A new recombinant procoagulant protein derived from the cDNA encoding human factor VIII

Meulien, Pierre; Faure, Thérèse; Mischler, Fabienne; Harrer, Huguette; Ulrich, P; Bouderbala, B.; Dott, Karin; Marie, Michelle; Mazurier, Claudine; Wiesel, M-L

doi:10.1093/protein/2.4.301

Cited by 59 publications

(36 citation statements)

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“…Previous studies on the requirements for functional activity of FVIII demonstrated that cleavage after Arg residues 372 and 1689 both were required for activation of factor VIII and that the B domain was not required for functional activity (13,(22)(23)(24). Deletion of residues 741-1689 yielded a functional molecule (90͞73) that displayed WT thrombin cleavage and activation, resulting in a heterotrimer similar to WT FVIIIa that was susceptible to rapid dissociation of the A2 subunit.…”

Section: Discussionmentioning

confidence: 99%

“…Cleavage after residue Arg-740 releases the heavily glycosylated B domain. Previous studies demonstrated that the B domain of FVIII is dispensable for FVIII cofactor activity (22)(23)(24). Genetically engineered FVIII molecules that have varying degrees of B domain deletion yield functional FVIII molecules that are more efficiently expressed in mammalian cells (22,23,25,26).…”

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confidence: 99%

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Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Pipe

Kaufman

1997

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

126

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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%

mentioning

confidence: 99%

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Pipe

Kaufman

1997

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

126

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“…Other authors have reported the construction and expression of recombinant factor VIII lacking most or all of the B domain (Eaton et al, 1986;Toole et al, 1986;Sarver et al, 1987;Lagner et al, 1988;Meulien et al, 1988;Bihoreau et al, 1991;Pittman et al, 1993). The products of the single cDNA gene constructs described either contained the light and heavy chains and, in addition, the single peptide chain representing the nonprocessed primary translation product as the main species or the latter peptide chain as most dominating component.…”

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confidence: 99%

Novel Forms of B‐Domain‐Deleted Recombinant Factor VIII Molecules

Lind¹,

Larsson²,

Spira³

et al. 1995

European Journal of Biochemistry

123

111

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Recombinant molecules similar to the smallest active plasma-derived factor VIII molecule, a complex of an 80-kDa and a 90-kDa polypeptide chain lacking the B domain, have been produced using various factor VIII cDNA constructs in order to obtain primary translation products which were efficiently processed into the 80+90-kDa complex. Three types of single-chain cDNAs encoding B-domain-deleted derivatives factor VIII were designed, taking account of sites at Arg740 and Glu1649, assumed to be important for processing factor VIII.In the type 1 constructs, either Arg747, Arg752, or Arg776 in the N-terminal region of factor VIII B domain was fused to the N-terminus (Glu1649) of the 80-kDa subunit. In the type 2 construct r-VIII SQ, Ser743 was fused to Gln1638, creating a link of 14 amino acids between the C-terminus (Arg740) of the 90-kDa chain and N-terminus of the 80-kDa chain, whereas in type 2 r-VIII RH, Arg747 was fused to His1646. In the type 3 constructs, the B-domain was completely removed or replaced with 1-4 Arg residues.After expression in Chinese hamster ovary cells, the type 1 derivatives and the type 3 derivatives with 0-2 Arg residues inserted were found to be only partially processed and contained a large amount of the 170-kDa primary translation product. In contrast, most of the type 2 derivatives r-VIII SQ and r-VIII RH and the type 3 derivatives r-VIII R4 and r-VIII R5 containing three or four extra Arg residues preceding the N-terminus of the 80-kDa chain were processed into the desired 80+90-kDa chain complexes. The feature common to the most efficiently processed factor VIII deletion derivatives was that they contained the recognition motif for proteolytic cleavage by the membrane-bound subtilisin-like protease furin, which is expressed in most types of cells; that is, basic amino acid residues at positions -1 and -4 relative to the cleavage site at Glu1649. Biochemical studies of r-VIII SQ and r-VIII R5, two of the most effectively processed factor VIII derivatives, showed that both proteins had a normal factor VIII cofactor function, and had N-and C-termini of the 80-kDa and 90-kDa chains corresponding to those found in plasma-derived factor VIII.

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“…Considering these structural requirements and the necessity for a therapeutic purpose to dispose of a greatly secreted protein, a second-generation antihaemophilia A factor, FVIIIAll, has been constructed [9]. This recombinant protein, lacking a large portion of the B domain (Pro771 -Asp1666) and the amino-terminal part of the light chain, contains the different thrombin-cleavage sites present in the heavy and light chains of the pd-FVIII.…”

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confidence: 99%

Copper‐atom identification in the active and inactive forms of plasma‐derived FVIII and recombinant FVIII‐ΔII

Bihoreau

Kersabiec

et al. 1994

European Journal of Biochemistry

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The plasma-derived factor VIII (pd-FVILI) circulates as different heterodimers of heavy and light chains associated by a metallic ion still present in the functional activated factor VIII trimer of molecular mass 50000-45000-70000 Da. The chelation of the metal leads to the dissociation of these complexes with a concomitant loss of the procoagulant activity. Until now, this ion has not been directly identified and its role in the structure/function relationships remains unclear. We report the first determination of the nature of this metal using atomic-absorption spectroscopy with Zeeman effect. A comparative identification was also performed with the new recombinant factor VIII, FVIII-AII. In the different active pd-FVIII heterodimers (of molecular mass ranging over 210000-80000-90000-80000 Da) and in FVIII-AII, copper was detected. This result is consistent with sequence similarities described between FVIII and copper-binding proteins. The quantification of the copper content in FVIII-A11 and in the corresponding pd-FVIII dimer of 90000-80000 Da indicated, for both proteins, the presence of one copper iordmol FVIII. Copper was also identified in the activated FVIII complex and remained in the dimer of 50000-70000 Da generated during FVIII inactivation. Further dissociation into isolated fragments of molecular masses 70000 Da and 50000 Da was concomitant with the loss of the copper ion. No copper was detected in the isolated fragment of molecular mass 45000 Da. These results suggest that the presence of the cation is not directly related to FVIII activity but is an essential structural prerequisite for FVIII heavy-lightchain association.Hemophilia A is related to the absence or to sequence modifications of factor VIII (FVIII), a glycoprotein which acts in the intrinsic coagulation pathway. The gene cloning [l] has greatly contributed to the understanding of the FVIII structure/function relationships. The amino acid sequence is predicted as a polypeptide of 2332 amino acids which exhibits a triplicated region of 330 residues (A domains), a unique region of 983 residues (B domain) and a duplicated region of 150 amino acids (C domains) arranged in the following order Al-A2-B-

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A new recombinant procoagulant protein derived from the cDNA encoding human factor VIII

Cited by 59 publications

References 0 publications

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Novel Forms of B‐Domain‐Deleted Recombinant Factor VIII Molecules

Copper‐atom identification in the active and inactive forms of plasma‐derived FVIII and recombinant FVIII‐ΔII

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