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.

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.

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.

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.

PARADOXICAL INCREASE IN HUMAN FACTOR VIII AFTER INFUSION OF PORCINE FACTOR VIII CONCENTRATE IN A PATIENT WITH ACQUIRED VARIANT VON WILLEBRAND'S DISEASE

1987 ◽  
Author(s):  
J Ball ◽  
R G Malía ◽  
M Greaves ◽  
F E Preston
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Human Factor ◽  
De Novo ◽  
Similar Time ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Human Factor Viii ◽  
Plasma Factor ◽  
Abnormal Pattern

A patient with acquired variant von Willebrand's disease was given an infusion of 2000 units of high purity porcine factor VIII (Hyate). Quantitative factor VIII parameters were assessed following infusion and human factor VIII multimers were analysed by radioimmunoelectrophoresis and autoradiography. We have previously described the patient to have acquired von Willebrand's disease due to a circulating inhibitor to the factor VIII complex (B. J. Haematol-, 54,233,1983). Prior to infusion plasma from the patient contained factor VIIIC, RRCo, and vWFAg at less than 10 u/dl- Plasma factor VIII multimers showed an abnormal pattern with no high molecular weight bands present despite a normal triplet structure in the low molecular weight forms. After the infusion of porcine factor VIII concentrate a large increase in the levels of plasma VIIIC was detected with a disappearance half-life of 3.5 hours. A specific non-crossreacting immunoradiometric assay (IRMA) showed that plasma levels of porcine vWFAg did not rise significantly after the infusion. Despite this, human vWFAg levels were notably elevated at 1 hour (40 u/dl by Laurell) and 2 hours (30 u/dl by IRMA) post infusion. Similarly, ristocetin induced platelet aggregation and plasma RRCo levels showed significant elevations , 2 hours after the infusion. Factor VIII multimers assessed on plasma samples taken over a similar time period revealed the transient appearance of a normal compliment of human factor VIII multimeric forms 2 hours after the infusion of porcine factor VIII concentrate. This study indicates that the abnormal pattern of factor VIII multimeric bands present in inhibitor-related variant acquired von Willebrand's disease can be transiently normalised by infused porcine factor VIII concentrate. Whether this represents antibody displacement or de novo synthesis is yet to be determined.

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.

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