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

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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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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von Willebrand Factor Activity in Plasmin Digests of Factor VIII

10.1055/s-0039-1680509 ◽

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.

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Microheterogeneity of Human Factor VIII

10.1055/s-0039-1680355 ◽

1977 ◽

Author(s):

J.A. van Mourik ◽

J. A. Hellings ◽

E. M. Hoorweg

Keyword(s):

Ionic Strength ◽

Factor Viii ◽

Von Willebrand Factor ◽

Human Factor ◽

Polyacrylamide Gel ◽

Gel Chromatography ◽

Large Pore ◽

Human Factor Viii ◽

Von Willebrand ◽

Willebrand Factor

Human factor VIII is a glycoprotein and tends to form a variety of large aggregates, the presence of which was demonstrated by a number of techniques including the use of large pore gel chromatography and electrophoresis using a polyacrylamide gel system designed for high molecular weight aggregates. Factor VIII aggregates can be fractionated in part according to molecular size. Reduction of these polymers by 2-mercaptoethanol results in presumably identical fragments suggesting that all polymers, though differing in size, are composed of identical subunits.Factor VIII aggregates are particularly sensitive to proteolytic breakdown by trypsin and plasmin as judged by large pore polyacrylamide gel electrophoresis. Short-term incubation of factor VIII with trace quantities (equivalent to plasmin- or trypsin-like activities present in normal plasma) of these enzymes respectively results in substantial fragmentation with concurrent loss of both factor VIII procoagulant and von Willebrand factor activity. However, factor VIII activity is lost prior to discernible protein fragmentation whereas von Willebrand factor inactivation is associated with advanced protein degradation. When the ionic strength of the medium is lowered the susceptibility of factor VIII to proteolytic breakdown by trypsin is increased dramatically. Interestingly, degradation of factor VIII by plasmin is not affected by the ionic strength. These data, then, provide conclusive evidence of microheterogeneity of normal human factor VIII and may account for the observed heterogeneity of factor VIII on crossed immunoelectrophoresis.

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Factor VIII/von Willebrand factor in subendothelium mediates platelet adhesion

Blood ◽

10.1182/blood.v65.4.823.bloodjournal654823 ◽

1985 ◽

Vol 65 (4) ◽

pp. 823-831 ◽

Cited By ~ 2

Author(s):

VT Turitto ◽

HJ Weiss ◽

TS Zimmerman ◽

II Sussman

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Vessel Wall ◽

Platelet Adhesion ◽

Human Factor ◽

Rabbit Aorta ◽

Human Factor Viii ◽

Plasma Factor ◽

Von Willebrand ◽

Willebrand Factor

The present studies were undertaken to determine whether factor VIII/von Willebrand factor (vWF) present in the vessel wall (in addition to that in plasma) may mediate the attachment of platelets to subendothelium. Subendothelium from everted rabbit aorta was exposed to human citrated blood flowing through an annular perfusion chamber at 40 mL/min (wall shear rate of 2,600 s-1 for five minutes). The vessel segments were incubated at 37 degrees C for one hour with various dilutions of either goat-anti-rabbit factor VIII/vWF serum or an IgG fraction prepared from the serum. Control segments were incubated with serum or IgG from a nonimmunized goat. Values of platelet contact (C), platelet adhesion (C + S), and thrombus formation (T) on the subendothelium were evaluated by a morphometric technique. Compared with vessels incubated with fractions prepared from a normal goat, a significant decrease in platelet adhesion (C + S), ranging from 45% to 65%, was observed on vessels incubated with various dilutions (1:5 to 1:50) of either serum or IgG fractions of goat-anti-rabbit factor VIII/vWF. A similar decrease in platelet adhesion was observed with vessels incubated with an F(ab')2 fragment against rabbit factor VIII/vWF prepared in the goat. When goat-anti-rabbit factor VIII/vWF IgG was added to rabbit blood (1:75 dilution), platelet adhesion was reduced to the same extent (65%) on normal rabbit vessels and on vessels pre-incubated with goat-anti-rabbit factor VIII/vWF. Immunofluorescence studies revealed the presence of rabbit factor VIII/vWF in the subendothelium of rabbit aorta and the continued binding of the goat-anti-factor VIII/vWF antibodies on subendothelium during the perfusion studies. No uptake of human factor VIII/vWF on the rabbit subendothelium was observed by this immunologic technique; human factor VIII/vWF was found to be entirely associated with the attached human platelets. Thus, factor VIII/vWF in the vessel wall may mediate platelet attachment to subendothelium in a manner similar to that of plasma factor VIII/vWF.

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