Von Willebrand factor (VWF) is a multimeric blood glycoprotein that is required for hemostasis. At sites of vascular injury, VWF binds connective tissue and then mediates platelet adhesion through binding to platelet membrane glycoprotein lb. The adhesive function of VWF depends on the largest multimers, and inability to assemble multimers causes bleeding associated with von Willebrand disease. Conversely, VWF multimers are cleaved at sites of thrombosis by ADAMTS13, a metalloprotease, and ADAMTS13 deficiency causes thrombotic thrombocytopenic purpura. Therefore, the control of VWF multimer size has substantial medical importance, and understanding the function of VWF depends on understanding the assembly and structure of VWF multimers. In the endoplasmic reticulum, proVWF subunits dimerize through their C-terminal CK domains, and Specific Aim 1 is to characterize the intersubunit disulfide structure of the dimeric VWF CK domain. ProVWF dimers are transported to the Golgi, where the propeptide promotes the formation of additional intersubunit disulfide bonds between N-terminal VWF D'D3 domains, thereby forming multimers, and Specific Aim 2 is to characterize the intrachain and interchain disulfide bonds of the multimerization site in the VWF D3 domain. The disulfide bonds of recombinant VWF CK dimers and recombinant VWF D'D3 dimers will be identified by partial reduction and alkylation, chemical and enzymatic fragmentation, protein sequencing, and mass spectrometry.
Specific Aim 3 is to characterize the propeptide-dependent mechanism of VWF multimerization. The VWF propeptide appears to function as an endogenous disulfide isomerase that promotes intersubunit disulfide bond formation not in the ER but in the Golgi, where disulfides otherwise do not form, and this remarkable mechanism is conserved in a family of multimeric epithelial mucins. The mechanism of VWF multimer assembly will be investigated with novel assays for disulfide bond formation between recombinant VWF D'D3 domains. Disulfide-linked intracellular intermediates between VWF propeptide and D'D3 domains have been identified and will be characterized. The function of Cys residues that form interdomain and intersubunit disulfides will be determined by mutagenesis. The structural requirements for VWF multimerization will be characterized with chimeric constructs of human VWF and homologous mucins, and of interspecies VWF chimeric proteins.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL072917-04
Application #
7017063
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Link, Rebecca P
Project Start
2003-04-01
Project End
2008-02-29
Budget Start
2006-03-01
Budget End
2007-02-28
Support Year
4
Fiscal Year
2006
Total Cost
$215,319
Indirect Cost
Name
Washington University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Sadler, J Evan (2017) Pathophysiology of thrombotic thrombocytopenic purpura. Blood 130:1181-1188
Obser, T; Ledford-Kraemer, M; Oyen, F et al. (2016) Identification and characterization of the elusive mutation causing the historical von Willebrand Disease type IIC Miami. J Thromb Haemost 14:1725-35
Parker, D'Andra N; Tasneem, Subia; Farndale, Richard W et al. (2016) The functions of the A1A2A3 domains in von Willebrand factor include multimerin 1 binding. Thromb Haemost 116:87-95
Deforche, L; Roose, E; Vandenbulcke, A et al. (2015) Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13. J Thromb Haemost 13:2063-75
Muia, Joshua; Zhu, Jian; Gupta, Garima et al. (2014) Allosteric activation of ADAMTS13 by von Willebrand factor. Proc Natl Acad Sci U S A 111:18584-9
Muia, J; Gao, W; Haberichter, S L et al. (2013) An optimized fluorogenic ADAMTS13 assay with increased sensitivity for the investigation of patients with thrombotic thrombocytopenic purpura. J Thromb Haemost 11:1511-8
Tarr, Phillip I; Sadler, J Evan; Chandler, Wayne L et al. (2012) Should all adult patients with diarrhoea-associated HUS receive plasma exchange? Lancet 379:516; author reply 516-7
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
Valentijn, Karine M; Sadler, J Evan; Valentijn, Jack A et al. (2011) Functional architecture of Weibel-Palade bodies. Blood 117:5033-43

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