Slowed Release of Thrombin-cleaved Factor VIII from von Willebrand Factor by a Monoclonal and a Human Antibody Is a Novel Mechanism for Factor VIII Inhibition

Saenko, Evgueni L.; Shima, Midori; Gilbert, Gary E.; Scandella, Dorothea

doi:10.1074/jbc.271.44.27424

Cited by 162 publications

(136 citation statements)

References 42 publications

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“…ESH8 is one anti-C2 domain inhibitory antibody that does not interfere with binding of FVIII to vWF and for which the mechanism of FVIII inactivation has been studied. Studies support that ESH8 stabilizes the FVIII-vWF interaction to delay release of FVIIIa from vWF by 4-fold, so that A2 domain dissociation occurs before FVIIIa release from vWF (40). The unique stability of IR8 after thrombin-activation provided a useful reagent to test this hypothesis that previously was derived from kinetic measurements.…”

Section: Discussionmentioning

confidence: 94%

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Pipe

Kaufman

1997

Proc. Natl. Acad. Sci. U.S.A.

122

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Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690-2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.Hemophilia A results from the quantitative or qualitative deficiency of coagulation factor VIII (FVIII), necessitating exogenous replacement by either plasma-or recombinant-derived FVIII preparations. FVIII has a domain organization of A1-A2-B-A3-C1-C2 and is synthesized as a 2,351-aa single-chain glycoprotein of 280 kDa from which a signal peptide is cleaved upon translocation into the lumen of the endoplasmic reticulum (1-3). Whereas the A and C domains exhibit 35-40% amino acid identity to each other and to the A and C domains of coagulation factor V, the B domain is not homologous to any known protein. Intracellular proteolytic processing within the B domain after residue Arg-1648 generates an 80-kDa light chain (domains A3-C1-C2) and a heterogeneous-sized heavy chain of 90-200 kDa (domains A1-A2-B). The heavy and light chains are associated as a heterodimer through a divalent metal-ion-dependent linkage between the A1 and A3 domains.In plasma, FVIII circulates in an inacti...

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Section: Discussionmentioning

confidence: 94%

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Pipe

Kaufman

1997

Proc. Natl. Acad. Sci. U.S.A.

122

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“…Gawryl and Hoyer demonstrated that some type II inhibitors compete with VWF for binding to FVIII [2]. Conversely, VWF is required for certain type II inhibitor antibodies to exert their activity, by binding exclusively to FVIII complexed with VWF [4] or by reducing the rate of dissociation of activated FVIII from VWF [3]. By contrast, Mab-LE2E9 inhibited FVIII effectively in the absence of VWF.…”

Section: Production and Characterization Of The Human Monoclonal Antimentioning

confidence: 99%

“…Competition with von Willebrand factor (VWF) has been demonstrated as one such mechanism [2]. Conversely, some inhibitors rely on the presence of VWF to inhibit FVIII activity [3,4].…”

Section: Introductionmentioning

confidence: 99%

Variable region heavy chain glycosylation determines the anticoagulant activity of a factor VIII antibody

Jacquemin

2010

Haemophilia

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Summary. The mechanism of action of antibodies inhibiting partially factor VIII (FVIII) activity (type II inhibitor) is still poorly understood. We produced an unusual type II monoclonal antibody, called LE2E9, derived from a patient with mild haemophilia A. The antibody displayed several unexpected structural and functional properties such as glycosylation in the variable region, binding to the FVIII C1 domain, inhibition of maximum 80-90% FVIII activity when in excess over FVIII, and prevention of FVIII binding to von Willebrand factor (VWF). Those unusual characteristics of the antibody prompted multidisciplinary studies to determine its mechanism of action and the role of the FVIII C1 domain. Enzymatic deglycosylation and site-directed mutagenesis indicated that the oligosaccharides do not determine the affinity of the antibody but enhanced its FVIII neutralizing activity. Modification of glycosylation in the variable region of antibodies therefore contributes to the diversity of FVIII type II inhibition and provides a novel strategy with which to modulate the functional activity of antibodies. Investigation of the FVIII C1 domain function led to identification of mutations located in that domain and impairing FVIII binding to VWF as a common cause of mild/moderate haemophilia A. Finally, the cloning of human monoclonal antibodies inhibiting partially FVIII activity opened the way to evaluate such antibodies as a novel type of anticoagulant drug. This concept was validated in animal models of thrombosis. Those studies are expected to have a significant impact on the optimization of treatments for patients with inhibitor as well as for patients with thrombophilia.

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“…The quantity of immobilized factor VIII was estimated by incubation with fluorescein-labeled ESH8, a mAb that binds to the C2 domain of factor VIII but does not interfere with binding to vWf or to phospholipid vesicles (37). Antibody binding curves were generated as described for ⌬Pro vWf, above.…”

Section: Normalization Of Binding Datamentioning

confidence: 99%

Four Hydrophobic Amino Acids of the Factor VIII C2 Domain Are Constituents of Both the Membrane-binding and von Willebrand Factor-binding Motifs

Gilbert¹,

Kaufman²,

Arena³

et al. 2002

Journal of Biological Chemistry

Self Cite

110

147

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Factor VIII binds to phospholipid membranes and to von Willebrand factor (vWf) via its second C domain, which has lectin homology. The crystal structure of the C2 domain has prompted a model in which membrane binding is mediated by two hydrophobic spikes, each composed of a pair of residues displayed on a ␤-hairpin turn, and also by net positive charge and specific interactions with phospho-L-serine. , and 91% reduction in specific activity in the activated partial thromboplastin time assay. In a phospholipidlimiting factor Xa activation assay, these mutants had a 65, 85, and 96% reduction in specific activity. Equilibrium binding of fluorescent, sonicated phospholipid vesicles to mutants immobilized on Superose beads was measured by flow cytometry. The affinities for phospholipid were reduced ϳ20-, 30-, and >35-fold for 2199/2200, 2251/2252, and 4-Ala, respectively. A dimeric form of mature vWf bound to immobilized factor VIII and the same mutants, but the affinities of the mutants were reduced ϳ5-, 10-, and >20-fold, respectively. In a competition, solution phase enzyme-linked immunosorbent assay, plasma vWf bound factor VIII and the same mutants with the affinities for the mutants reduced >5-, >5-, and >50-fold, respectively. We conclude that the two hydrophobic spikes are constituents of both the phospholipidbinding and vWf-binding motifs. In plasma, vWf apparently binds the inherently sticky membrane-binding motif, preventing nonspecific interactions.Factor VIII is a phosphatidyl-L-serine (PS) 1 binding cofactor (1, 2) for the vitamin K-dependent serine protease, factor IXa, that also binds to PS-containing membranes (3, 4). The membrane-bound factor VIIIa-factor IXa complex cleaves the zymogen, factor X, to factor Xa, which is then responsible for catalyzing prothrombin activation (5). The importance of this enzyme complex is illustrated by hemophilia, a disease in which a deficiency of either factor VIII (hemophilia A) or factor IX (hemophilia B) leads to life-threatening bleeding. Factor IXa gains more than 100,000-fold greater efficiency in activating factor X by assembling with factor VIIIa on a PS-containing membrane than when free in solution (6). We have recently found that the predominant effect of PS-containing membranes on the factor VIIIa-factor IXa complex is to increase the k cat by more than 1000-fold (7). These membranes also increase the affinity of factor IXa for factor VIIIa and for factor X. The central importance of the membrane binding function of factor VIII motivates studies to define the membrane-binding motif.Factor VIII, with M r 280,000, is homologous to another procoagulant protein, factor V, in amino acid sequence (8 -10) and in function, as a membrane-bound enzyme cofactor (5, 11-13). The proteins share a repeating domain structure of A1-A2-B-A3-C1-C2 in which the A domains are homologous with ceruloplasmin, the B domain is unique to each protein, and the C domains are homologous with discoidin I, a phospholipid-binding lectin (14), and with a murine milk fat globule membrane ...

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Slowed Release of Thrombin-cleaved Factor VIII from von Willebrand Factor by a Monoclonal and a Human Antibody Is a Novel Mechanism for Factor VIII Inhibition

Cited by 162 publications

References 42 publications

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Variable region heavy chain glycosylation determines the anticoagulant activity of a factor VIII antibody

Four Hydrophobic Amino Acids of the Factor VIII C2 Domain Are Constituents of Both the Membrane-binding and von Willebrand Factor-binding Motifs

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