Molecular weight of human factor VIII procoagulant activity

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.

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Structural basis for the decreased procoagulant activity of human factor VIII compared to the porcine homolog

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)98924-6 ◽

1991 ◽

Vol 266 (19) ◽

pp. 12481-12486

Author(s):

P. Lollar ◽

E.T. Parker

Keyword(s):

Factor Viii ◽

Human Factor ◽

Procoagulant Activity ◽

Structural Basis ◽

Human Factor Viii

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Immunologic studies of native and modified human factor VIII/von Willebrand factor

Blood ◽

10.1182/blood.v54.2.310.310 ◽

1979 ◽

Vol 54 (2) ◽

pp. 310-321 ◽

Cited By ~ 5

Author(s):

ME Switzer ◽

PA McKee

Keyword(s):

Molecular Weight ◽

Factor Viii ◽

Von Willebrand Factor ◽

Proteolytic Enzymes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Procoagulant Activity ◽

Human Factor Viii ◽

Von Willebrand ◽

Willebrand Factor

Abstract Factor VIII/von Willebrand factor (FVIII/vWF) is a glycoprotein with a molecular weight greater than one-million daltons. Two activities are associated with this large molecule: FVIII procoagulant activity and vWF activity. Incubation of FVIII/vWF with proteolytic enzymes causes rapid inactivation of the FVIII procoagulant activity but has little effect on the vWF activity or antigenicity. In an attempt to gain insight into the structural features required for these two activities, antisera were raised in rabbits to normal, thrombin-inactivated, and plasmin-inactivated FVIII/vWF. All of these proteolytically modified forms of FVIII/vWF cross-reacted with each of the rabbit antisera; each blocked the ability of a human inhibitor to inactivate native active FVIII/vWF. Each of the antisera was a potent inhibitor of vWF activity and inactivated vWF activity at the same titer. The antisera were much less potent inhibitors of FVIII activity than of vWF activity. Antibodies to thrombin-inactivated FVIII/vWF or normal FVIII/vWF had about the same ability to inactivate FVIII procoagulant activity. Surprisingly, those to plasmin-inactivated FVIII/vWF still retained about 50% of this inhibitory capacity. A comparison of the three types of antigens by polyacrylamide gel electrophoresis in sodium dodecyl sulfate-6 M urea demonstrated that the structure of thrombin- inactivated FVIII/vWF was indistinguishable from that of normal FVIII/vWF, while plasmin-inactivated FVII/vWF was completely cleaved to lower molecular weight fragments. Some of the reported variations in the ability of rabbit antibodies to inhibit procoagulant activity may be due to partial degradation of the starting antigen. The retention by FVIII/vWF protein of its immunologic properties even after extensive proteolytic degradation suggests that under nondenaturing conditions, the conformation of the native and degraded molecules are very similar.

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Concentration-dependent dissociation of factor VIII in 1 M NaCl

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1976.230.2.434 ◽

1976 ◽

Vol 230 (2) ◽

pp. 434-440 ◽

Cited By ~ 7

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.

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Immunologic studies of native and modified human factor VIII/von Willebrand factor

Blood ◽

10.1182/blood.v54.2.310.bloodjournal542310 ◽

1979 ◽

Vol 54 (2) ◽

pp. 310-321

Author(s):

ME Switzer ◽

PA McKee

Keyword(s):

Molecular Weight ◽

Factor Viii ◽

Von Willebrand Factor ◽

Proteolytic Enzymes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Procoagulant Activity ◽

Human Factor Viii ◽

Von Willebrand ◽

Willebrand Factor

Factor VIII/von Willebrand factor (FVIII/vWF) is a glycoprotein with a molecular weight greater than one-million daltons. Two activities are associated with this large molecule: FVIII procoagulant activity and vWF activity. Incubation of FVIII/vWF with proteolytic enzymes causes rapid inactivation of the FVIII procoagulant activity but has little effect on the vWF activity or antigenicity. In an attempt to gain insight into the structural features required for these two activities, antisera were raised in rabbits to normal, thrombin-inactivated, and plasmin-inactivated FVIII/vWF. All of these proteolytically modified forms of FVIII/vWF cross-reacted with each of the rabbit antisera; each blocked the ability of a human inhibitor to inactivate native active FVIII/vWF. Each of the antisera was a potent inhibitor of vWF activity and inactivated vWF activity at the same titer. The antisera were much less potent inhibitors of FVIII activity than of vWF activity. Antibodies to thrombin-inactivated FVIII/vWF or normal FVIII/vWF had about the same ability to inactivate FVIII procoagulant activity. Surprisingly, those to plasmin-inactivated FVIII/vWF still retained about 50% of this inhibitory capacity. A comparison of the three types of antigens by polyacrylamide gel electrophoresis in sodium dodecyl sulfate-6 M urea demonstrated that the structure of thrombin- inactivated FVIII/vWF was indistinguishable from that of normal FVIII/vWF, while plasmin-inactivated FVII/vWF was completely cleaved to lower molecular weight fragments. Some of the reported variations in the ability of rabbit antibodies to inhibit procoagulant activity may be due to partial degradation of the starting antigen. The retention by FVIII/vWF protein of its immunologic properties even after extensive proteolytic degradation suggests that under nondenaturing conditions, the conformation of the native and degraded molecules are very similar.

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Von Willebrand Activity of Low Molecular Weight Human Factor VIII Increases by Binding to Gold Granules

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650179 ◽

1981 ◽

Vol 45 (03) ◽

pp. 242-246 ◽

Cited By ~ 8

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.

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

10.1055/s-0038-1644110 ◽

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.

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