Von Willebrand factor (VWF) is a multimeric blood glycoprotein that is required for hemostasis. At sites of vascular injury, VWF binds connective tissue and mediates platelet adhesion. The adhesive function of VWF requires the largest multimers, and inability to assemble them causes bleeding associated with von Willebrand disease. Conversely, ADAMTS13, a metalloprotease, cleaves VWF multimers at sites of thrombosis, and ADAMTS13 deficiency causes thrombotic thrombocytopenic purpura (TTP). Thus, the normal balance between the assembly and catabolism of VWF multimers has substantial medical importance, and understanding the function of VWF depends on understanding the assembly and structure of VWF multimers.
Specific Aim 1 will characterize the propeptide-dependent mechanism of VWF multimer assembly. Multimer assembly depends on the N-terminal propeptide (D1D2 domains) and adjacent D'D3 region of the mature VWF subunit, which together promote disulfide bond formation under the acidic conditions of the Golgi. The structural features that enable the N-terminus of VWF to form intersubunit disulfide bonds will be identified by mutagenesis and functional studies in transfected cells. Intersubunit and intrasubunit disulfide bonds within the dimeric D'D3 segment of VWF will be determined by mass spectrometry.
Specific Aim 2 will characterize the mechanism of VWF multimer packing within and extrusion from Weibel-Palade bodies. Noncovalent pH-dependent and Ca2+- dependent interactions between D1D2 and D'D3 domains are required to reversibly condense VWF multimers into tubular arrays within Weibel-Palade bodies. These interactions will be characterized by analyzing the targeting, storage and secretion of recombinant VWF in transfected cells by fluorescence and electron microscopy. Homotypic and heterotypic contacts between VWF propeptide and D'D3 domains will be characterized structurally and biochemically. The cleavage of the VWF propeptide by furin also is regulated by pH, and the role of propeptide cleavage in VWF storage and secretion will be characterized.
Specific Aim 3 will determine the three-dimensional structure and arrangement of VWF domains within VWF tubules. Under conditions of low pH and high Ca2+, recombinant D1D2 and dimeric D'D3 fragments assemble in vitro into tubules like those in Weibel-Palade bodies. Three-dimensional reconstructions from electron microscopy images show that VWF tubules contain a repeating unit of one D'D3 dimer and two propeptides, in a right- handed helix with 4.2 units per turn. The symmetry and location of interdomain contacts suggests a mechanism for how decreasing pH along the secretory pathway coordinates the disulfide-linked assembly of VWF multimers with their tubular packaging. Three-dimensional reconstructions will be generated for tubules assembled from progressively larger VWF constructs, up to the complete VWF subunit, to build a molecular model for intact VWF multimers. Factor VIII can be stored with VWF in Weibel-Palade bodies, and these studies will also demonstrate how factor VIII affects the assembly of VWF tubules and where it binds to them. Project Narrative: VWF is blood protein that is required to stop bleeding at sites of injury, and this function depends on the assembly of very large VWF multimers from relatively small, identical subunits. The goal of the proposed studies is to understand how VWF multimers are assembled, packaged in the correct cell compartments, and secreted into the blood. This knowledge should be translatable into better treatment for disorders of bleeding and thrombosis.
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|Huang, Jing; Haberichter, Sandra L; Sadler, J Evan (2012) The B subunits of Shiga-like toxins induce regulated VWF secretion in a phospholipase D1-dependent manner. Blood 120:1143-9|
|Gao, Weiqiang; Zhu, Jian; Westfield, Lisa A et al. (2012) Rearranging exosites in noncatalytic domains can redirect the substrate specificity of ADAMTS proteases. J Biol Chem 287:26944-52|
|Liu, Fang; Huang, Jing; Sadler, J Evan (2011) Shiga toxin (Stx)1B and Stx2B induce von Willebrand factor secretion from human umbilical vein endothelial cells through different signaling pathways. Blood 118:3392-8|
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