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

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.

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Dissociation of Factor VIII in the Presence of Proteolytic Inhibitors

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648665 ◽

1978 ◽

Vol 40 (02) ◽

pp. 316-325 ◽

Cited By ~ 5

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.

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

10.1055/s-0039-1680464 ◽

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.

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

Blood ◽

10.1182/blood.v47.2.253.253 ◽

1976 ◽

Vol 47 (2) ◽

pp. 253-264 ◽

Cited By ~ 8

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.

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Influence of high ionic strength buffers on factor VIII/von Willebrand factor from different species

Thrombosis Research ◽

10.1016/0049-3848(78)90127-5 ◽

1978 ◽

Vol 13 (3) ◽

pp. 409-418 ◽

Cited By ~ 2

Author(s):

J.M. Lavergne ◽

D. Meyer ◽

C.S.P. Jenkins ◽

B. Obert ◽

M.J. Larrieu

Keyword(s):

Ionic Strength ◽

Factor Viii ◽

Von Willebrand Factor ◽

High Ionic Strength ◽

Von Willebrand ◽

Willebrand Factor

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The Separation of Willebrand Factor from Factor VIII Related Antigen

10.1055/s-0039-1680426 ◽

1977 ◽

Author(s):

E. S. Barrow ◽

H. M. Reisner ◽

J. B. Graham

Keyword(s):

Ionic Strength ◽

Factor Viii ◽

Gel Filtration ◽

High Ionic Strength ◽

Human Antibody ◽

Rabbit Antibody ◽

Clotting Time ◽

High Affinity ◽

Willebrand Factor ◽

Free Plasma

Factor VIII (F. VIII) in normal plasma: a) shortens the prolonged clotting time of hemophilic plasma (F. VIII coagulant, VIII:C), b) precipitates with heterologous antisera (F. VIII related antigen, VIIIR:AG), and c) together with the antibiotic Ristocetin, aggregates platelets (F. VIII related Willebrand factor, VIIIR:WF). VIII:C has been shown by others to be separable from VIIIR:AG-WF proteins by gel filtration with buffers of high ionic strength. No one has, to our knowledge, clearly separated VIIIR:WF from VIIIR:AG. We seem to have accomplished this by sequential use of two antibodies to F. VIII. The IgG fractions of a precipitating rabbit anti-human VIII and of a human, non-precipitating anti-VIII were separately bound covalently to CNBr-activated Sepharose. The rabbit antibody had a high affinity for VIIIR: AG, but a low affinity for VIII:C and VIIIR:WF. Passage of human plasma over the rabbit antibody column completely removed VIIIR:AG, but not the VIII:C or VIIIR:WF. The VIIIR:AG-free plasma was then sent over the human antibody column which removed only VIII:C. The effluent retained 60% of the original VIIIR:WF activity but had no measurable VIII:C or VIIIR:AG. Bio-Gel A-15 filtration of the VIIIR:WF resulted in elution of the activity in the Vo fractions. These data suggest that the VIII:C, VIIIR: AG and VIIIR:WF activities may exist in plasma as separate entities.

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Bovine Factor VIII. Antigens and Biological Activities

10.1055/s-0039-1682506 ◽

1977 ◽

Author(s):

G. Casillas ◽

C. Simonetti ◽

A. Pavlovsky

Keyword(s):

Factor Viii ◽

Hemophilia A ◽

Biological Activities ◽

Gel Filtration ◽

Species Specificity ◽

Procoagulant Activity ◽

Rabbit Antibody ◽

Von Willebrand ◽

The Relationship ◽

Bovine Factor

The procoagulant (PcgF) and the platelet aggregating (PAF) activities of bovine F. VIII were studied in order to establish their relation to the antigens A1 and A2, antigens similar to those synthetized by von Willebrand and hemophilia A patients respectively. Studies of stability to different agents (temperature. EDTA, thrombin, etc.) allowed us to establish that VIII (PcgF) and VIII (PAF) are not mutually dependenteAn homologous antibody of low species specificity against human F. VIII binds specifically with VIII (A,) antigenic moiety of bovine F. VIII. The complex so formed is purified by gel filtration and has VIII (PAF) activity but not VIII (PcgF) activity indicating that VIII(A1) and VIM (PcgF) activities associate,, Another complex, formed by bovine factor VIII and a rabbit antibody against the VIII (A2) moiety, was prepared,, This complex has VIII (PcgF) activity but not VIII (PAF) activity, indicating that VIII (A2) and VIII (PAF) associate,, The complex and its procoagulant activity sediment after centrifugation and may by re-covered by suspension of the precipitate. The following scheme showing the relationship between the antigenic moieties and the activities of bovine factor VIII is proposed: VIII(A1-PcgF) (A2-PAF).

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

Blood ◽

10.1182/blood.v47.2.253.bloodjournal472253 ◽

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.

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Selective absence of large forms of factor VIII/von Willebrand factor in acquired von Willebrand's syndrome. Response to transfusion

Blood ◽

10.1182/blood.v54.3.600.600 ◽

1979 ◽

Vol 54 (3) ◽

pp. 600-606 ◽

Cited By ~ 34

Author(s):

D Meyer ◽

D Frommel ◽

MJ Larrieu ◽

TS Zimmerman

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Von Willebrand Disease ◽

Procoagulant Activity ◽

Factor Viii Related Antigen ◽

Cofactor Activity ◽

Von Willebrand ◽

Willebrand Factor ◽

Ristocetin Cofactor ◽

Ristocetin Cofactor Activity

Abstract A previously healthy elderly man with mucocutaneous bleeding was found to have a benign monoclonal IgG gammapathy associated with criteria for severe von Willebrand disease (Factor VIII procoagulant activity, Factor-VIII-related antigen, and ristocetin cofactor activity, less than 10% of normal). Associated qualitative abnormalities of factor VIII/von Willebrand factor were demonstrated by radiocrossed immunoelectrophoresis and immunoradiometric assay. The late clinical onset and negative family history are in favor of an acquired form of vWD. The monoclonal gammapathy and abnormalities of factor VIII/von Willebrand factor have been stable over a 10-yr period. No inhibitor to Factor VIII procoagulant activity, ristocetin cofactor activity, or Factor-VIII-related antigen could be demonstrated. Following transfusion of cryoprecipitate (with a normal cross immunoelectrophoretic pattern), there was a rapid removal of the large forms of Factor.-VIII-related antigen, paralleled by a decay of ristocetin cofactor activity. The transfusion study of this patient with acquired von Willebrand disease type II (variant of von Willebrand disease) serves to emphasize the relationship between polydispersity of Factor VIII/von Willebrand Factor and functional heterogeneity.

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Thrombin Binding To Factor VIII/Von Willebrand Factor Protein

10.1055/s-0038-1652744 ◽

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.

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Studies of the human factor VIII/von Willebrand's factor protein. II. Identification and characterization of the von Willebrand protein

Blood ◽

10.1182/blood.v46.3.417.bloodjournal463417 ◽

1975 ◽

Vol 46 (3) ◽

pp. 417-430

Author(s):

HR Gralnick ◽

BS Coller

Keyword(s):

Factor Viii ◽

Human Factor ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Related Protein ◽

Human Factor Viii ◽

Von Willebrand’S Factor ◽

Von Willebrand ◽

Identification And Characterization

The purified factor VIII-related protein we have previously characterized from normal cryoprecipitate possesses both procoagulant activity and vWf activity. We have attempted to isolate and characterize this protein from three patients with severe vWd. This protein is absent or markedly diminished in amount in these vWd patients, as judged by gel filtration, polyacrylamide-gel electrophoresis, and immunoprecipitation assays. Likewise, the procoagulant and vWf activities are deficient. As vWf activity is one of the major biologic functions of either the normal or hemophilic factor VIII-related protein, the purified protein should be designated the f VIII/vWf protein.

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