Chromatography of Antihemophilic Factor on Diaminoalkane- and Aminoalkane-Derivatized Sepharose

1982 ◽  
Vol 47 (02) ◽  
pp. 124-127 ◽  
Author(s):  
J-J Morgenthaler
Keyword(s):  
Ionic Strength ◽  
Ethylene Glycol ◽  
Factor Viii ◽  
Homologous Series ◽  
Procoagulant Activity ◽  
Antihemophilic Factor

SummaryAntihemophilic factor was chromatographed on a homologous series of diaminoalkane- and aminoalkane-modified Sepharose beads. Both factor VIII procoagulant activity (VIII:C) and protein are retarded on these columns when compared to their elution on a column made of unmodified Sepharose. Longer chains bind VIII:C and protein more tightly than shorter chains. No bound activity could be eluted with ethylene glycol. Increasing ionic strength eluted VIII:C and protein from aminoalkane- as well as from alkane-Sepharose. It seems likely that hydrophobic, rather than ionic forces, are responsible for the binding of VIII:C to the latter carriers.

Importance of Protease Inhibition in Studies on Purified Factor VIII (Antihaemophilic Factor)

1976 ◽  
Vol 35 (01) ◽  
pp. 186-190 ◽  
Author(s):  
Eugen A. Beck ◽  
Peter Bachmann ◽  
Peter Barbier ◽  
Miha Furlan
Keyword(s):  
Ionic Strength ◽  
Factor Viii ◽  
Gel Filtration ◽  
High Ionic Strength ◽  
Procoagulant Activity ◽  
Carrier Protein ◽  
Protease Inhibition ◽  
Functional Sites ◽  
Molecular Weight Peak ◽  
Von Willebrand

SummaryAccording to some authors factor VIII procoagulant activity may be dissociable from carrier protein (MW~ 2 × 106) by agarose gel filtration, e.g. at high ionic strength. We were able to reproduce this phenomenon. However, addition of protease inhibitor (Trasylol) prevented the appearance of low molecular weight peak of factor VIII procoagulant activity both at high ionic strength and elevated temperature (37°C). We conclude from our results that procoagulant activity and carrier protein (von Willebrand factor, factor VIII antigen) are closely associated functional sites of native factor VIII macro molecule. Consequently, proteolytic degradation should be avoided in functional and structural studies on factor VIII and especially in preparing factor VIII concentrate for therapeutic use.

scholarly journals Immunologic Studies of Antihemophilic Factor (AHF, Factor VIII) II. Properties of Cross-reacting Material

Blood ◽  
1970 ◽  
Vol 35 (6) ◽  
pp. 809-820 ◽  
Author(s):  
LEON W. HOYER ◽  
ROBERT T. BRECKENRIDGE
Keyword(s):  
Calcium Phosphate ◽  
Physical Properties ◽  
Factor Viii ◽  
Barium Sulfate ◽  
Genetic Variant ◽  
Hemophilia A ◽  
Procoagulant Activity ◽  
Normal Plasma ◽  
Fraction I ◽  
Antihemophilic Factor

Abstract Patients with a genetic variant of hemophilia A have in their plasmas material which has antigenic characteristics similar to those of antihemophilic factor (AHF). The physical properties of the biologically inactive cross-reacting material (CRM) are like those of AHF from normal plasma. The CRM is concentrated in Cohn fraction I and in cryoprecipitates and is not adsorbed from plasma by calcium phosphate or barium sulfate. It is inactivated by heating to 56° for 30 minutes. The CRM is less sensitive to thrombin inactivation than AHF and is recovered in serum. The similar properties of AHF and CRM support the hypothesis that patients with this genetic variant of hemophilia A synthesize a material similar to AHF but lacking procoagulant activity.

Dissociation of Factor VIII in the Presence of Proteolytic Inhibitors

1978 ◽  
Vol 40 (02) ◽  
pp. 316-325 ◽  
Author(s):  
Ira I Sussman ◽  
Harvey J Weiss
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Factor Viii ◽  
Direct Evidence ◽  
Gel Filtration ◽  
High Ionic Strength ◽  
Procoagulant Activity ◽  
Low Molecular Weight ◽  
Bean Trypsin Inhibitor ◽  
Benzamidine Hydrochloride

SummaryWhen gel filtration of factor VIII is performed with buffers of high ionic strength (1.0 M NaCl or 0.25 M CaCl2), the procoagulant activity elutes with proteins of relatively low molecular weight. It has been suggested that in the presence of proteolytic inhibitors, the procoagulant activity would appear at the void volume. To test this hypothesis, chromatography with buffers of high ionic strength was performed in the presence of benzamidine hydrochloride, soy bean trypsin inhibitor, heparin, DFP, and aprotinin. Under all of these conditions, the procoagulant activity continued to elute with proteins of low molecular weight. Similar findings were obtained after chromatographing cryoprecipitate prepared from the plasma of a normal subject who had received heparin. Thus, at present there is no direct evidence to suggest that proteolysis is involved in the dissociation of factor VIII by buffers of high ionic strength.

scholarly journals The Binding of Bovine Factor VIII of Human Platelets

1977 ◽  
Author(s):  
Edward P. Kirby ◽  
Sha May Tang
Keyword(s):  
Factor Viii ◽  
Evans Blue ◽  
Sodium Iodide ◽  
Dextran Sulfate ◽  
Human Platelets ◽  
Procoagulant Activity ◽  
Human Fibrinogen ◽  
Total Binding ◽  
Antihemophilic Factor ◽  
Bovine Factor

Highly purified bovine Factor VIII (FVIII) was iodinated by the lactoperoxidase method. Subsequent chromatography on agarose and extensive dialysis against sodium iodide solutions was required to remove non-covalently bound 1-125 which adhered tightly to FVIII. Iodination destroyed the procoagulant activity (Antihemophilic Factor-AHF) of FVIII, but did not affect its Platelet Aggregating Factor (PAF) activity. Binding to human platelets was determined by incubating radio!abelled FVIII with formalin-treated platelets, centrifuging, and measuring both bound and free radioactivity. Results obtained by this method were much more precise than those obtained by measuring disappearance of unlabelled AHF, PAF, or FVIII-related antigen from the supernatant, although the estimates of total binding obtained were comparable. Binding of FVIII to formal in-treated platelets was approximately the same as to unfixed platelets, and the binding could be saturated by adding an excess of unlabelled FVIII. Maximal binding occurred within 1-2 minutes at 37° and binding could be blocked by dextran sulfate, Evans Blue or other inhibitors of FVIII-induced platelet agglutination. Treatment of platelets with trypsin inhibited binding of labelled F-VIII. Binding was not affected by the presence of plasma, or high levels of purified human fibrinogen or FVIII.

scholarly journals Immunologic studies on human factor VIII (anti-hemophilic factor A, AHF) components produced by low-ionic-strength dialysis

Blood ◽  
1976 ◽  
Vol 47 (2) ◽  
pp. 253-264 ◽  
Author(s):  
BN Bouma ◽  
JA van Mourik ◽  
S de Graaf ◽  
JM Hordijk-Hos ◽  
JJ Sixma
Keyword(s):  
Ionic Strength ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Human Factor ◽  
Procoagulant Activity ◽  
Factor Activity ◽  
Human Factor Viii ◽  
Low Ionic Strength ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Since dialysis of human factor VIII against buffers of low ionic strength yielded two distinct components, and since the factor VIII fraction isolated from normal plasma showed von Willebrand factor activity as defined by the corrective effect on abnormal platelet retention and ristocetin aggregation in von Willebrand's disease, the present studies were performed to determine if the correcting activities could be attributed to one or both of the components. Dialysis of factor VIII against buffers of low ionic strength led, however, to a decrease in factor VIII procoagulant activity and the reduction of the correcting activities, which suggested that the intact aggregate was required for procoagulant activity and for von Willebrand factor activity. In this respect dialysis of factor VIII at low ionic strength differed from dissociation at high salt concentrations. The two low ionic strength components were identified by the use of a rabbit antiserum against factor VIII, and could be distinguished on the basis of specific antigenic structures. Dialysis of factor VIII at low ionic strength led to a decrease in antigenic determinants closely related to factor VIII function. Specific antibodies to the low ionic strength components inhibited factor VIII activity in normal plasma, but the residual factor VIII was higher than that after inhibition with antibodies against intact factor VIII. Both antibodies interfered with von Willebrand factor activity.

scholarly journals Immunologic Studies of Antihemophilic Factor (AHF, Factor VIII). V. Immunologic Properties of AHF Subunits Produced by Salt Dissociation

Blood ◽  
1973 ◽  
Vol 42 (5) ◽  
pp. 737-747 ◽  
Author(s):  
Margaret E. Rick ◽  
Leon W. Hoyer
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Factor Viii ◽  
Plasma Protein ◽  
Gel Filtration ◽  
Agarose Gel ◽  
Antibody Neutralization ◽  
Antihemophilic Factor ◽  
Immunologic Properties

Abstract Human antihemophilic factor (AHF, factor VIII), a large plasma protein with a molecular weight of approximately two million, is dissociated by changes in ionic strength. The immunologic properties of subunits obtained by sucrose density ultracentrifugation in 1 M NaCl and by agarose gel filtration in 0.24 M CaCl2 have been determined using human and rabbit anti-AHF. Asymmetric dissociation of AHF has been identified with formation of two subunits in these separations: a nonfunctional highmolecular-weight (HMW) subunit similar in size to plasma AHF which is identified by immunoprecipitation and radioimmunoassay for AHF antigen, and an active lowmolecular-weight (LMW) subunit which is not detected by these antigen assays. The LMW subunit retains AHF antigens, however, for it is inactivated by both human and rabbit anti-AHF. Antibody neutralization studies verify the presence of AHF antigens on the HMW subunit. These immunologic studies provide constraints which must be incorporated into models of AHF structure.

Monoclonal Antibodies Against Factor Viii Procoagulant Activity

1981 ◽  
Author(s):  
Y Sultan ◽  
B Sola ◽  
Ph Avner ◽  
P Maisonneuve ◽  
C Jeanneau
Keyword(s):  
Monoclonal Antibodies ◽  
Factor Viii ◽  
Inhibitory Activity ◽  
Solid Phase ◽  
Procoagulant Activity ◽  
Hybridoma Cells ◽  
Commercial Preparation ◽  
Neutralizing Activity ◽  
Von Willebrand ◽  
Antihemophilic Factor

Monoclonal antibodies against the factor VIII/Von Willebrand molecule were obtained using the hybridoma technique. Splenocytes of mice immunized with a commercial preparation of high purity antihemophilic factor were fiised with a non secreting myeloma cell line SP 2/0. Hybrid cells were cloned twice by limiting dilution. Among 8 antibodies reacting with purified F.VIII/VW factor in a solid phase radio immunoassay, two of them showed an inhibitory activity against the procoagulant activity of the antihemophilic factor. This inhibitory activity was not detected in culture supernatants. Presumably in corelation with subtances of the culture medium interfering with the coagulation process and masking the neutralizing activity. However the anti VIIIc activity was demonstrated in the ascitic fluid obtained from mice injected with hybridoma cells.The specific anti VIIIc neutralizing activity of F4 115 monoclonal antibody is dependent on Ig concentration, time of incubation, temperature and Ph. In contrast with most of anti VIIIc inhibitors occuring in multitransfured hemophiliac, neutralization was more rapid and highier at 4° than 37°C. This neutralizing activity is absorbed by normal plasma or purified F.VIII preparation, however it is not absorbed by plasmas of a limited number of severe hemophilic and severe Von Willebrand patients. The study of a larger patients population is in progress.The second antibody F4 415 showed different inhibitory properties. Inhibition of the procoagulant activity is independent of incubation time butdependent on temperature and similarly more active at 4°C.These monoclonal antibodies provide new tools to investigate the procoagulant piece of the F.VIII molecule

scholarly journals Immunologic studies on human factor VIII (anti-hemophilic factor A, AHF) components produced by low-ionic-strength dialysis

Blood ◽  
1976 ◽  
Vol 47 (2) ◽  
pp. 253-264
Author(s):  
BN Bouma ◽  
JA van Mourik ◽  
S de Graaf ◽  
JM Hordijk-Hos ◽  
JJ Sixma
Keyword(s):  
Ionic Strength ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Human Factor ◽  
Procoagulant Activity ◽  
Factor Activity ◽  
Human Factor Viii ◽  
Low Ionic Strength ◽  
Von Willebrand ◽  
Willebrand Factor

Since dialysis of human factor VIII against buffers of low ionic strength yielded two distinct components, and since the factor VIII fraction isolated from normal plasma showed von Willebrand factor activity as defined by the corrective effect on abnormal platelet retention and ristocetin aggregation in von Willebrand's disease, the present studies were performed to determine if the correcting activities could be attributed to one or both of the components. Dialysis of factor VIII against buffers of low ionic strength led, however, to a decrease in factor VIII procoagulant activity and the reduction of the correcting activities, which suggested that the intact aggregate was required for procoagulant activity and for von Willebrand factor activity. In this respect dialysis of factor VIII at low ionic strength differed from dissociation at high salt concentrations. The two low ionic strength components were identified by the use of a rabbit antiserum against factor VIII, and could be distinguished on the basis of specific antigenic structures. Dialysis of factor VIII at low ionic strength led to a decrease in antigenic determinants closely related to factor VIII function. Specific antibodies to the low ionic strength components inhibited factor VIII activity in normal plasma, but the residual factor VIII was higher than that after inhibition with antibodies against intact factor VIII. Both antibodies interfered with von Willebrand factor activity.

The Factor VIII Bypassing Activity of Prothrombin Complex Concentrates: The Roles of Factor VIla and of Endothelial Cell Tissue Factor

1991 ◽  
Vol 66 (05) ◽  
pp. 559-564 ◽  
Author(s):  
Jerome M Teitel
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Procoagulant Activity ◽  
Tissue Factor Expression ◽  
Prothrombin Complex Concentrates ◽  
Cell Tissue ◽  
Factor Expression ◽  
Necrosis Factor

SummaryAn experimental model incorporating cultured endothelial cells (EC) was used to study the "factor VIII bypassing" activity of prothrombin complex concentrates (PCC), a property exploited in the treatment of hemophiliacs with alloantibodies to factor VIII. Two PCC preparations were ineffective as stimuli of tissue factor expression by EC. However, incubation with a combination of PCC plus endotoxin (lipopolysaccharide, LPS) or tumor necrosis factor (TNF) induced much greater tissue factor expression than was seen in response to either substance alone. PCC expressed an additional direct procoagulant activity at the EC surface, which could not be attributed to either thrombin or factor Xa, and which was diminished by an anti-tissue factor antibody. Therefore factor VIIa, which was detectable in both PCC preparations, likely provided this additional direct procoagulant activity at the EC surface. We also excluded the possibility that coagulation proteases contained in or generated in the presence of PCC are protected from inactivation by AT III. Therefore, PCC can indirectly bypass factor VIII by enhancing induced endothelial tissue factor expression, and also possess direct procoagulant activity, probably mediated by factor VIIa.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

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