Factor VIII binds to von Willebrand factor via its Mr-80000 light chain

Hamer, R.J.; Koedam, J. A.; Beeser‐Visser, Nel H.; Sixma, Jan J.; Bertina, Rogier M.; Mourik, Jan A. van

doi:10.1111/j.1432-1033.1987.tb13480.x

Cited by 90 publications

(68 citation statements)

References 28 publications

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“…We now speculate that the protective effect of von Willebrand factor on activation and inactivation of factor VIIl by factor Xa is caused by its ability to block the phospholipid-dependent binding of factor Xa to factor VIII. von Willebrand factor, which binds to the light chain of factor VIII [29,311, has been shown to compete with phospholipids in binding to factor VIII [32]; we have recently described a similar effect of von Willebrand factor on the inactivation of factor VIII by activated protein C [27], a reaction which is also dependent on phospholipid. Further studies are required to characterize further the mechanism and physiological relevance of the phenomenon described in this study.…”

Section: Discussionmentioning

confidence: 99%

“…1251-factor VIII (I0000 cpm/well) was bound to the immobilized von Willebrand factor for 2 h at 37°C and the wells were washed with 0.3% BSA/O.l% Tween-20. This binding system has been described and characterized before [29]. To study the release of proteolytic products of factor VIII from von Willebrand factor, the wells were incubated at 37°C with factor Xa (10 pg/ml, 200 nM) in a 50 4 1 2 3 Fig.…”

Section: Binding Of Factor VIII To Von Willebrand Factor and Elution mentioning

confidence: 99%

“…Polystyrene microtiter wells (Removawell strips, Greiner B.V., Alphen a/d Rijn, The Netherlands) were coated with von Willebrand factor (100 pl, 1 pg/ml in Michaelis buffer) for 2 h at 3 7 T , followed by blocking of the wells with 3% BSA/O.l% Tween-20 [29]. 1251-factor VIII (I0000 cpm/well) was bound to the immobilized von Willebrand factor for 2 h at 37°C and the wells were washed with 0.3% BSA/O.l% Tween-20.…”

Section: Binding Of Factor VIII To Von Willebrand Factor and Elution mentioning

confidence: 99%

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The effect of von Willebrand factor on activation of factor VIII by factor Xa

Koedam¹,

Hamer²,

Beeser‐Visser³

et al. 1990

European Journal of Biochemistry

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Factor VIII has to be activated before it can serve efficiently as a cofactor in the intrinsic pathway of blood coagulation. This activation occurs through specific proteolytic cleavages in the molecule by either thrombin or factor Xa. In this study, we show that von Willebrand factor inhibits the activation of factor VIII by factor Xa. Incubation of factor VIII (30 Ujml) with 0.1 pg/ml factor Xa resulted in a 1.6-fold activation followed by a decay of coagulant activity. In the presence of 10 pg/ml von Willebrand factor, activation and inactivation of factor VIII was completely inhibited. In contrast, the activation of factor VIII by thrombin was not influenced by von Willebrand factor. At high concentrations of factor Xa (10 pg/ml), von-Willebrand-factor-bound factor VIII could be cleaved and activated. The generated proteolytic fragments were identical to the fragments produced in the absence of von Willebrand factor and all fragments were released from von Willebrand factor. The major products were light-chain-derived fragments of molecular mass 66/68 kDa and 60 kDa and heavy-chain-derived fragments of 40 and 42 kDa. Also minor products of 12,20/21,23,27 and 30 kDa were observed, most of which were specific for cleavage of factor VIII by factor Xa.Factor VIII is an important plasma protein which acts as a cofactor in the intrinsic pathway of coagulation. It does so by dramatically increasing the V,,, for the activation of factor X by the enzyme factor IXa in the presence of calcium and a phospholipid surface [I]. The critical role of factor VIII in hemostasis is illustrated by the severe bleeding tendency (known as hemophilia A) in its absence [2]. It is a key protein in the regulation of the coagulation cascade, since its cofactor activity can be enhanced through feed-back mechanisms by thrombin and factor Xa. Factor VIII activity is subsequently destroyed by the regulatory-enzyme-activated protein C. Both activation and inactivation of factor VIII occur through specific proteolytic cleavages within the molecule [3 -51.Factor VIII is synthesized as a single-chain precursor molecule of 2332 amino acids [6, 71 and purified from plasma as a carboxy-terminal light chain of 80 kDa which is associated with an amino-terminal heavy chain ranging in molecular mass between 90 -210 kDa [4, 8 -111. In many studies, the kinetics of factor-VIII and factor-X activation [I, 12-181, as well as the changes that occur within the factor VIII molecule upon activation by thrombin or factor Xa [3,4, 17, 191, have been described. These studies have ignored, however, that factor VIII circulates as a non-covalent complex with von Willebrand factor [20]. Formation of this complex is of great physiological importance for factor VIII, since it prolongs its half-life in the circulation [21,22]. Since von Willebrand factor is a very large glycoprotein which represents 99% of the mass of the factor-VIII -von-Willebrand-factor complex, it is conceivable that it modulates the activation and inactivation of factor VIII.In a previous paper ...

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Section: Discussionmentioning

confidence: 99%

Section: Binding Of Factor VIII To Von Willebrand Factor and Elution mentioning

confidence: 99%

Section: Binding Of Factor VIII To Von Willebrand Factor and Elution mentioning

confidence: 99%

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The effect of von Willebrand factor on activation of factor VIII by factor Xa

Koedam¹,

Hamer²,

Beeser‐Visser³

et al. 1990

European Journal of Biochemistry

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“…When those experiments were performed, the binding of FVIII to VWF was attributed to two FVIII regions: the carboxy-terminal part of the C2 domain and the acidic part of the A3 domain [15][16][17][18]. It was therefore unexpected that Mab-LE2E9, which recognizes an epitope in the C1 domain, could interfere with FVIII binding to VWF.…”

Section: Role Of the C1 Domain In Fviii Binding To Vwfmentioning

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 binding of FVIII to vWF is important for the survival of FVIII as the plasma concentration of FVIII is considerably reduced in the absence of vWF (6)(7)(8). FVIII binds to vWF involving A3 and C2 domains of the light chain (9)(10)(11)(12)(13). It has been shown that the A2, A3, and C2 domains of FVIII are involved in the interaction with LRP (14,15).…”

Section: Introductionmentioning

confidence: 99%

Phosphatidylinositol Containing Lipidic Particles Reduces Immunogenicity and Catabolism of Factor VIII in Hemophilia A Mice

Peng

Straubinger

Balu‐Iyer

2010

AAPS J

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Abstract. Factor VIII (FVIII) is an important cofactor in blood coagulation cascade. It is a multidomain protein that consists of six domains, NH 2 -A1-A2-B-A3-C1-C2-COOH. The deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. Replacement therapy using recombinant FVIII (rFVIII) is the first line of therapy, but a major clinical complication is the development of inhibitory antibodies that abrogate the pharmacological activity of the administered protein. FVIII binds to anionic phospholipids (PL), such as phosphatidylinositol (PI), via lipid binding region within the C2 domain of FVIII. This lipid binding site not only consists of immunodominant epitopes but is also involved in von Willebrand factor binding that protects FVIII from degradation in vivo. Thus, we hypothesize that FVIII-PL complex will influence immunogenicity and catabolism of FVIII. The biophysical studies showed that PI binding did not alter conformation of the protein but improved intrinsic stability as measured by thermal denaturation studies. ELISA studies confirmed the involvement of the C2 domain in binding to PI containing lipid particles. PI binding prolonged the in vivo circulation time and reduced catabolism of FVIII in hemophilia A mice. FVIII-PI complex reduced inhibitor development in hemophilia A mice following intravenous and subcutaneous administration. The data suggest that PI binding reduces catabolism and immunogenicity of FVIII and has potential to be a useful therapeutic approach for hemophilia A.

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Factor VIII binds to von Willebrand factor via its Mr-80000 light chain

Cited by 90 publications

References 28 publications

The effect of von Willebrand factor on activation of factor VIII by factor Xa

The effect of von Willebrand factor on activation of factor VIII by factor Xa

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

Phosphatidylinositol Containing Lipidic Particles Reduces Immunogenicity and Catabolism of Factor VIII in Hemophilia A Mice

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