The interaction between human blood-coagulation factor VIII and von Willebrand factor. Characterization of a high-affinity binding site on factor VIII

Leyte, Anja; Verbeet, Martin Ph.; Brodniewicz-Proba, Teresa; Mourik, Jan A. van; Mertens, Koen

doi:10.1042/bj2570679

Cited by 145 publications

(93 citation statements)

References 29 publications

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“…This Initial studies supported that vWF interacts with the acidic amino acid-rich amino terminus of the FVIII light chain. mAbs to residues 1670-1689 inhibit interaction with vWF (46)(47)(48) and deletion of a portion of this region destroys high-affinity vWF interaction (47). However, the isolated acidic peptide 1648-1689 was incapable of binding vWF, suggesting the vWF interaction requires other regions of the FVIII molecule.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Pipe

Kaufman

1997

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

125

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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: 99%

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Pipe

Kaufman

1997

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

125

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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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“…[1][2][3] Upon proteolytic activation, FVIIIa is released from VWF as a heterotrimer composed of the A1 and A2 domains plus the FVIIIa light chain, A3-C1-C2. Activated platelet membranes expose negatively charged phosphatidylserine (PS), which increases from 2% to 10% or more of the surface phospholipids upon activation.…”

Section: Introductionmentioning

confidence: 99%

The factor VIII C1 domain contributes to platelet binding

Hsu

Pratt

Thompson

2008

Blood

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Activated factor VIII (FVIIIa) forms a procoagulant complex with factor IXa on negatively charged membranes, including activated platelet surfaces. Membrane attachment involves the FVIII C2 domain; involvement of the adjacent C1 domain has not been established. Binding of recombinant FVIII C1C2 and C2 proteins to platelets was detected by flow cytometry using (1) anti-C2 monoclonal antibody ESH8 followed by a phycoerythrinlabeled secondary antibody; (2) biotinylated C1C2 detected by phycoerythrinlabeled streptavidin, and (3) C1C2 and C2 site-specifically labeled with fluorescein. Highest binding and lowest background were obtained using fluoresceinconjugated proteins. More than 90% of activated platelets bound C1C2, compared with approximately 50% for equimolar C2. Estimates using fluorescent microbeads indicated approximately 7000 C1C2-binding sites per platelet, approximately 1400 for C2, and approximately 3000 for fluorescein-labeled FVIIIa. Unlike C2 or FVIII(a), C1C2 bound to approximately 700 sites/platelet before activation. C1C2 binding to activated platelets appeared independent of von Willebrand factor and was competed effectively by FVIII(a), but only partially by excess C2. Fluorescein-labeled FVIIIa was competed much more effectively by C1C2 than C2 for binding to activated platelets. Two monoclonal antibodies that inhibit C2 binding to membranes competed platelet binding of C2 more effectively than C1C2. Thus, the C1 domain of FVIII contributes to platelet-binding affinity. IntroductionFactor VIII (FVIII) circulates in plasma in a noncovalent complex with von Willebrand factor (VWF); this interaction is mediated by the FVIII C2 domain and an acidic sequence prior to the A3 domain. [1][2][3] Upon proteolytic activation, FVIIIa is released from VWF as a heterotrimer composed of the A1 and A2 domains plus the FVIIIa light chain, A3-C1-C2. Activated platelet membranes expose negatively charged phosphatidylserine (PS), which increases from 2% to 10% or more of the surface phospholipids upon activation. 4,5 FVIIIa forms a complex with FIXa and calcium on negatively charged phospholipid membranes, enhancing FIXa catalysis 100 000-to 200 000-fold. [6][7][8] Although FVIII can bind to FIXa on phospholipids, 9 or directly to activated platelets, 10 FVIIIa is required for procoagulant activity. 9 A hydrophobic surface on the FVIII(a) C2 domain 11 becomes buried in the phospholipid membrane upon binding, 12,13 and basic C2 residues make favorable charge-charge interactions with negatively charged PS head groups. Although FVIIIa and the light chain bind to PS-containing vesicles and activated platelets with similar affinities, the affinity of the recombinant C2 domain is 5-to 100-fold lower, 10,14,15 suggesting possible roles for the C1 and/or A3 domains.To address the potential role of the C1 domain in FVIII(a) attachment to platelets, a recombinant human FVIII C1C2 protein (residues 2020-2332) was produced in Escherichia coli, refolded, and characterized. The binding of C1C2 to platelet surfaces both before ...

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The interaction between human blood-coagulation factor VIII and von Willebrand factor. Characterization of a high-affinity binding site on factor VIII

Cited by 145 publications

References 29 publications

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

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

The factor VIII C1 domain contributes to platelet binding

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