The primary hemostatic Von Willebrand Factor (vWF) sequesters platelets to arrest bleed- ing. Subject to the rheological shear of blood ?ow, multimeric ?bers 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 classi?cations of von Willebrand disease (vWD), the most common inherited human bleeding dis- order, causing quantitative de?ciencies of vWF in plasma and functional ?aws in platelet adhesion. The central paradigm of vWF function in disease is that mutations alter vWF's response to the rheological effects of blood ?ow, 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 unidenti?ed A1-GPIB? binding sites The overall objective of this application is to decipher the role of A1 conformational disorder in GPIb? af?nity 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? af?nity and it employs new RNA aptamer molecular probes that speci?cally 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 scienti?c 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.
The proposed research is relevant to public health because it will identify phenotype-speci?c mechanisms by which inherited Von Willebrand Disease mutations alter the structure and function of the platelet adhesive A1 domain of Von Willebrand Factor. The project is congruent with the strategic vision of the NHLBI to develop and optimize diagnostics and therapeutics to prevent, treat and cure Heart, Lung, Blood and Sleep diseases because it provides novel strategies to detect and inhibit Von Willebrand Factor disease states and meets a declared need for accurate functional phenotyping.
