The goal of this proposal is to understand the structural basis of ADAMTS13 specificity and to translate this knowledge into better treatment for thrombotic thrombocytopenic purpura, a medical emergency that strikes mainly women between 30 and 50 years of age. ADAMTS13 is a metalloprotease found in the blood that cleaves von Willebrand factor (VWF), a multimeric protein that mediates the adhesion of platelets at sites of vascular injury. Thrombotic thrombocytopenic purpura usually is caused by autoantibodies against ADAMTS13 that prevent it from cleaving VWF. The resulting ADAMTS13 deficiency leads to unchecked platelet adhesion and widespread microvascular thrombosis, demonstrating that normal hemostasis depends on the inhibition of VWF function by ADAMTS13. This wonderfully precise feedback inhibitory mechanism involves the recognition and cleavage of VWF specifically when it is subjected to tensile stress by the force of flowing blood, as occurs in a growing thrombus. Several experimental strategies are proposed to investigate ADAMTS13 and make use of the results to advance patient care.
Specific Aim 1 is to characterize the regulation of ADAMTS13 by fluid shear stress, cofactors, and VWF structure. Platelet membrane GPIb or heparin can cooperate with fluid shear stress to promote VWF cleavage. The corresponding biochemical mechanisms will be characterized under conditions of fluid shear stress in platelet suspensions and on endothelial cell surfaces. Participating structural domains of VWF and ADAMTS13 will be identified by mutagenesis and analysis of recombinant proteins.
Specific Aim 2 is to characterize the structural basis for the recognition of VWF by ADAMTS13. When subjected to tensile stress, VWF displays a unique combination of features that interact with multiple exosites on ADAMTS13. These extensive contacts enforce the strict specificity of ADAMTS13 for VWF in vivo. A comprehensive biochemical approach will define the location, structure and energetics of binding between complementary sites on VWF and ADAMTS13.
Specific Aim 3 is to develop improved clinical assays of ADAMTS13 activity. Prompt diagnosis of thrombotic thrombocytopenic purpura is essential because almost all patients die if untreated and plasma exchange therapy increases survival to >80%. Understanding how ADAMTS 13 recognizes and cleaves VWF will enable the development of rapid clinical ADAMTS 13 assays, which will facilitate the diagnosis and treatment of patients with thrombotic thrombocytopenic purpura caused by ADAMTS13 deficiency.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL089746-04
Application #
7881537
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Kindzelski, Andrei L
Project Start
2007-08-15
Project End
2012-06-30
Budget Start
2010-07-01
Budget End
2011-06-30
Support Year
4
Fiscal Year
2010
Total Cost
$458,155
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
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
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
Huang, Jing; Motto, David G; Bundle, David R et al. (2010) Shiga toxin B subunits induce VWF secretion by human endothelial cells and thrombotic microangiopathy in ADAMTS13-deficient mice. Blood 116:3653-9
Gézsi, A; Budde, U; Deák, I et al. (2010) Accelerated clearance alone explains ultra-large multimers in von Willebrand disease Vicenza. J Thromb Haemost 8:1273-80
Huang, Ren-Huai; Fremont, Daved H; Diener, John L et al. (2009) A structural explanation for the antithrombotic activity of ARC1172, a DNA aptamer that binds von Willebrand factor domain A1. Structure 17:1476-84
Huang, Jing; Roth, Robyn; Heuser, John E et al. (2009) Integrin alpha(v)beta(3) on human endothelial cells binds von Willebrand factor strings under fluid shear stress. Blood 113:1589-97

Showing the most recent 10 out of 15 publications