immunoglobulin. Using the IRMT a monoclonal antibody to VIIIRAg was detectable at a dilution of 5 × 105. Mice with the highest serum antibody titre were selected, hyper- immunised and used as spleen donors. Spleen cells were harvested, fused with homologous NS-1 myeloma cells and cultured in HAT selective medium. Hybrids producing specific antibody to VIIIRAg were detected by the IRMT and cloned by the limiting dilution method. Antibodies were tested for their activity against VIIIRAg and factor VIII related ristocetin cofactor activity, and also for any activity against VIIICAg and procoagulant factor VIII.

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 Microheterogeneity of Human Factor VIII

1977 ◽  
Author(s):  
J.A. van Mourik ◽  
J. A. Hellings ◽  
E. M. Hoorweg
Keyword(s):  
Ionic Strength ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Human Factor ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Large Pore ◽  
Human Factor Viii ◽  
Von Willebrand ◽  
Willebrand Factor

Human factor VIII is a glycoprotein and tends to form a variety of large aggregates, the presence of which was demonstrated by a number of techniques including the use of large pore gel chromatography and electrophoresis using a polyacrylamide gel system designed for high molecular weight aggregates. Factor VIII aggregates can be fractionated in part according to molecular size. Reduction of these polymers by 2-mercaptoethanol results in presumably identical fragments suggesting that all polymers, though differing in size, are composed of identical subunits.Factor VIII aggregates are particularly sensitive to proteolytic breakdown by trypsin and plasmin as judged by large pore polyacrylamide gel electrophoresis. Short-term incubation of factor VIII with trace quantities (equivalent to plasmin- or trypsin-like activities present in normal plasma) of these enzymes respectively results in substantial fragmentation with concurrent loss of both factor VIII procoagulant and von Willebrand factor activity. However, factor VIII activity is lost prior to discernible protein fragmentation whereas von Willebrand factor inactivation is associated with advanced protein degradation. When the ionic strength of the medium is lowered the susceptibility of factor VIII to proteolytic breakdown by trypsin is increased dramatically. Interestingly, degradation of factor VIII by plasmin is not affected by the ionic strength. These data, then, provide conclusive evidence of microheterogeneity of normal human factor VIII and may account for the observed heterogeneity of factor VIII on crossed immunoelectrophoresis.

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.

Plasmin Digestion of factor VIII: Characterization of the Breakdown Products with Respect to Antigenicity and von Willebrand Activity

1978 ◽  
Vol 40 (02) ◽  
pp. 302-315 ◽  
Author(s):  
J A Guisasola ◽  
C G Cockburn ◽  
R M Hardisty
Keyword(s):  
Factor Viii ◽  
Biological Activities ◽  
Molecular Size ◽  
Progressive Increase ◽  
Gel Chromatography ◽  
Molecular Weights ◽  
Von Willebrand ◽  
Ristocetin Cofactor

SummaryHighly purified factor VIII was incubated for up to 24 hours in the presence of plasmin, and the biological activities and peptide structure of the digestion products determined at intervals. Procoagulant activity (VIIIC) was rapidly lost, but 17-32% of the initial ristocetin cofactor (VIIIR: WF) activity remained after 24 hours. Immunoelectrophoresis showed a progressive increase in rocket height and a concomitant increase in electrophoretic mobility of the factor-VIII-related antigen (VIIIR: AG). Crossed immunoelectrophoresis of the 24- hour digest showed three distinct precipitin arcs, of which the major one, with intermediate anodal mobility, gave reactions of non-identity with the other two. On sepharose gel chromatography the 24-hour digest gave three peaks: peak II contained about 80% of the residual VIIIR: WF and resolved on SDS-polyacrylamide gels into a series of peptides with apparent molecular weights between 125,000 and 185,000; these were reduced by mercaptoethanol to fragments of 15,000-80,000 daltons, a 65,000 dalton fragment being particularly strongly PAS positive. We conclude that large molecular size is not a prerequisite for VIIIR :WF activity, and that the presence of factor-VIII breakdown products may be a cause of misleading results in the determination of VIIIR:AG by immunoelectrophoresis.

T Lymphocyte Proliferative Responses Induced by Recombinant Factor VIII in Hemophilia A Patients with Inhibitors

1996 ◽  
Vol 76 (01) ◽  
pp. 017-022 ◽  
Author(s):  
Sylvia T Singer ◽  
Joseph E Addiego ◽  
Donald C Reason ◽  
Alexander H Lucas
Keyword(s):  
T Lymphocytes ◽  
Factor Viii ◽  
Cellular Proliferation ◽  
Mononuclear Cells ◽  
Hemophilia A ◽  
Normal Subjects ◽  
Normal Male ◽  
Cell Fractionation ◽  
Severe Hemophilia ◽  
Human Factor Viii

SummaryIn this study we sought to determine whether factor VUI-reactive T lymphocytes were present in hemophilia A patients with inhibitor antibodies. Peripheral blood mononuclear cells (MNC) were obtained from 12 severe hemophilia A patients having high titer inhibitors, 4 severe hemophilia A patients without inhibitors and 5 normal male subjects. B cell-depleted MNC were cultured in serum-free medium in the absence or presence of 2 µg of recombinant human factor VIII (rFVIII) per ml, and cellular proliferation was assessed after 5 days of culture by measuring 3H-thymidine incorporation. rFVIII induced marked cellular proliferation in cultures of 4 of 12 inhibitor-positive hemophilia patients: fold increase over background (stimulation index, SI) of 7.8 to 23.3. The remaining 8 inhibitor-positive patients, the 4 hemophilia patients without inhibitors and the 5 normal subjects, all had lower proliferative responses to rFVIII, SI range = 1.6 to 6.0. As a group, the inhibitor-positive subjects had significantly higher proliferative responses to rFVIII than did the inhibitor-negative and normal subjects (p < 0.05 by t-test). Cell fractionation experiments showed that T lymphocytes were the rFVIII-responsive cell type, and that monocytes were required for T cell proliferation. Thus, rFVIII-reactive T lymphocytes are present in the peripheral circulation of some inhibitor-positive hemophilia A patients. These T cells may recognize FVIII in an antigen-specific manner and play a central role in the regulation of inhibitor antibody production

Factor VIII-Related Activities in Normal, Haemophilic and von Willebrand’s Disease Platelet Fractions

1978 ◽  
Vol 40 (02) ◽  
pp. 288-301 ◽  
Author(s):  
P Meucci ◽  
I R Peake ◽  
A L Bloom
Keyword(s):  
Factor Viii ◽  
Electrophoretic Mobility ◽  
Concanavalin A ◽  
Freezing And Thawing ◽  
Von Willebrand's Disease ◽  
Factor Viii Related Antigen ◽  
Von Willebrand’S Disease ◽  
Normal Plasma ◽  
Significant Difference ◽  
Ristocetin Cofactor

SummaryFactor VIII-related activities have been studied in platelet fractions in order to try to reconcile the conflicting findings of other workers, and to extend the studies. In platelets from 16 normal subjects procoagulant factor VIII was not detected. The amount of factor VIII-related antigen (FVIIIR: AG) in the cytosol per mg of protein was about twice that in the membrane fraction and about ten times that in the debris fraction. There was no significant difference between the amount of FVIIIR: AG and ristocetin cofactor (RistCof) activity in each fraction. The findings in haemophilic platelets were similar. In von Willebrand’s disease (vWd) one serverely affected patient had no detectable factor VIII related activities in any platelet fraction. In 5 patients with intermediate vWd results were normal. In a further 5, with more prolonged bleeding times, no FVIIIR: RistCof was detected in platelets, despite a normal amount of FVIIIR: AG in the cytosol and debris. The electrophoretic mobility of cytosol FVIIIR: AG was increased in all normals and patients, while that in the membrane and debris fractions had normal mobility. Cytosol FVIIIR: AG eluted later than normal FVIIIR: AG on gel filtration on Sepharose 2B, and also showed reduced antibody binding in an immunoradiometric assay. Precipitation of FVIIIR: AG by concanavalin A was incomplete in all platelet fractions from normals, and even more reduced in vWd platelet fractions. The results suggest the possibility of two types of platelet FVIIIR: AG.A factor VIII-related antigen was shown to be associated with normal washed platelets by immunofluorescence techniques (Bloom et al. 1973). Since then, several studies have been reported on the localisation of factor VIII related antigen (FVIIIR: AG), factor VIII procoagulant activity (FVIII: C) and factor VIII related ristocetin cofactor activity (FVIIIR: RistCof) within the platelets. Initially, Howard et al. (1974) indicated that FVIIIR: AG was firmly bound to the platelet membrane, and noted that in lysed platelets the level of FVIIIR: AG as measured by electroimmunodiffusion was higher than that in whole platelet suspensions. However, further studies by Nachman and Jaffe (1975) showed that FVIIIR: AG was also present to a considerable extent in the granules, and they detected none in the platelet cytosol. Bouma and colleagues (1975) were, however, able to find FVIIIR: AG and FVIIIR: RistCof in the cytosol upon freezing and thawing platelets. This FVIIIR: AG had an electrophoretic mobility comparable to that of normal plasma. They also noted that platelets which were air dried apparently had a granular FVIIIR:AG localisation by immunfluorescence; however, intact platelets in suspension did not stain by this method.Recently Ruggeri et al. (1977) and Sultan et al. (1977) have also found FVIIIR: AG in the cytosol, and the former authors reported it to have increased electrophoretic mobility when compared to normal plasma FVIIIR:AG. Results concerning the localisation of FVIIIR: AG in normal platelets have thus been conflicting. Similarly, in the few reports available concerning platelet FVIIIR: AG in von Willibrand’s disease variable results have also been obtained (Ruggeri et al. 1977, Howard et al. 1974, Shearn et al. 1974 and Bouma et al. 1975).In this study we report on the localisation of factor VIII-related activities in normal, haemophilic and von Willebrand’s disease platelets using available standard techniques as well as precipitation of FVIIIR: AG with the plant lectin concanavalin A, a procedure which has been shown to detect abnormal forms of FVIIIR:AG in certain types of von Willebrand’s disease (Peake and Bloom 1977).

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.

Impaired Prothrombin Consumption in Bernard-Soulier Syndrome Is Corrected In Vitro by Human Factor VIII

1997 ◽  
Vol 77 (02) ◽  
pp. 383-386 ◽  
Author(s):  
S Bellucci ◽  
J P Girma ◽  
M Lozano ◽  
D Meyer ◽  
J P Caen
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Platelet Membrane ◽  
Giant Platelets ◽  
Prolonged Bleeding Time ◽  
Human Factor Viii ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Bernard Soulier Syndrome

SummaryThe Bernard-Soulier syndrome (BSS) is characterized by thrombocytopenia with giant platelets, a prolonged bleeding time with defective platelet adhesion to the subendothelium related to a defect in platelet membrane glycoprotein lb (GPIb) and a decreased prothrombin consumption. The mechanism of the latter abnormality remains unknown. In this study, we showed that this defect was corrected by the addition of purified human factor VIII (FVIII) to blood from four patients with BSS. The correction of prothrombin consumption was almost complete at concentrations between 1.5 and 3 IU/ml of FVIII procoagulant activity (VIII.'C) and partially abolished by a monoclonal antibody which neutralizes VIII:C. This correction was specific for FVIII and was not observed after addition of purified human FIX. It was obtained, in the same magnitude range, with FVIII complexed to von Willebrand factor (vWF) but not with free vWF. These data provide a new insight into the knowledge of the physiological interaction between the platelet membrane and the vWF-FVIII complex facilitating plasma coagulation activation and may lead to helpful therapeutic advances.

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