Structure-Resolved Mechanistic Phenotyping of Von Willebrand Disease

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PROJECT SUMMARY: The primary hemostatic Von Willebrand Factor (vWF) sequesters platelets to arrest bleed- ing. Subject to the rheological shear of blood flow, multimeric fibers of vWF unravel exposing A1 domain hooks which capture platelets through the binding of platelet GPIb↵ receptors. Mutations within the A1 domain occur in all clinical classifications of von Willebrand disease (vWD), the most common inherited human bleeding dis- order, causing quantitative deficiencies of vWF in plasma and functional flaws in platelet adhesion. The central paradigm of vWF function in disease is that mutations alter vWF's response to the rheological effects of blood flow, but the the structural basis for how these mutations alter the mechanics of platelet adhesion to vWF is not understood. vWD mutations in A1 induce conformational changes that unfold local regions of the A1 domain structure in both type 2B (gain-of-function) and type 2M (loss-of-function) vWD phenotypes which are proposed to alter two previously unidentified A1-GPIB↵ binding sites The overall objective of this application is to decipher the role of A1 conformational disorder in GPIb↵ affinity recognition. The central hypothesis is that the mechanism of dysfunction in vWD is not shear dependent, but rather, determined by the intrinsic conformational dynamics of these putative binding sites. To accomplish our objective, we will 1) identify the structural determinants for gain and loss of vWF-platelet function, 2) decipher the binding mechanism, and 3) develop novel technologies for the detection of pathological conformations of vWF in plasma. Our approach is innovative because it utilizes hydrogen-deuterium exchange and cross-linking mass spectrometry to attain high-resolution map of how struc- tural disorder predetermines GPIb↵ affinity and it employs new RNA aptamer molecular probes that specifically bind and inhibit disordered conformations of the A1 domain within vWD patient plasma vWF. This research project addresses explicit needs, stated by the NHLBI, to enhance knowledge of vWD mechanisms, improve vWD diag- nostics, and it establishes novel methods for the phenotyping of vWD. The proposed studies are expected to enhance a basic scientific understanding of how vWD affects the linkage between folding and function of vWF and to improve interpretation of current diagnostics leading to better informed treatment recommendations and enhanced patient care.