Von Willebrand factor (VWF), a multimeric polymer glycoprotein secreted from vascular endothelial cells and activated platelets, functions to sequester and adhere platelets to the subendothelium and initiate coagulation. Since the initial observation that the function of VWF is conformationally regulated by the rheological shear stress of blood flow, the prevailing idea has been that exposure to elevated vascular shear stress unfolds VWF to an activated conformation that increases its binding affinity of the A1 domain for the platelet GPIba surface receptor. When we identified the presence of a thermodynamically distinct unfolding intermediate in the """"""""platelet GPIba binding"""""""" A1 domain of VWF, it set forth numerous studies of the functional role that this conformation plays in health and disease. VWF has merited extensive study because of its association with the most common inherited bleeding disorder in man, Von Willebrand Disease (VWD). Two subtypes of type 2 VWD are characterized by either enhanced (type 2B) or deficient (type 2M) platelet-VWF interactions due to point mutations located specifically in the A1 domain of VWF. Our studies have established that these phenotypically opposite mutations differentially affect the thermodynamic stability of the A1 domain, the binding affinity of the A1 domain for platelet GPIba, and the force-dependent catch-slip bonding between A1 and GPIba, a property that regulates platelet-rolling velocities on VWF as shear flow is increased. The degree to which these mutations affect these properties of the A1 domain have revealed that 1) the binding affinity is coupled to this native to intermediate (N?I) conformational equilibrium and 2) the force/shear stress dependent properties of the A1-GPIba interaction are a direct consequence of the thermodynamic linkage between the native low affinity and intermediate high affinity conformations. These studies have resulted in the first quantitative working model for the mechanism of VWF function that forms the basis for the central hypothesis of this application that the intermediate conformation of the A1 domain forms an integral part of the structure that comprises the active state of VWF. Our objective is to identify how A1 stability and domain association within the A1A2A3 tri-domain fragment of VWF are thermodynamically coupled and how this regulation is linked to the severity of bleeding in clinical disease. To accomplish this objective we will test the Conformational Model through the investigation of a comprehensive clinical database of type 2B and 2M mutations that cause Von Willebrand disease and establish the effects of these clinical mutations on the quaternary domain interactions within the A1A2A3 tri-domain. The proposed work will be of significant value to the clinical diagnosis of VWD as it has the potential to classify mutations according to their conformational effects on VWF structure and function. A thorough understanding of the breadth of conformational defects caused by inherited mutations will be essential for the development of quantitative structure-activity relationships in VWF as well as the development of new treatments for cardiovascular disease.

Public Health Relevance

Von Willebrand factor (VWF) is a main responder to vascular injury, and functions to sequester and adhere platelets to the subendothelium and initiate coagulation. The objective of the proposed investigation is to test a novel model for how inherited abnormalities of VWF result in clinical disorders of hemostasis. The principles learned from this research project will provide a unique perspective into the mechanisms of disease that can potentially be translated to modern medical approaches for diagnosis and treatment of cardiovascular disorders.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
7R01HL109109-03
Application #
8497717
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Link, Rebecca P
Project Start
2011-07-15
Project End
2016-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
3
Fiscal Year
2013
Total Cost
$335,223
Indirect Cost
$123,706
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
Tischer, Alexander; Machha, Venkata R; Rösgen, Jörg et al. (2018) ""Cooperative collapse"" of the denatured state revealed through Clausius-Clapeyron analysis of protein denaturation phase diagrams. Biopolymers 109:e23106
Machha, Venkata R; Tischer, Alexander; Moon-Tasson, Laurie et al. (2017) The Von Willebrand Factor A1-Collagen III Interaction Is Independent of Conformation and Type 2 Von Willebrand Disease Phenotype. J Mol Biol 429:32-47
Tischer, Alexander; Machha, Venkata R; Frontroth, Juan P et al. (2017) Enhanced Local Disorder in a Clinically Elusive von Willebrand Factor Provokes High-Affinity Platelet Clumping. J Mol Biol 429:2161-2177
Tischer, Alexander; Campbell, James C; Machha, Venkata R et al. (2016) Mutational Constraints on Local Unfolding Inhibit the Rheological Adaptation of von Willebrand Factor. J Biol Chem 291:3848-59
Campbell, James C; Tischer, Alexander; Machha, Venkata et al. (2016) Data on the purification and crystallization of the loss-of-function von Willebrand disease variant (p.Gly1324Ser) of the von Willebrand factor A1 domain. Data Brief 7:1700-1706
Lea, Wendy A; O'Neil, Pierce T; Machen, Alexandra J et al. (2016) Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins. Biochemistry 55:4885-908
Zimmermann, Michael T; Tischer, Alexander; Whitten, Steven T et al. (2015) Structural origins of misfolding propensity in the platelet adhesive von Willebrand factor A1 domain. Biophys J 109:398-406
McNaughton, Candace D; Cawthon, Courtney; Kripalani, Sunil et al. (2015) Health literacy and mortality: a cohort study of patients hospitalized for acute heart failure. J Am Heart Assoc 4:
Perez, Romel B; Tischer, Alexander; Auton, Matthew et al. (2014) Alanine and proline content modulate global sensitivity to discrete perturbations in disordered proteins. Proteins 82:3373-84
Tischer, Alexander; Madde, Pranathi; Blancas-Mejia, Luis M et al. (2014) A molten globule intermediate of the von Willebrand factor A1 domain firmly tethers platelets under shear flow. Proteins 82:867-78

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