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Dimerization and multimerization defects of von Willebrand factor due to mutated cysteine residues
P Tjernberg1, H L Vos, G Castaman
1Department of Hematology, Hemostasis and Thrombosis Research Center, Leiden University Medical Center, Leiden, the Netherlands. H.C.J.Eikenboom@LUMC.nl
Insights
Mutations affecting cysteine residues in the von Willebrand factor (VWF) disrupt its structure, leading to reduced secretion and impaired multimerization, causing von Willebrand disease. These findings explain quantitative VWF deficiency in patients.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Von Willebrand disease (VWD) is a bleeding disorder caused by defects in the von Willebrand factor (VWF).
- Missense mutations leading to loss of cysteine residues in VWF's D3 and carboxy-terminal domains have been identified in patients with type 1 and type 3 VWD.
- Understanding how these structural changes impact VWF function is crucial for explaining VWF deficiency.
Purpose of the Study:
- To investigate how missense mutations affecting cysteine residues in the D3-domain (multimerization) and carboxy-terminus (dimerization) of VWF lead to quantitative VWF deficiency.
- To elucidate the interference of these mutations with VWF dimerization and multimerization processes.
Main Methods:
- Human recombinant VWF with mutations in the multimerization (C1130F, C1149R) and dimerization (C2671Y, C2739Y, C2754W) areas were created.
- Transient transfection assays in 293T cells were used to analyze the secretion, intracellular retention, and multimeric patterns of the mutant VWF.
Main Results:
- All VWF mutants exhibited reduced secretion and intracellular retention.
- Amino-terminal mutants (C1130F, C1149R) showed impaired multimerization, lacking high molecular weight (HMW) multimers, mimicking heterozygous type 1 VWD.
- Carboxy-terminal mutants (C2739Y, C2754W) displayed severely reduced secretion, consistent with type 3 VWD, and showed defects in dimerization, indicated by absent HMW multimers and an excess of monomers.
- The C2671Y mutant showed milder defects, with reduced secretion and intermediate retention but a normal multimerization pattern.
Conclusions:
- Cysteine mutations in VWF impair secretion and dimerization/multimerization, explaining quantitative VWF deficiency.
- While in vitro studies show reduced VWF levels, the more severe deficiency in patients suggests additional in vivo clearance mechanisms.
- The distinct phenotypes of different mutants highlight the specific roles of cysteine residues in VWF structure and function.
Abstract:
In patients classified with type 1 and type 3 von Willebrand disease missense mutations resulting in the loss of cysteine residues in the D3-domain (multimerization area) and in the carboxy-terminus (dimerization area) of the von Willebrand factor (VWF) have been identified. We have investigated how these structural changes result in a quantitative VWF deficiency and how they interfere with the dimerization and multimerization processes. The effect of mutations in the multimerization area (C1130F, C1149R) and in the dimerization area (C2671Y, C2739Y, C2754W) of human recombinant VWF were investigated in transient transfection assays in 293T cells. All mutations resulted in reduced secretion of VWF in the medium and in intracellular retention. The amino-terminal mutants C1130F and C1149R showed impaired multimerization by lacking high molecular weight (HMW) multimers, in cotransfection experiments with wild-type (wt) VWF, the multimeric pattern was consistent with the pattern in the heterozygous type 1 patients. The carboxy-terminal mutants C2739Y and C2754W showed strongly reduced to nearly absent secretion of VWF, consistent with type 3 VWD. The multimeric pattern of C2739Y and C2754W is characterized by the absence of HMW multimers, an excess of monomers and intervening odd-numbered multimeric bands, indicating a dimerization defect. The carboxy-terminal mutant C2671Y is different, with mildly reduced secretion, intermediate intracellular retention and a normal multimerization pattern. We conclude that, in accordance with a phenotype of quantitative VWF deficiency, all cysteine mutants show impaired secretion, although the decrease of VWF in vitro appears lower than in the patients, suggesting additional, possibly heightened clearance, mechanisms in vivo.
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