Isolation and characterization of human factor VIII: molecular forms in commercial factor VIII concentrate, cryoprecipitate, and plasma.

Factor VIII was purified 1200-fold from commercial concentrates (Centre National de Transfusion Sanguine) by immunoaffinity chromatography using an anti-(80-kDa light chain) monoclonal antibody. The different molecular forms isolated were subsequently separated and analyzed using Fast Protein Liquid Chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis. The different factor-VI11 forms obtained, consisted of 80-kDa light chains, each being associated with one more-or-less fragmented heavy chain ranging over 90-210 kDa. The specific activity of these different forms was 7000 U/mg.At different stages of activation of factor VIII by thrombin, various forms were separated and identified. Activated complexes were found to result from the association of the 70-kDa light chain (generated from the SO-kDa light chain) with heavy chains ranging over 90-210 kDa. Two different thrombin activation steps were characterized. The first step corresponding to the cleavage of the 80-kDa light chain led to a sixfold increase in the procoagulant activity, and yielded a stable activated intermediate form. Compared with normal f-actor VIII, the ratio of von Willebrand activity to factor-Vlll activity, measured in the activated fractions, decreased, indicating that von Willebrand factor dissociates from factor VIII after proteolysis of the light chain by thrombin.In the second step, the 90-kDa heavy chain was cleaved into two polypeptides of 45 kDa and 50 kDa, which were associated with the 70-kDa proteolyzed light chain, generating the final activated complex (45 -50 ~ 70 kDa).The new intermediate forms we described imply a new scheme for the multistep activation process of factor VIII.Factor VIII is a glycoprotein present in the plasma at a very low concentration (0.1 pg/ml). Its absence or deficiency characterizes hemophilia A. It drives, as a cofactor, the activation of factor X mediated by factor IXa in the presence of phospholipids and calcium, in the blood coagulation intrinsic pathway [l]. The primary structure of f k t o r VIII contains 2332 amino acids and exhibits a triplicated region of 330 amino acids (A domains), a unique region of 983 amino acids (B domain), and a carboxy-terminal duplicated region of 150 amino acids (C domain), that are arranged in the order AlFactor VIII is susceptible to proteolysis by thrombin, plasmin and other serine proteases [3, 41. Factor VIII purified from plasma fractions of intermediate purity, consists of variable active forms with molecular masses ranging over 290-170 kDa, though a single chain precursor of 330 kDa has been reported [5]. Continuous proteolysis of the 210-kDa proteins yields a series of polypeptides of 210-90 kDa [6, 71. The polypeptides of 210 kDa and 80 kDa represent the N-terminal and C-terminal parts of factor VIII, respectively [2,8, 91. Fay et al. [lo] have identified the heterodimeric structure of the active factor VIII as the association, by a metal ion, of lightCorrespondence to H. Van de Pol, Centre National de Transfusion Sanguine...

Section: Discussionsupporting

confidence: 65%

Isolation and characterization of different activated forms of factor VIII, the human antihemophilic A factor

Bihoreau

Sauger

Yon

et al. 1989

“…More or less proteolytically degraded forms of factor VIII have been found as active molecules in factor VIII material purified from therapeutic factor VIII concentrates (Andersson et al, 1986). The different forms of factor VIII, having molecular masses between 170-280kDa were shown to consist of one heavy chain, between 90-200 kDa, in combination with one 80-kDa light chain.…”

mentioning

confidence: 99%

“…Human factor VIII is synthesized as a single-chain molecule of approximately 300kDa and consists of the structural domains Al-A2-B-A3-CI-C2 Wood et al, 1984;Vehar et al, 1984;Toole et al, 1984). In newly drawn plasma prepared in the presence of protease inhibitors, factor VIII appears as a complex of two polypeptide chains of 200 and 80 kDa (Andersson et al, 1986). It has since been found that processing of the precursor product into these two polypeptide chains takes place in the Golgi apparatus (Kaufman et al, 1988).…”

mentioning

confidence: 99%

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

Lind¹,

Larsson²,

Spira³

et al. 1995

123

111

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.

“…The B region, delimited by the amino acid residues 740 and 1648, is proteolyzed at different cleavage sites to generate active FVIII heterodimers of molecular mass ranging over 210000-80000 Da (including the total B domain) to 90000-80000 Da (without the B domain). Andersson et al [3] have shown that these different complexes have the same specific activity, suggesting that the B region is dispensable for FVIII procoagulant activity [4]. Nevertheless, the more efficient secretion observed for the different B-domain-deleted recombinant FVIII compared to the corresponding complete protein, suggests that this B region may be essential to regulate the FVIII biosynthesis [S].…”

mentioning

confidence: 99%

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

Bihoreau

Kersabiec

et al. 1994

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-