Force Sensitivity of the Von Willebrand Factor A2 Domain

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Force Sensitivity of the Von Willebrand Factor A2 Domain

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dc.contributor.advisor Springer, Timothy A.
dc.contributor.author Xu, Amy Jia
dc.date.accessioned 2012-11-13T17:10:18Z
dash.embargo.terms 2014-10-05 en_US
dash.embargo.terms 2014-10-05
dc.date.issued 2012-11-13
dc.date.submitted 2012
dc.identifier.other http://dissertations.umi.com/gsas.harvard:10538 en
dc.identifier.uri http://nrs.harvard.edu/urn-3:HUL.InstRepos:9888978
dc.description.abstract Von Willebrand factor (VWF) is a multimeric glycoprotein that critically supports platelet aggregation in hemostasis. Disordered VWF function causes both thrombotic and bleeding disorders, and genetic defects in VWF are responsible for von Willebrand’s disease (VWD), the most common inherited bleeding disorder in humans. Very large VWF multimers exhibit the greatest thrombogenic activity, which is attenuated by ADAMTS13 cleavage in the A2 domain. A2 cleavage is regulated by mechanical force, and pathologically high shear forces are known to enhance proteolysis and cause bleeding in patients. Enhanced cleavage is also described in patients with VWD 2A mutations. In contrast, VWF A2 is stabilized against cleavage by a calcium binding site within A2. Single molecule studies have demonstrated that mechanical unfolding is required for A2 cleavage to expose the scissile bond. In this dissertation, we aim to better understand the mechanosensitivity of A2 cleavage by characterizing the force sensitivity of A2 unfolding and refolding. We first characterized the interaction between VWF A2 and calcium using bulk isothermal calorimetry and thermal denaturation assays. In parallel, we used single molecule optical tweezers to characterize A2 unfolding and refolding. Calcium was found to bind A2 with high affinity, stabilize A2 against thermal denaturation, and enhance domain refolding. In contrast, we found that VWD 2A mutations destabilize the A2 domain against thermal denaturation. R1597W, the most common VWD 2A mutation, lies within the calcium binding loop and exhibited diminished calcium stabilization against thermal denaturation. Using optical tweezers, we found that R1597W also diminished A2 refolding. R1597W refolding in the presence of calcium was similar to that of wild-type A2 in the absence of calcium, suggesting that loss of calcium stabilization contributes to the disease mechanism of R1597W. Other VWD 2A mutations lying outside the calcium binding loop also destabilized A2, but retained calcium mediated stabilization. These studies provide a better understanding of VWD 2A pathophysiology and offer structural insights into A2 unfolding and refolding pathways. By exploring the role of mechanical force in regulating VWF cleavage, this work moves towards a better understanding of how hydrodynamic forces within the vasculature regulate VWF function in hemostasis and thrombosis. en_US
dc.language.iso en_US en_US
dash.license LAA
dc.subject A2 en_US
dc.subject optical tweezers en_US
dc.subject protein folding en_US
dc.subject single molecule en_US
dc.subject biophysics en_US
dc.subject von Willebrand factor en_US
dc.subject von Willebrand's disease en_US
dc.title Force Sensitivity of the Von Willebrand Factor A2 Domain en_US
dc.type Thesis or Dissertation en_US
dash.depositing.author Xu, Amy Jia
dc.date.available 2014-10-06T07:30:37Z
thesis.degree.date 2012 en_US
thesis.degree.discipline Biophysics en_US
thesis.degree.grantor Harvard University en_US
thesis.degree.level doctoral en_US
thesis.degree.name Ph.D. en_US
dc.contributor.committeeMember Blacklow, Stephen en_US
dc.contributor.committeeMember Golan, David en_US
dc.contributor.committeeMember Hogle, James en_US

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