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

Blood ◽  
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.

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

Blood ◽  
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

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.

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 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.

scholarly journals Effect of Thrombin on Factor VIII/VON Willebrand Factor (FVIII/vWF)

1977 ◽  
Author(s):  
Mary Ellen Switzer ◽  
P.A. McKee
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Protein Concentration ◽  
Sodium Dodecyl ◽  
Activity Peak ◽  
Thrombin Activation ◽  
Von Willebrand ◽  
Willebrand Factor

The sodium dodecyl sulfate (SDS) gels and immunological properties of FVIII/vWF and thrombin-activated (thr-act) FVIII/vWF are identical. Hence thrombin must either modify FVIII/vWF by very minor proteolysis or cleave only a few FVIII/vWF molecules. The procoagulant (PC) activity of FVIII/vWF and thr-act FVIII/vWF eluted sharply in the void volume (Vo) from 4% agarose in 0.15 M NaCl, with >65% loss of the PC activity of the thr-act FVIII/vWF by 3 hrs. The PC activity of FVIII/vWF and thr-act FVIII/vWF was stabilized by 0.25 M CaCl2. When FVIII/vWF and thr-act FVIII/vWF were filtered on 4% agarose in 0.25 M CaCl2, the protein eluted in the Vo, but most of the PC activity eluted later in a region of little detectable protein; however, the delayed PC peak from thr-act FVIII/vWF was greatly enhanced above control levels. Nonreduced, neither activity peak protein entered a 7.5% SDS gel. After reduction, the gel pattern for the PC activity peak protein from FVIII/vWF showed major bands of 195x, 79x, 61x, 51x and 18x (103) molecular weight (MW) and several minor bands >100,000 MW. The reduced PC protein from thr-act FVIII/vWF lacked all bands >100,000 MW, but the four lower MW bands were present and well-resolved. Thrombin activation did not affect the vWF activity which was proportional to protein concentration throughout any chromatogram; the 195,000-dalton subunit was not necessary for vWF activity. FVIII/vWF, which had the peak PC activity in the Vo and no other PC activity peak on 4% agarose-0.25 M CaCl2, was thr-act before filtration under the same conditions. Then, the greatly enhanced PC activity eluted well after the Vo, with <10% PC activity in the Vo. We conclude that modification of a few FVIII/vWF molecules by thrombin and their stabilization by 0.25 M CaCl2 causes the PC peak that elutes aberrantly from 4% agarose-0.25 M CaCl2.

scholarly journals Factor VIII/von Willebrand factor in subendothelium mediates platelet adhesion

Blood ◽  
1985 ◽  
Vol 65 (4) ◽  
pp. 823-831 ◽  
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.

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

Blood ◽  
1979 ◽  
Vol 54 (3) ◽  
pp. 600-606 ◽  
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.

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.

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