Selective Measurement of Human Factor VIII Activity in the Citrated Plasma of Laboratory Animals

2019 ◽  
Author(s):  
A. Engelmaier ◽  
C. Zlabinger ◽  
A. Weber
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Laboratory Animals ◽  
Factor Viii Activity ◽  
Human Factor Viii

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.

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.

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.

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.

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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