Visualization of an N-terminal fragment of von Willebrand factor in complex with factor VIII

Abstract The mechanisms responsible for the low factor VIII (fVIII) activity in the plasma of patients with mild/moderate hemophilia A are poorly understood. In such patients, we have identified a series of fVIII mutations (Ile2098Ser, Ser2119Tyr, Asn2129Ser, Arg2150His, and Pro2153Gln) clustered in the C1 domain and associated with reduced binding of fVIII to von Willebrand factor (vWf). For each patient plasma, the specific activity of mutated fVIII was close to that of normal fVIII. Scatchard analysis showed that the affinity for vWf of recombinant Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII mutants was reduced 8-fold, 80-fold, and 3-fold, respectively, when compared with normal fVIII. Given the importance of vWf for the stability of fVIII in plasma, these findings suggested that the reduction of fVIII binding to vWf resulting from the above-mentioned mutations could contribute to patients' low fVIII plasma levels. We, therefore, analyzed the effect of vWf on fVIII production by Chinese hamster ovary (CHO) cells transfected with expression vectors for recombinant B domain-deleted normal, Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII. These 3 mutations impaired the vWf-dependent accumulation of functional fVIII in culture medium. Analysis of fVIII production by transiently transfected CHO cells indicated that, in addition to the impaired stabilization by vWf, the secretion of functional Ile2098Ser and Arg2150His fVIII was reduced about 2-fold and 6-fold, respectively, by comparison to Ser2119Tyr and normal fVIII. These findings indicate that C1-domain mutations resulting in reduced fVIII binding to vWf are an important cause of mild/moderate hemophilia A.

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von Willebrand factor and factor VIII levels after desmopressin are associated with bleeding phenotype in type 1 VWD

Blood Advances ◽

10.1182/bloodadvances.2019000863 ◽

2019 ◽

Vol 3 (24) ◽

pp. 4147-4154 ◽

Cited By ~ 1

Author(s):

Ferdows Atiq ◽

Lisette M. Schütte ◽

Agnes E. M. Looijen ◽

Johan Boender ◽

Marjon H. Cnossen ◽

...

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Key Points ◽

Von Willebrand ◽

Willebrand Factor

Key Points VWF and FVIII levels after desmopressin, which mimic hemostatic response, are associated with the bleeding phenotype of type 1 VWD patients. Variability in VWF and FVIII response to hemostatic challenges may partly explain heterogeneity in bleeding phenotype of VWD patients.

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A novel cause of mild/moderate hemophilia A: mutations scattered in the factor VIII C1 domain reduce factor VIII binding to von Willebrand factor

Blood ◽

10.1182/blood.v96.3.958.015k13_958_965 ◽

2000 ◽

Vol 96 (3) ◽

pp. 958-965 ◽

Cited By ~ 4

Author(s):

Marc Jacquemin ◽

Renaud Lavend'homme ◽

Abdellah Benhida ◽

Beatrijs Vanzieleghem ◽

Roseline d'Oiron ◽

...

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Cho Cells ◽

Hemophilia A ◽

Chinese Hamster ◽

Expression Vectors ◽

Scatchard Analysis ◽

C1 Domain ◽

Von Willebrand ◽

Willebrand Factor

The mechanisms responsible for the low factor VIII (fVIII) activity in the plasma of patients with mild/moderate hemophilia A are poorly understood. In such patients, we have identified a series of fVIII mutations (Ile2098Ser, Ser2119Tyr, Asn2129Ser, Arg2150His, and Pro2153Gln) clustered in the C1 domain and associated with reduced binding of fVIII to von Willebrand factor (vWf). For each patient plasma, the specific activity of mutated fVIII was close to that of normal fVIII. Scatchard analysis showed that the affinity for vWf of recombinant Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII mutants was reduced 8-fold, 80-fold, and 3-fold, respectively, when compared with normal fVIII. Given the importance of vWf for the stability of fVIII in plasma, these findings suggested that the reduction of fVIII binding to vWf resulting from the above-mentioned mutations could contribute to patients' low fVIII plasma levels. We, therefore, analyzed the effect of vWf on fVIII production by Chinese hamster ovary (CHO) cells transfected with expression vectors for recombinant B domain-deleted normal, Ile2098Ser, Ser2119Tyr, and Arg2150His fVIII. These 3 mutations impaired the vWf-dependent accumulation of functional fVIII in culture medium. Analysis of fVIII production by transiently transfected CHO cells indicated that, in addition to the impaired stabilization by vWf, the secretion of functional Ile2098Ser and Arg2150His fVIII was reduced about 2-fold and 6-fold, respectively, by comparison to Ser2119Tyr and normal fVIII. These findings indicate that C1-domain mutations resulting in reduced fVIII binding to vWf are an important cause of mild/moderate hemophilia A.

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A human antibody directed to the factor VIII C1 domain inhibits factor VIII cofactor activity and binding to von Willebrand factor

Blood ◽

10.1182/blood.v95.1.156 ◽

2000 ◽

Vol 95 (1) ◽

pp. 156-163 ◽

Cited By ~ 86

Author(s):

Marc Jacquemin ◽

Abdellah Benhida ◽

Kathelijne Peerlinck ◽

Benoı̂t Desqueper ◽

Luc Vander Elst ◽

...

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Rare Complication ◽

Cell Epitope ◽

Human Antibody ◽

Mutation Site ◽

C1 Domain ◽

Cofactor Activity ◽

Von Willebrand ◽

Willebrand Factor

Abstract The occurrence of factor VIII (fVIII) inhibitory antibodies is a rare complication of fVIII substitution therapy in mild/moderate hemophilia A patients. fVIII mutations in certain regions such as the C1 domain are, however, more frequently associated with inhibitor, for reasons which remain unclear. To determine whether inhibitors could map to the mutation site, we analyzed at the clonal level the immune response of such a patient with an inhibitor to wild-type but not self-fVIII and an Arg2150His substitution in the C1 domain. Immortalization of the patient B lymphocytes provided a cell line producing an anti-fVIII IgG4κ antibody, LE2E9, that inhibited fVIII cofactor activity, following type 2 kinetics and prevented fVIII binding to von Willebrand factor. Epitope mapping with recombinant fVIII fragments indicated that LE2E9 recognized the fVIII C1 domain, but not the Arg2150His-substituted C1 domain. Accordingly, LE2E9 did not inhibit Arg2150His fVIII activity. These observations identify C1 as a novel target for fVIII inhibitors and demonstrate that Arg2150His substitution alters a B-cell epitope in the C1 domain, which may contribute to the higher inhibitor incidence in patients carrying such substitution. (Blood. 2000; 95:156-163)

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A human antibody directed to the factor VIII C1 domain inhibits factor VIII cofactor activity and binding to von Willebrand factor

Blood ◽

10.1182/blood.v95.1.156.001k50_156_163 ◽

2000 ◽

Vol 95 (1) ◽

pp. 156-163 ◽

Cited By ~ 1

Author(s):

Marc Jacquemin ◽

Abdellah Benhida ◽

Kathelijne Peerlinck ◽

Benoı̂t Desqueper ◽

Luc Vander Elst ◽

...

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Rare Complication ◽

Cell Epitope ◽

Human Antibody ◽

Mutation Site ◽

C1 Domain ◽

Cofactor Activity ◽

Von Willebrand ◽

Willebrand Factor

The occurrence of factor VIII (fVIII) inhibitory antibodies is a rare complication of fVIII substitution therapy in mild/moderate hemophilia A patients. fVIII mutations in certain regions such as the C1 domain are, however, more frequently associated with inhibitor, for reasons which remain unclear. To determine whether inhibitors could map to the mutation site, we analyzed at the clonal level the immune response of such a patient with an inhibitor to wild-type but not self-fVIII and an Arg2150His substitution in the C1 domain. Immortalization of the patient B lymphocytes provided a cell line producing an anti-fVIII IgG4κ antibody, LE2E9, that inhibited fVIII cofactor activity, following type 2 kinetics and prevented fVIII binding to von Willebrand factor. Epitope mapping with recombinant fVIII fragments indicated that LE2E9 recognized the fVIII C1 domain, but not the Arg2150His-substituted C1 domain. Accordingly, LE2E9 did not inhibit Arg2150His fVIII activity. These observations identify C1 as a novel target for fVIII inhibitors and demonstrate that Arg2150His substitution alters a B-cell epitope in the C1 domain, which may contribute to the higher inhibitor incidence in patients carrying such substitution. (Blood. 2000; 95:156-163)

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LOCALIZATION OF A FACTOR VIII BINDING DOMAIN ON THE N-TERMINAL PORTION (FRAGMENT SpIII) OF VON WILLEBRAND FACTOR

10.1055/s-0038-1642876 ◽

1987 ◽

Author(s):

Y Takahashi ◽

J P Girma ◽

M Kalafatis ◽

K Sewerin ◽

L O Andersson ◽

...

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Inhibitory Effect ◽

Binding Domain ◽

Dependent Manner ◽

Terminal Portion ◽

Terminal Fragment ◽

Von Willebrand ◽

Willebrand Factor ◽

Dose Dependent

A new domain has been identified on the von Willebrand Factor (vWF) subunit. vWF binds to platelet glycoproteins GPIb and GPIIb/IIIa as well as to collagen and corresponding domains have been isolated. vWF also binds to Factor VIII (F.VIII). We show here that the corresponding domain is located on the N-terminal portion of the vWF subunit (residues 1 to 1,365). For this purpose, F.VIII was tested for its ability to bind to purified vWF degradation fragments obtained by digestion with S.aureus V-8 protease, ie a dimeric N-terminal fragment of 320 kd (SpIII) and a dimeric C-terminal fragment of 220 kd (SpII). Human F.VIII was purified from cryoprecipitate by immunoadsorption of F.VIII/vWF onto a monoclonal antibody (MAb) to vWF coupled to Sepharose, followed by elution using 0.25 M CaCl2. The F.VIII preparation contained 100 U/ml VIII:C and less than 0.001 XJ/ml vWFAg. The binding assay was performed using polystyrene tubes coated with 2 ug/ml of purified vWF, SpIII or SpII. Coated albumin, fibrinogen or fibronectin were used as controls. Purified F.VIII (0.1 to 2 U/ml VIII:C) was incubated in the coated tubes for 1 h at 37°C. Following washing, bound F.VIII was estimated in situ by one-stage clotting and chromogenic assays. Immunoradiometric assay with 125 I-MAbs to SpII or SpIII demonstrated that the amount of coated protein remained constant throughout the experiments. F.VIII bound in a dose-dependent manner to coated vWF and SpIII but not to SpII. Binding was specific for F.VIII as demonstrated by inhibition experiments. Bound F.VIII could be removed with 0.25 M CaCl2 and its coagulant activity inhibited by a MAb or an oligoclonal (homologous) antibody neutralising VIII:C. Binding of F.VIII to coated vWF or SpIII was also inhibited in a dose-dependent way by vWF or SpIII. In contrast, addition of SpII had no effect upon the binding. Binding of F.VIII to SpIII was confirmed using MAbs to vWF. Among 28 MAbs which bound SpIII and had no anti VIII:C activity, 12 inhibited binding of F.VIII whereas the others had no effect. Among the latter, 3 MAbs blocked binding of vWF or SpIII to GPIb and 6 MAbs inhibited binding of vWF or SpIII to collagen. Ten MAbs to SpII had no inhibitory effect upon binding of F.VIII. These results indicate that a F.VIII binding domain of vWF is located in the N-terminal portion of vWF (residues 1 to 1,365) and that it is distinct from the GPIb and collagen binding domains. The 12 MAbs to SpIII which block binding of F.VIII to vWF or SpIII should allow the precise localization of this new domain.

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A von Willebrand factor fragment containing the D′D3 domains is sufficient to stabilize coagulation factor VIII in mice

Blood ◽

10.1182/blood-2013-11-540534 ◽

2014 ◽

Vol 124 (3) ◽

pp. 445-452 ◽

Cited By ~ 37

Author(s):

Andrew Yee ◽

Robert D. Gildersleeve ◽

Shufang Gu ◽

Colin A. Kretz ◽

Beth M. McGee ◽

...

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Coagulation Factor ◽

Coagulation Factor Viii ◽

Key Points ◽

Von Willebrand ◽

Willebrand Factor

Key Points The D′D3 domains of VWF are sufficient to stabilize FVIII in vivo. The prolongation of VWF D′D3 survival in vivo by Fc fusion elevates FVIII levels in the setting of VWF but not FVIII deficiency.

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Patterns of expression of factor VIII and von Willebrand factor by endothelial cell subsets in vivo

Blood ◽

10.1182/blood-2015-12-684688 ◽

2016 ◽

Vol 128 (1) ◽

pp. 104-109 ◽

Cited By ~ 36

Author(s):

Junliang Pan ◽

Thanh Theresa Dinh ◽

Anusha Rajaraman ◽

Mike Lee ◽

Alexander Scholz ◽

...

Keyword(s):

Endothelial Cell ◽

Factor Viii ◽

Von Willebrand Factor ◽

Blood Capillary ◽

High Endothelial Venules ◽

Key Points ◽

Von Willebrand ◽

Willebrand Factor

Key Points Subsets of ECs, including lymphatic and fenestrated ECs, but not conventional blood capillary ECs, synthesize FVIII. von Willebrand factor and FVIII are coexpressed in postcapillary high endothelial venules but not in most other ECs.

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