First determination of the secondary structure of purified factor VIII light chain

Bihoreau, Nicolas; Fontaine‐Aupart, Marie‐Pierre; Lehegarat, A; Desmadril, Michel; Yon, Jeannine

doi:10.1042/bj2880035

Cited by 9 publications

(4 citation statements)

References 32 publications

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“…Recent CD studies and computer analyses by Bihoreau et al (1992) of human factor Villa light chain have shown that the light-chain structure contains a fair amount of secondary structure. Approximately 5 5-5 8% of the light chain is ordered with about 31-36% 0 sheet and 22-24% a helix.…”

Section: Resultsmentioning

confidence: 99%

Determination of the disulfide bridges in factor Va light chain

Xue¹,

Kalafatis²,

Mann³

1993

Biochemistry

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The 74-kDa light chain of bovine factor Va is composed of three domains: the NH2-terminal A3 domain and the COOH-terminal C1 and C2 domains. In total, the light chain has eight cysteines: two in the A3 domain and three in each C domain. To determine the locations of the disulfide bridges, peptides were obtained from factor Va and iodo[1-14C]acetamide-labeled factor Va light chains by digestion with trypsin, activated protein C, lysylendopeptidase, and V8 protease. After HPLC purification, amino acid sequence and composition analyses showed that each domain of bovine Va light chain possesses a disulfide bond. The sites are Cys1684-Cys1710 (A3), Cys1866-Cys2020 (C1), and Cys2025-Cys2180 (C2). One free cysteine is located in each C domain, i.e., Cys1953 and Cys2100. The locations of the disulfide bonds in human Va and VIIIa light chains are anticipated to be similar to those of bovine Va light chain, because the cysteines involved are conserved.

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Section: Resultsmentioning

confidence: 99%

Determination of the disulfide bridges in factor Va light chain

Xue¹,

Kalafatis²,

Mann³

1993

Biochemistry

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“…The secondary structure of factor VIII is partially elucidated by c.d. spectroscopy, which revealed that the light chain contains both alphahelixes (22%) and beta-strand structures (36%) (81). Scanning transmission electron microscopy studies with porcine factor VIII indicated that the heavy and light chains of the factor VIII molecule are closely associated and constitute a globular core structure with a diameter of 10-12 nm (82).…”

Section: Biosynthesis and Structure Of Factor VIIImentioning

confidence: 99%

Factor VIII and von Willebrand Factor

Vlot¹,

Koppelman²,

Bouma³

et al. 1998

Thromb Haemost

118

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IntroducationFactor VIII and von Willebrand factor are plasma glycoproteins whose deficiency or structural defects cause hemophilia A and von Willebrand disease, respectively (1). These diseases are the most common inherited bleeding disorders of man. Factor VIII and vWF are synthesized by different cell types and circulate in plasma as a tightly bound complex. Factor VIII is synthesized in the liver (2), and functions as a cofactor for activated factor IX in the intrinsic activation of factor X on a membrane surface (3). vWF is synthesized in endothelial cells (4, 5) and megakaryocytes (6). vWF has a dual role in hemostasis: it promotes platelet adhesion to subendothelium after vessel injury (7, 8) and it acts as a carrier protein of factor VIII (1).The distinction between factor VIII and vWF was unclear for many years, because severe Von Willebrand disease is associated with factor VIII deficiency and because early preparations of factor VIII concentrates contained vWF and were therefore effective in correcting the platelet adhesion defects in patients with von Willebrand disease (9). Since factor VIII and vWF form a tightly bound non-covalent complex in plasma, both proteins are copurified when isolated from plasma, unless special measures are taken (1). The stoichiometry of factor VIII and vWF in plasma is approximately 1:50 and factor VIII and monomeric vWF have similar molecular weights of approximately 240 kDa. Therefore, vWF represents 98% of the molecular mass of the factor VIII-vWF complex (10) and almost all the antibodies raised against the complex react to vWF. In the 1980’s, factor VIII and vWF have each been purified to homogeneity and the genes for these proteins have been cloned. This set the stage for studies with purified proteins which have elucidated structure-function relationships for both proteins. Also, the interaction between both proteins could be studied using proteolytic fragments, small peptides, and monoclonal antibodies. In the last few years, the construction of recombinant mutants and fragments of both factor VIII (11-13) and vWF (14-16) has proven to be a powerful tool in the elucidation of the structure and function of both proteins.Binding of factor VIII to vWF is essential for the survival of factor VIII in vivo (17, 18). The underlying mechanism is probably that factor VIII bound to vWF is protected from phospholipid dependent proteolysis by activated protein C and factor Xa (19, 20). The binding site for factor VIII has been located at the amino terminus of vWF (21, 22). A tryptic fragment containing this binding site was not sufficient to protect factor VIII against activated protein C-mediated degradation according to some groups (23, 24). In contrast, a recent study using comparable vWF fragments showed protection of factor VIII equivalent to mature vWF (16).In 1989, a new variant of von Willebrand disease was discerned (type Normandy or 2N), distinct from the more than 20 subtypes known, characterized by a mutant vWF that is structurally and functionally normal, except that it does not bind to and stabilize factor VIII (25, 26). Since then, several mutations in the factor VIII binding site on vWF have been found (27). A number of reports have shown that factor VIII binds vWF via a high affinity binding site on its light chain (28-30). Two recent studies suggest that this binding site consists of two separate binding sites (31, 32).This review summarizes current knowledge on the interaction between factor VIII and vWF. Emphasis will be laid on the biological importance of, and the domains involved in binding, and on the stoichiometry and kinetics of complex formation.

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“…1. Peak 1, eluted at 0.36 M NaC1, corresponds to free FVIII light chain of molecular mass 80000 Da [27]. The clotting activity was further eluted over 0.49-0.57 M NaCl in two different peaks (peaks 2 and 3).…”

Section: Copper Atomic Absorption Of Purified Plasma-derived Fviiimentioning

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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First determination of the secondary structure of purified factor VIII light chain

Cited by 9 publications

References 32 publications

Determination of the disulfide bridges in factor Va light chain

Determination of the disulfide bridges in factor Va light chain

Factor VIII and von Willebrand Factor

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

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