Dissociation of “Factor VIII Complex” in Various Animal Species.

1975 ◽  
Author(s):  
J. M. Lavergne ◽  
Dominique Meyer ◽  
C. S. P. Jenkins ◽  
Marie-José Larrieu
Keyword(s):  
Factor Viii ◽  
Animal Species ◽  
Volume Fraction ◽  
Gel Filtration ◽  
High Salt ◽  
Void Volume ◽  
Factor Activity ◽  
Step Procedure ◽  
Sepharose 4B ◽  
Willebrand Factor

Under conditions of high salt concentration, “Factor VIII complex” (Factor VHI activity, Willebrand Factor activity – measured using a washed platelet system and ristocetin – and Willebrand antigen) may be dissociated into a high (M. W. > 106) and a low molecular weight fragment. The dissociation of “Factor VIII complex” was studied by a two step procedure. Human or animal plasma or cryoprecipitate was submitted to gel filtration on Sepharose 4B, using 0.15 M NaCl, Imidazole or Tris-HCl Buffer as eluant. The void volume fraction, containing the three entities of “Factor VIII complex” was concentrated and submitted to a second gel filtration using a dissociating buffer as eluant (1 M NaCl or 0.25 M CaCl2). The three entities of “Factor VIII complex” were measured in the eluted fractions. “Factor VIII complex” was found to dissociate using the high salt buffer in some but not all animal species. When dissociation occurred, Willebrand Factor activity and antigen eluted in the void volume, and Factor VIII activity in later fractions. Heterologous antisera were raised against the different fractions and the reactivity towards human and animal plasma was studied.

Thrombin Binding To Factor VIII/Von Willebrand Factor Protein

1981 ◽  
Author(s):  
M E P Switzer ◽  
P A McKee
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Substrate Binding ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Void Volume ◽  
Peptidase Activity ◽  
Sds Page ◽  
Von Willebrand ◽  
Willebrand Factor

Thrombin (IIa) both activates and inactivates the procoagulant activity of Factor VIII/von Willebrand Factor (FVIII/vWF). The level of activation increases as the IIa: FVIII/vWF ratio approaches 1:1, suggesting that IIa might bind stoichiometrically to FVIII/vWF either during or after activation. We approached this question by gel filtration and ultracentrifugation studies of FVIII/vWF and l25I-IIa, which activated FVIII/vWF as well as unlabeled IIa. When the mixture of 125I-IIa and FVIII/vWF was chromatographed on 4% agarose a peak of 125I-IIa was eluted with the FVIII/ vWF in the void volume (V0). Similarly, when 125I-IIa was ultracentrifuged with FVIII/vWF, a peak of radioactivity sedimented with the FVIII/vWF protein. 125I-aibumin, used to approximate a control, did not bind to FVIII/vWF. The 125I-IIa-FVIII/vWF complex isolated from the 4% agarose filtration retained ∼50% peptidase activity. The ability to activate additional FVIII/vWF or to clot fibrinogen was <10% of that of free IIa isolated from the same chromatogram. Both the FVIII and vWF moieties appear to be important in binding, since VD protein isolated from the gel filtration of FVIII/vWF on 4% agarose in 0.25 M CaCl2 binds about 24% as much 125I-IIa as native FVIII/vWF. When the isolated 125I-IIa-FVIII/vWF complex was rechromatographed on 4% agarose in 0.15 M NaCl, essentially no dissociation occurred. When these experiments were repeated in 4 M guanidine hydrochloride (GnHCl), ∼30% of the IIa remained bound. When the 125I-IIa-FVIII/vWF complex was isolated from the GnHCl chromatography and analyzed by SDS-PAGE, 58% of the IIa remained bound to the FVIII/vWF before reduction and 43% of the IIa remained bound even after reduction with β-mercaptoethanol for 3 hours at 37°. Thus FVIII/vWF binds at least some of the IIa very tightly. Since FVIII/vWF-bound thrombin is essentially inactive toward macromolecular substrate, binding of thrombin to FVIII/vWF is most likely a mechanism for removing active thrombin from the circulation.

scholarly journals Structure-Function Studies of Factor VIII/Von Willebrand Protein(s)

1977 ◽  
Author(s):  
Patrick A. McKee
Keyword(s):  
Factor Viii ◽  
Single Molecule ◽  
Gel Filtration ◽  
Protein S ◽  
Fold Increase ◽  
Procoagulant Activity ◽  
Void Volume ◽  
Pas Staining ◽  
Von Willebrand ◽  
Willebrand Factor

Factor VIII (FVIII) procoagulant activity was initially thought to be a glycoprotein with a molecular weight (MW) >1 million and composed of disulfide-1inked ~200,000 MW subunits. A protein with similar properties, except lacking procoagulant activity, is in hemophilic plasma; it was identical to normal FVIII by SDS-gel analyses, isoelectric focusing, and PAS staining. Subsequently it was shown that the FVIII glycoprotein also has von Willebrand factor (vWF) activity, suggesting that both FVIII and vWF activities might be properties of the same molecule. When the FVIII/vWF protein(s) is rechromatographed on 4% agarose and 0.25 M CaCl2, virtually all the protein and vWF activity elute in the void volume, but most of the FVIII procoagulant activity elutes much later. The extent of separation of the two activities depends on the amount of protein applied to the column. Also, exposure of the FVIII/vWF to thrombin before gel filtration strikingly accentuates separation of the two activities. The reduced SDS-gel pattern of the void volume protein peak showed the 200,000 MW subunit while that of the procoagulant peak contained several subunit bands which ranged from ~30,000–100,000 MW. Removal of sialic acid from FVIII/vWF is associated with reduced ristocetin induced platelet aggregation and causes a 50-fold increase in the rate of clearance of protein from the circulation by the hepatocyte. Currently, our data suggest that FVIII procoagulant and vWF activities are properties of a single molecule composed of disulfide-bound identical subunits. Cleavage by thrombin then results in FVIII procoagulant activity. Additional cleavages, to which the molecule appears very sensitive, results in FVIII inactivation. The vWF activity is very stable—even to proteolysis—and it appears to be a function of the carbohydrate side chains of the molecule.

Interaction of Ristocetin (R), yon Willebrand Factor (vWF), Factor VIII Clotting Activity (VIIIc) and Platelets

1975 ◽  
Author(s):  
M. B. Zucker ◽  
J. McPherson ◽  
S.-J. Kim ◽  
V. E. Macdonald ◽  
D. Taylor ◽  
...  
Keyword(s):  
Factor Viii ◽  
Void Volume ◽  
Normal Plasma ◽  
Sepharose 4B ◽  
Willebrand Factor ◽  
Formalin Fixed

Formalin-fixed platelets were resuspended in a 1 in 3 dilution of plasma and aggregated with 1.5 mg 3H-R per ml. The supernatant contained over 90% of the R, about 25% of the vWF (assessed as R co-factor or by radioimmunoassay of VIIIag) and 50% of the VIIIc. The aggregates contained 6-9% 3H-R and only 1-2% of trapped 14C-albumin. They dispersed in buffer in the absence of R, and about 35% of the initial vWF but only 16% of the initial VIIIc was recovered in the resuspending buffer. Thus the ratio of vWF to VIIIc activity was < 1 in the initial supernatant and > 1 in the buffer in which the aggregates had been dispersed. Platelets in PRP behaved similarly but bound only a small percentage of plasma vWF during aggregation, and less R than formalin-fixed platelets. The latter bound R in the absence of plasma. Bernard-Soulier platelets did not aggregate; they bound R but not vWF. When 3H-R was added to fresh normal plasma and passed through a Sepharose-4B column, R failed to appear in the void volume (Vo) but emerged earlier than when chromatographed without plasma. vWF and VIII emerged together in Vo when plasma alone was used, but VIIIc peaked later than vWF in the presence of R. In summary, (1) R separates VIIIc from vWF, (2) platelets are passively involved, (3) R binds reversibly to normal as well as to Bernard-Soulier platelets, (4) in the presence of R, vWF binds in part reversibly to normal but not to Bernard-Soulier platelets. Thus separation of vWF and VIIIc by R may produce altered vWF which alone cannot bind R or aggregate platelets, but can bind to and hence aggregate platelets in the presence of R.

scholarly journals von Willebrand Factor Activity in Plasmin Digests of Factor VIII

1977 ◽  
Author(s):  
J. A. Guisasola ◽  
C. Cockburn ◽  
R. M. Hardisty
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Human Factor ◽  
Factor Activity ◽  
Group Iii ◽  
Molecular Weights ◽  
Human Factor Viii ◽  
Group Ii ◽  
Von Willebrand ◽  
Willebrand Factor

Purified human factor VIII was incubated for up to 24 hours with plasmin, and the activity of the breakdown products studied at intervals. Factor VIII coagulant activity was lost within the first hour, but von Willebrand factor activity (FVIIIR:WF) was retained for two hours, and then declined slowly during the subsequent incubation. Analysis of the 24-hour breakdown products by immuno-electrophoresis, sepharose 4B chromatography and SDS Polyacrylamide electrophoresis revealed three main groups of fragments recognised by rabbit anti-human factor VIII anti-serum, and having molecular weights in the following ranges: Group 1 300,000=500,000; Group II, 150–200,000; Group III, 100,000. FVIIIR:WF activity, which was found only in Group II, appeared to be associated with glycopeptide(s) of up to 155,000 daltons.

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.

Dissociation of Factor VIII Procoagulant Antigen VIII : CAg and Factor VIII Related Antigen VIIIR : Ag by EDTA - Influence of Divalent Cation on the Binding of VIII: CAg and VIIIR :Ag

1983 ◽  
Vol 50 (02) ◽  
pp. 547-551 ◽  
Author(s):  
T H Tran ◽  
F Duckert
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Divalent Cation ◽  
Steric Hindrance ◽  
Gel Filtration ◽  
Void Volume ◽  
Antigenic Determinants ◽  
Elution Buffer ◽  
Edta Plasma ◽  
Internal Volume

SummaryAssuming 1 U/ml in titrated plasma, the VIII: CAg concentration was found 1.66 U/ml in EDTA-plasma, 1.09 U/ml in heparinized plasma and 0.67 U/ml in serum. Addition of 10 mmol/1 EDTA to titrated and heparinized plasmas increased VIII: CAg 1.5fold. There was no increase of VIII: CAg in serum. Gel filtration of plasmas on different anticoagulants showed an elution of VIII: CAg in the void volume Vo and in the later fractions. The VIII: CAg amount detected in the internal volume increased following the series heparin < citrate < EDTA. Serum VIII: CAg was eluted at 2.2 Vo. Presence of EDTA in the elution buffer or incubation of plasma with EDTA prior to chromatography caused a displacement of practically all VIII: CAg amount in the internal volume with a peak at 2.2-2.3 Vo. VIIIR: Ag was exclusively detected in the void volume.Removal of divalent cation by chelation likely exposes more antigenic determinants of VIII: CAg, which are otherwise masked by steric hindrance due to VIIIR: Ag in citrated and heparinized milieu. Moreover gel filtration of plasma in the presence of EDTA completely dissociates VIII: CAg from VIIIR :Ag. The VIII: CAg fragment, having an estimated molecular weight of 70,000, might also be present in serum.

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