We will employ a biophysical approach combined with mutagenesis to study the structural bases and biophysical mechanisms that regulate the molecular interaction between von Willebrand factor (VWF) and VWF-cleaving metalloprotease ADAMTS-13 (A Disintegrin And Metalloprotease with a ThromboSpondin type 1 motifs). We will focus on the processes of VWF domain unfolding, conformational changes, binding to and proteolytic cleavage by ADAMTS-13 at the level of single molecules or single pairs of molecules. The objective is to elucidate how mechanics regulate these molecular processes in order to understand how their functions are regulated by the blood flow in the circulation. These studies are organized into two specific aims.
Aim 1 is to elucidate the regulatory mechanisms and structural bases of ADAMTS-13/VWF binding. Our hypotheses include: The CUB domains and the spacer domain of ADAMTS-13 bind to separate sites on the A domains of VWF. Initial binding involves either the CUB domains or the spacer domain and is regulated by separation distance. Subsequent binding of the second site is induced by the binding of the first site and regulated by applied force. We will determine how distance regulates formation, and how force regulates dissociation, of ADAMTS-13/VWF bonds, measure the effects of structural variations on distance-dependent formation and force-dependent dissociation of ADAMTS-13/VWF bonds, and develop a multi-site and multi-state binding model for the ADAMTS-13/VWF interaction.
Aim 2 is to investigate the structural stability of VWF, its structural determinants, its regulation by ADAMTS-13 binding, and its regulation of ADAMTS-13 proteolysis. Our hypotheses include: Force regulates VWF-cleavage by ADAMTS-13 via disrupting noncovalent interactions between and/or within the A domains. This destabilizes the protein structure and induces catastrophic structural changes, which exposes the cryptic cleavage site in the A2 domain, allowing proteolysis by ADAMTS-13. The forced-induced structural changes in the A domains may also be regulated by binding of ADAMTS-13 to the A domains, which may change the A domain conformations. We will determine the kinetics of force-induced structural changes in A domains, its regulation by ADAMTS-13 binding, and its regulation of ADAMTS-13 proteolysis. We will also measure the effects of structural variations on forced-destabilization of A domains and on the force-regulated VWF proteolytic cleavage by ADAMTS-13. This project will clarify how mechanics regulates the chemistry of binding and cleavage of VWF by ADAMTS-13 to meet the stringent requirements for them to carry out their biological functions in the stressful environment of the circulation of rapidly flowing blood. Decoding how molecular structures determine these regulatory mechanisms will provide crucial insights into vascular physiology and pathology. Information thus obtained will also help develop new therapeutic approaches to inhibiting pathological platelet adhesion during thrombosis and/or intervention to thrombotic thrombocytopenic purpura (TTP) and/or the bleeding disorder von Willebrand diseases (VWD).

Public Health Relevance

We propose to study binding and cleaving of von Willebrand factor by metalloprotease ADAMTS-13, which regulates platelet adhesion to von Villebrand factor by regulating its size. This regulation is crucial because insufficient adhesion cannot stop bleeding to maintain hemostasis but excessive adhesion results in thrombosis. The data may offer new therapeutic approaches to inhibiting pathological platelet adhesion during thrombosis and/or intervention to thrombotic thrombocytopenic purpura and/or to the bleeding disorder von Willebrand diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL093723-04
Application #
8274715
Study Section
Special Emphasis Panel (ZRG1-HEME-C (02))
Program Officer
Kindzelski, Andrei L
Project Start
2009-06-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2014-05-31
Support Year
4
Fiscal Year
2012
Total Cost
$367,660
Indirect Cost
$88,129
Name
Georgia Institute of Technology
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
097394084
City
Atlanta
State
GA
Country
United States
Zip Code
30332
Zhu, Cheng (2014) Mechanochemitry: a molecular biomechanics view of mechanosensing. Ann Biomed Eng 42:388-404
Fiore, Vincent F; Ju, Lining; Chen, Yunfeng et al. (2014) Dynamic catch of a Thy-1-?5?1+syndecan-4 trimolecular complex. Nat Commun 5:4886
Lee, Cho-yin; Lou, Jizhong; Wen, Kuo-kuang et al. (2013) Actin depolymerization under force is governed by lysine 113:glutamic acid 195-mediated catch-slip bonds. Proc Natl Acad Sci U S A 110:5022-7
Zarnitsyna, Veronika I; Zhu, Cheng (2011) Adhesion frequency assay for in situ kinetics analysis of cross-junctional molecular interactions at the cell-cell interface. J Vis Exp :e3519
Wu, Tao; Lin, Jiangguo; Cruz, Miguel A et al. (2010) Force-induced cleavage of single VWFA1A2A3 tridomains by ADAMTS-13. Blood 115:370-8
McEver, Rodger P; Zhu, Cheng (2010) Rolling cell adhesion. Annu Rev Cell Dev Biol 26:363-96
Chen, Wei; Zhu, Cheng (2010) A model for single-substrate trimolecular enzymatic kinetics. Biophys J 98:1957-65