title: Differential effects of the loss of intrachain- versus interchain-disulfide bonds in the cystine-knot domain of von Willebrand factor on the clinical phenotype of von Willebrand disease.
abstract: Von Willebrand factor (VWF) contains a large number of cysteine residues, which all form disulfide bonds. Mutations of cysteines located in the cystine-knot (CK) domain of VWF have been identified in both qualitative type 2A (IID) and quantitative type 3 von Willebrand disease (VWD). Our objective was to test the hypothesis that the difference in phenotype is related to whether the mutated cysteine residue is involved in either interchain- or intrachain-disulfide-bond formation. The effects of three cysteine mutations which are all located in the CK-domain of VWF, C2773S (type 2A(IID)), C2739Y (type 3), and C2754W (type 3), were studied by transient expression in 293T cells. Cotransfection of wild-type (wt) and C2773S VWF constructs reproduced the plasma phenotype of heterozygous type 2A(IID) patients, with normal to high levels of VWF antigen (VWF:Ag), absence of high-molecular-weight multimers, and the presence of intervening bands between the normal multimers. In contrast, single transfections of C2739Y or C2754W resulted in a quantitative VWF defect with low VWF:Ag levels, and co-transfections of wt and mutant constructs resulted in a 50% reduction of VWF:Ag and only a minor effect on VWF multimerization. We demonstrated N-terminal dimerization of VWF-C2773S and both N- and C-terminal dimerization of VWF-C2754W. Our data suggest that loss of a single disulfide bond in the CK-domain of VWF leads to a recessive, quantitative VWF deficiency if an intrachain-disulfide bond is involved, and to a dominant-negative, qualitative defect of VWF if an interchain-disulfide bond is involved.
authors.affiliation: Hemostasis and Thrombosis Research Center, Department of Hematology, C2-R, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, the Netherlands.
authors.forename: Pernilla
authors.initials: P
authors.lastname: Tjernberg
authors.forename: Hans L
authors.initials: HL
authors.lastname: Vos
authors.forename: Caroline C
authors.initials: CC
authors.lastname: Spaargaren-van Riel
authors.forename: Brenda M
authors.initials: BM
authors.lastname: Luken
authors.forename: Jan
authors.initials: J
authors.lastname: Voorberg
authors.forename: Rogier M
authors.initials: RM
authors.lastname: Bertina
authors.forename: Jeroen C J
authors.initials: JC
authors.lastname: Eikenboom
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism; D000071120:ADAMTS13 Protein; D001777:Blood Coagulation; D002460:Cell Line; D003545:Cysteine; D047168:Cystine Knot Motifs; D004252:DNA Mutational Analysis; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008297:Male; D020125:Mutation, Missense; D010375:Pedigree; D010641:Phenotype; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D014162:Transfection; D014842:von Willebrand Diseases / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004220:Disulfides; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human; D003545:Cysteine