scholarly journals Disulfide Bonds and the Quaternary Structure of Factor VIII/von Willebrand Factor

1978 ◽  
Vol 62 (3) ◽  
pp. 702-709 ◽  
Author(s):  
Richard B. Counts ◽  
Stefan L. Paskell ◽  
Susan K. Elgee
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Disulfide Bonds ◽  
Quaternary Structure ◽  
Von Willebrand ◽  
Willebrand Factor

Binding of Factor VIII to von Willebrand Factor Is Enabled by Cleavage of the von Willebrand Factor Propeptide and Enhanced by Formation of Disulfide-Linked Multimers

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 529-538 ◽  
Author(s):  
Ana Victoria Bendetowicz ◽  
Jill A. Morris ◽  
Robert J. Wise ◽  
Gary E. Gilbert ◽  
Randal J. Kaufman
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Posttranslational Modifications ◽  
Binding Sites ◽  
Disulfide Bonds ◽  
Free System ◽  
N Terminus ◽  
A Cell ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.

THE INTERACTION BETWEEN FACTOR VIII AND VON WILLEBRAND FACTOR

1987 ◽  
Author(s):  
Joost A Koedam ◽  
Rob J Hamer ◽  
Nel H Beeser-Visser ◽  
Etienne Jap Tjoen San ◽  
Kees Schippers ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Heavy Chain ◽  
Light Chain ◽  
Disulfide Bonds ◽  
Factor Xa ◽  
Exchange Chromatography ◽  
Solid Matrix ◽  
Von Willebrand ◽  
Willebrand Factor

Factor VIII (FVIII) circulates in plasma as a non-covalent complex with von Willebrand factor (VWF), a large multimeric adhesive glycoprotein. VWF serves as a carrier for FVIII and is thought to stabilize FVIII. The interaction between the two proteins was studied by binding purified human 125I-FVIII to VWF which was coated on a solid matrix. Experiments employing isolated heavy and light chains of FVIII and monoclonal antibodies indicated that binding occurred through the carboxyterminal 80kDa light chain of factor VIII. Treatment of VWF-bound 125I-FVIII with thrombin resulted in the release of a light chain-derived 70kDa fragment and a heavy chain-derived 50kDa fragment. A 42kDa heavy chain-derived fragment was found in the fraction which remained bound to VWF. Treatment with factor Xa (FXa) resulted in the release of 63, 50, 45, and 42kDa fragments. No phospholipids were required for proteolysis of FVIII by either of these enzymes. In solution, the activation of FVIII by FXa, but not by thrombin, was inhibited by VWF. Neither activation, nor cleavage or release from VWF were observed when FVIII was incubated with factor IXa. Activation of FVIII was parallelled by its release from VWF. We conclude that the thrombin-activated form of FVIII consists of a complex between the 70kDa and 50kDa fragments. Inactivation of FVIII by activated protein C (APC) was inhibited when FVIII was complexed to VWF. This protective effect of VWF was abolished upon activation of FVIII and its subsequent release from VWF.In order to locate the binding site for FVIII on the VWF molecule, we digested VWF with Staphylococcal V8 protease (Sp). Digestion products were isolated with Mono Q ion-exchange chromatography and identified as Spl (39 kDa), SpII dimers (220 kDa) and Spill dimers (a triplet ranging from 210-280 kDa) by their molecular weight and chromatographic behaviour (J.-P. Girma et al.. Biochemistry 1986, 25:3156-3163). Purified VWF or digestion products were spotted on nitrocellulose paper, followed by blocking with an albumin solution. Binding of FVIII was studied by incubating the filters with 125I-FVIII, followed by autoradiography. Fifty ng of VWF was sufficient in order to detect FVIII binding. No binding was observed to partially reduced dimeric undigested VWF. Of the isolated digestion products, only the SpIII dimer was able to bind 125I-FVIII. After Western blotting of VWF-fragments from SDS-polyacrylamide gels, 125I-FVIII bound only to the bands which represented SpIII. Therefore, the domain on VWF responsible for the binding of FVIII seems to be located on its aminoterminal SpIII fragment. The integrity of internal disulfide bonds and dimerisation of VWF are required for FVIII binding.

Binding of Factor VIII to von Willebrand Factor Is Enabled by Cleavage of the von Willebrand Factor Propeptide and Enhanced by Formation of Disulfide-Linked Multimers

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 529-538 ◽  
Author(s):  
Ana Victoria Bendetowicz ◽  
Jill A. Morris ◽  
Robert J. Wise ◽  
Gary E. Gilbert ◽  
Randal J. Kaufman
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Posttranslational Modifications ◽  
Binding Sites ◽  
Disulfide Bonds ◽  
Free System ◽  
N Terminus ◽  
A Cell ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.

Factor VIII Inhibitor Antibodies with C2 Domain Specificity Are Less inhibitory to Factor VIII Complexed with von Willebrand Factor

1996 ◽  
Vol 76 (05) ◽  
pp. 749-754 ◽  
Author(s):  
Suzuki Suzuki ◽  
Morio Arai ◽  
Kagehiro Amano ◽  
Kazuhiko Kagawa ◽  
Katsuyuki Fukutake
Keyword(s):  
Complex Formation ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Domain Specificity ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.

Molecular Biology of Factor VIII/von Willebrand Factor

1978 ◽  
Vol 40 (02) ◽  
pp. 245-251 ◽  
Author(s):  
D Meyer ◽  
P A Mc Kee ◽  
L W Hoyer ◽  
T S Zimmerman ◽  
H R Gralnick
Keyword(s):  
Molecular Biology ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Von Willebrand ◽  
Willebrand Factor

Comparative Study of Intranasal, Subcutaneous and Intravenous Administration of Desamino-D-Arginine Vasopressin (DDAVP)

1986 ◽  
Vol 55 (01) ◽  
pp. 108-111 ◽  
Author(s):  
M Köhler ◽  
P Hellstern ◽  
C Miyashita ◽  
G von Blohn ◽  
E Wenzel
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Factor Xii ◽  
Additional Advantage ◽  
Mean Values ◽  
Routes Of Administration ◽  
The Mean ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Factor Antigen

SummaryThis study was performed to evaluate the influence of different routes of administration on the efficacy of DDAVP treatment. Ten healthy volunteers received DDAVP intranasally (i.n.), subcutaneously (s.c.) and intravenously (i.v.) in a randomized cross-over trial. Factor XII and high molecular weight (HMW)-kininogen levels increased only slightly after DDAVP administration. The mean increase of factor VIII: C was 3.1 (i. v.), 2.3 (s. c.), and 1.3 (i.n.) - fold over baseline. Ristocetin cofactor (von Willebrand factor antigen) increased 3.1 (2.5), 2.0 (2.3) and 1.2 (1.2) - fold over baseline mean values after i.v., s.c. and i.n. DDAVP, respectively. The half-disappearance time of factor VIII and von Willebrand factor (vWF) after DDAVP ranged from five (factor VIII: C) to eight hours (vWF). The mean increase of fibrinolytic activity was more pronounced after i.v. DDAVP. The antidiuretic effect was moderate with no apparent differences between the routes of application. This study provides further evidence that both i.v. and s.c. DDAVP administration result in an appropriate and reliable stimulation of haemostasis. An additional advantage of s. c. administration is its suitability for home treatment.

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