The Specificity of Antibodies to Factor VIII Produced in the Rabbit after Immunization with Human Cryoprecipitate

1973 ◽  
Vol 29 (03) ◽  
pp. 652-660 ◽  
Author(s):  
P. B. A Kernoff ◽  
C. R Rizza
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Neutralizing Antibody ◽  
Normal Subjects ◽  
Factor Viii Activity ◽  
Marked Rise ◽  
Antigenic Sites ◽  
Level Of Activity ◽  
Human Factor Viii ◽  
The Relationship

SummaryThe relationship between the activity-neutralizing and precipitating activities of rabbit antibody to human factor VIII was studied by measuring the changes in levels of the two activities in two sensitized rabbits after stimulation with cryoprecipitates prepared from the plasmas of normal subjects, haemophiliacs and patients with von Wil- lebrand’s disease. Injection of cryoprecipitates prepared from plasmas with a detectable level of factor VIII clotting activity was followed by a marked rise in the level of activity-neutralizing antibody, whereas there was no rise or a continued fall in level after injection of cryoprecipitates prepared from haemophilic plasmas without detectable factor VIII activity. The level of precipitating antibody rose markedly after injection of normal and haemophilic cryoprecipitates, but little or not at all after cryoprecipitates prepared from the plasma of the patients with von Willebrand’s disease. It is suggested that the specific antigenic sites associated with factor VIII clotting activity were not present in cryoprecipitates prepared from haemophilic plasmas without detectable factor VIII activity, and also that antibodies of two different specificities could be detected.

scholarly journals The Activation of Factor VIII by Thrombin

Blood ◽  
1965 ◽  
Vol 26 (4) ◽  
pp. 500-509 ◽  
Author(s):  
A. H. ÖZGE-ANWAR ◽  
G. E. CONNELL ◽  
J. F. MUSTARD
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Experimental Approach ◽  
Factor Viii Activity ◽  
Clotting Factors ◽  
Activation Effect ◽  
Activation Reaction ◽  
Human Factor Viii

Abstract The activation of human factor VIII by thrombin has been demonstrated by a new experimental approach. This method permitted investigation of the interaction of thrombin and factor VIII in the absence of most other clotting factors. The activation effect of thrombin is susceptible to inhibition by diisopropylfluorophosphate. Trypsin cannot replace thrombin in the activation reaction, and it destroys factor VIII activity rapidly.

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.

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 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.

Studies on the Neutralization of Rabbit Antibodies to Human Factor VIII

1977 ◽  
Vol 38 (03) ◽  
pp. 0707-0716 ◽  
Author(s):  
P. B. A Kernoff ◽  
A. C Kaelin
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Quantitative Estimation ◽  
Anticoagulant Effect ◽  
Human Origin ◽  
Antigenic Sites ◽  
Human Factor Viii ◽  
Neutralizing Capacity

SummaryThe capacity of normal and haemophilic cryoprecipitates to neutralize the anticoagulant effect of rabbit antibodies to human factor VIII (anti-VIII) was assessed using a quantitative estimation of antibody. About 4 times as much anti-VIII could be neutralized by normal factor VIII as was required to neutralize clotting activity. This suggests that there are probably several antigenic sites intimately associated with factor VIII clotting activity, quite apart from any antigenic sites which may be detected using antibodies directed against other components of the factor VIII complex. The neutralizing capacity of factor VIII was only slightly greater for the rabbit antibodies employed in this study than has been previously reported for antibodies of human origin, thus indicating only minor differences in specificities. Additional evidence in support of this concept was the finding that cryoprecipitates prepared from haemophilic plasmas previously recognized as either lacking or possessing the capacity to neutralize antibodies of human origin neutralized least or most quantities of rabbit antibodies, respectively.

ACTIVATION AND INACTIVATION OF PURIFIED HUMAN FACTOR VIII BY THROMBIN

1987 ◽  
Author(s):  
K Sewerin ◽  
A Lundin ◽  
E Hellström ◽  
K Larsen ◽  
H Sandberg
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Human Factor ◽  
Peptide Chain ◽  
Factor Viii Activity ◽  
High Concentration ◽  
Human Factor Viii ◽  
Human Thrombin ◽  
Peptide Composition ◽  
High Concentrations

Highly purified human factor VIII was activated by human thrombin. The factor VIII material used showed variation in molecular weight and was separated into one heterogeneous form with a molecular weight ranging from 280 to 185 kDa and into one homogeneous 170 kDaform. Incubation of the (280-185) kDa form of factor VIII with thrombin resulted in proteolytic degradation of the heavy chain and formation of a 90 kDa peptide chain. This chain was further split intoone 52 kDa and one 43 kDa peptide chain, as was the 90 kDa chain pesent in the 170 kDa form of factor VIII. The light chain of 80 kDa was split into a 70 kDa peptide chain. The change in peptide composition was followed by scanning and integration of SDS-PAGE gels on samples from the activation of both forms of factor VIII with thrombin. No strict relationship between factor VIII activity and degradation or formation of a new peptide chain was seen.When factor VIII bound to von Willebrand factor matrix was incubated with thrombin, activated factor VIII was released. The released material consisted mainly of a 90 kDa and a 70 kDa peptide chain, but the presence of the 52 and 43 kDa peptide chains increased with the passage of time. The concentration of Ca2+ ions was of great importance for the degree and rate of activation. A high concentration of CaCl2 resulted in a lower degree of activation but also slower inactivation of the factor Villa formed. However, no permanent stabilization of factor Villa was noted at either low or high concentrations of CaCl2.Inhibition or removal of thrombin from the factor VIII system, by the use of protease inhibitors or matrix bound thrombin, did not prevent the loss of factor VIII activity following the activation. Thepeptide composition was stabilized, however, when thrombin was removed. This indicates that the inactivation of factor Villa is due to an intramolecular change in the factor VIII molecule.

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