VON WILLEBRAND FACTOR STRUCTURE AND FUNCTION UNDER FLUID FLOW
NEELAMEGHAM, SRIRAM Principal Investigator
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DESCRIPTION (provided by applicant): In models of vascular injury, the engagement of blood platelets by substrate-immobilized von Willebrand Factor (vWF) under fluid flow leads to platelet activation and subsequent thrombus formation. Experiments carried out in suspension assays that apply fluid forces on platelets and blood proteins also demonstrate that mechanical forces cause changes in platelet membrane phospholipid distribution, and they augment shear-induced platelet activation and aggregation. In our recent studies on platelet activation, we proposed a two-step mechanism of cell activation to explain these observations based on the relative roles of platelet receptor Gplb, plasma vWF and fluid forces. As opposed to traditional scaling arguments, we developed rigorous computational methods to estimate the magnitude and nature of fluid forces applied on cells and molecules under these conditions. Further, we observed that vWF undergoes self-association or aggregation when mixed under defined conditions. This suggests that protein conformation may change under fluid shear flow and this may have functional consequences. Based on these observations, the specific goals of this project are: 1) To determine the physiological fluid shear conditions under which vWF unimers may self-associate. For this aspect, light scattering, chromatography, western blot analysis and surface plasmon resonance are employed to detect vWF self-association, and to determine the kinetics/affinity of this process. Studies of shear- induced platelet activation are also conducted to establish a mechanistic link between vWF self- association and platelet activation. 2) To demonstrate that the size of the vWF molecule and length of platelet Gplb receptor are critical parameters regulating platelet activation rates under fluid shear. In order to do this, we create microspheres and nanoparticles of varying sizes bearing immobilized antibodies and recombinant forms of vWF at varying densities. The ability of these particles to bind and activate cells is quantified. 3) To characterize conformational changes in vWF under physiological fluid shear conditions. Here, the solution structure of vWF and protein conformational changes under fluid shear are measured using light, neutron and X-ray scattering spectroscopy. New mathematical theories are developed to quantitatively guide the interpretation of the above experiments. Successful completion of this work will establish that fluid shear may regulate bio-molecule structure and function. Results linking self-association and platelet activation, in the long run, may also prompt in vivo examination of this phenomenon and stimulate new drug development against self-association.