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The Structural and Functional Study of Atherothrombosis Related Protein Von Willebrand Factor A2 Domain

Author: ChenHua
Tutor: LuoXinPing
School: Fudan University
Course: Cardiology
Keywords: Atherothrombosis vWF A2 calcium molecular simulation ADAMTS-13
CLC: R543.5
Type: Master's thesis
Year: 2011
Downloads: 72
Quote: 0
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Abstract


Atherosclerotic cardiovascular disease is a common condition which is defined as myocardial ischemia caused by the imbalance between myocardial oxygen supply and demand. Of note, the incidence of this disease is increasing in our country. It is found that the main pathophysiology of myocardial ischemia is atherothrombosis. Atherothrombosis is the major cause of death and premature disability in coronary heart disease. von Willebrand Factor(vWF) which serves as a key protein in atherothrombosis, plays an important role in primary hemostasis through mediating platelet adhesion. vWF is cleaved by the specific vWF cleaving protease ADAMTS-13. The A2 domain of vWF becomes the focus of many studies because of the existence of the specific cleavage site. In the present study, we determined the crystal structure of an engineered A2 domain, which exhibits significant difference in theα3-β4 loop compared with the previously reported structure of the wild-type A2. In addition, a metal ion was detected at a site formed mainly by the C-terminal region of theα3-β4 loop, and later identified to be Ca2+ with various biophysical and biochemical studies. Force probe molecular dynamics simulations of a modeled structure of the wild-type A2 that features the discovered Ca2+-binding site revealed that an increase of the force is needed to unfold strandβ4 when Ca2+ is bound. Consistently, cleavage assays demonstrated that the Ca2+ binding stabilizes the A2 domain and impedes its unfolding, and consequently protects it from cleavage by ADAMTS-13.Finally, the mechanism proposed by our study will provide new insights into the regulation of vWF cleavage by ADAMTS-13 in atherothrombosis.The study of expression, purification, and crystallization of an engineered A2 domain and the structure-based cleavage assays in vitroObjective:In order to understand the underlying cleavage mechanism of vWF A2 domain, the expression, purification, and crystallization of an engineered A2 domain were performed. The difference between the engineered A2 domain and wild type A2 domain was also compared in this study, and finally the function of calcium in the process of cleavage was explored.Methods:Firstly, we constructed the A2 mutant vector by means of conventional molecular cloning technique. Then, we transformed this vector into the prokaryotic expression system and expressed the target protein in E. coli strain. Thirdly, techniques such as affinity chromatography, gel fitration, dialysis and so on were carried out before obtaining the purified protein. Following the purification, the hanging drop vapor diffusion method was used to screening and optimization for the crystal. Lastly, force probe molecular dynamics simulations and in vitro digestion experiments were explored.Results:We expressed and purified vWF A2 mutant domain successfully in vitro. The crystals of A2 mutants were grown in drops of crystallization solution at 4℃. The A2 mutant structure was solved using the molecular replacement method, and a metal ion was detected at a site formed mainly by the C-terminal region of theα3-β4 loop, and later identified to be Ca2+ with various biophysical and biochemical studies. Subsequently, force probe molecular dynamics simulations and cleavage assays demonstrated that the Ca2+ binding stabilizes the A2 domain and impedes its unfolding, and consequently protects it from cleavage by ADAMTS-13.Conclusion:For the first time, we have revealed a novel Ca2+-binding site at the A2 domain of VWF and demonstrated interplay of Ca2+ and force in the regulation of VWF and in atherothrombosis

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CLC: > Medicine, health > Internal Medicine > Heart, blood vessels ( circulatory ) disease > Vascular disease > Artery disease
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