Corrigendum to "Characterization of Zebrafish von Willebrand Factor Reveals Conservation of Domain Structure, Multimerization, and Intracellular Storage".

[This corrects the article DOI: 10.1155/2012/214209.].

Characterization of zebrafish von Willebrand factor reveals conservation of domain structure, multimerization, and intracellular storage.

von Willebrand disease (VWD) is the most common inherited human bleeding disorder and is caused by quantitative or qualitative defects in von Willebrand factor (VWF). VWF is a secreted glycoprotein that circulates as large multimers. While reduced VWF is associated with bleeding, elevations in overall level or multimer size are implicated in thrombosis. The zebrafish is a powerful genetic model in which the hemostatic system is well conserved with mammals. The ability of this organism to generate thousands of offspring and its optical transparency make it unique and complementary to mammalian models of hemostasis. Previously, partial clones of zebrafish vwf have been identified, and some functional conservation has been demonstrated. In this paper we clone the complete zebrafish vwf cDNA and show that there is conservation of domain structure. Recombinant zebrafish Vwf forms large multimers and pseudo-Weibel-Palade bodies (WPBs) in cell culture. Larval expression is in the pharyngeal arches, yolk sac, and intestinal epithelium. These results provide a foundation for continued study of zebrafish Vwf that may further our understanding of the mechanisms of VWD.

The role of the D1 domain of the von Willebrand factor propeptide in multimerization of VWF.

While studying patient plasma containing an unusual pattern of von Willebrand factor (VWF) multimers, we discovered a previously unreported phenomenon: heavy predominance of dimeric VWF. Genomic analysis revealed a new congenital mutation (Tyr87Ser) that altered the final stages of VWF biosynthesis. This mutation in the propeptide (VWFpp) resulted in synthesis of dimeric VWF with an almost complete loss of N-terminal multimerization. The multimer pattern in patient plasma appears to result from separate alleles' synthesizing wild-type or mutant (dimeric) VWF, with homodimers composing the predominant protomeric species. We have expressed VWF protein containing the Tyr87Ser mutation and analyzed the intracellular processing and resulting VWF biological functions. The expressed dimeric VWF displayed a loss of several specific functions: collagen binding, factor VIII binding, and ristocetin-induced platelet binding. However, granular storage of dimeric VWF was normal, demonstrating that the lack of multimerization does not preclude granular storage. Although the tertiary structure of the VWFpp remains unknown, the mutant amino acid is located in a region that is highly conserved across several species and may play a major role in the multimerization of VWF. Our data suggest that one function of the highly cysteine-rich VWFpp is to align the adjacent subunits of VWF into the correct configuration, serving as an intramolecular chaperone. The integrity of the VWFpp is essential to maintain the proper spacing and alignment of the multiple cysteines in the VWFpp and N-terminus of the mature VWF.

The von Willebrand factor propeptide (VWFpp) traffics an unrelated protein to storage.

The von Willebrand factor (VWF) propeptide (VWFpp) is critical for the targeting of VWF multimers to storage granules. VWFpp alone efficiently navigates the storage pathway in AtT-20 and endothelial cells and chaperones mature VWF multimers to storage granules when the two proteins are expressed in cis or in trans. To further define the role of VWFpp in granular sorting, we examined its ability to sort an unrelated protein, C3alpha into the regulated secretory pathway. Chimeric constructs of VWFpp and the alpha-chain of C3 were developed. The C3alpha protein expressed alone did not sort to granules in AtT-20 cells. The trans expression of C3alpha and VWFpp resulted in granular storage of VWFpp but no corresponding storage of C3alpha. When C3alpha is expressed as a single chain molecule with VWFpp that was rendered uncleavable by furin, C3alpha is re-routed to storage and is colocalized with VWFpp. The uncleavable protein was expressed in bovine aortic endothelial cells where it sorted to Weibel-Palade bodies, colocalized with bovine VWF, and was released when agonist stimulated. We now demonstrate that VWFpp re-routes a constitutively secreted protein to the regulated storage pathway. Furthermore, our studies suggest that the VWFpp storage signal is contained within amino acids 201 to 741.