Von Willebrand factor (VWF) is a long, polymeric blood protein. It adheres to platelets at sites of injury to form plugs that stops bleeding. The lack or inadequacy of VWF function causes von Willebrand disease, the most common hereditary bleeding disorder, which affects 1% of the US population. On the other hand, if platelet plug formation occurs at the wrong time or place, it can lead to thrombosis, which can cut off circulation, causing a stroke or heart attack. To precisely turn on VWF-platelet adhesion only when injuries are detected, nature has programmed VWF to sense changes in blood flow and to respond by activating its adhesion to GPIb?, the receptor protein on platelet surface. I propose to combine novel microfluidic systems, single-molecule methods and special fluorescence techniques to improve our understanding of the biochemical and mechanical factors regulating VWF function by flow. Using advanced techniques that I have developed, we have stretched single VWF multimers by flow and directly visualized their force-activated conformational transitions and activation for the first time. These measurements revealed that VWF multimers first elongate and then activate their binding sites for GPIb? with force under flow. Building on this, I will answer three questions related to the mechanical regulation of VWF. First, I will measure how flow-induced force in VWF regulates its degradation by ADAMTS13 protease. This degradation process limits the size of VWF to lower the clotting potential of VWF. I will test a ?molecular zipper? binding model, in which one end of the ADAMT13 scarcely binds to the D4-CK domains of globular VWF, with further binding propagating to the proteolysis site once the A2 domain is unfolded. I will also determine the amount of ADAMTS13 cleavage in the presence of GPIb? or platelets. Second, I will test the hypothesis that reactive oxygen species (ROS) and cell-free hemoglobin directly integrate with the flow sensing capability of VWF to regulate its function. I will measure the enhancement of flow-induced VWF adhesion in the presence of these chemical cues. Third, I will reveal how elongational flow found in ruptured or narrowed blood vessels activates the adhesion between freely circulating VWF and GPIb? to augment thrombosis. I will accomplish this by combining a high flow-rate cross-slot microfluidic system, a confocal microscope and special fluorescence techniques to measure the time-dependent response of VWF to changes in elongational flow. This proposed work will improve the understanding of VWF regulation by biochemical and mechanical cues in blood vessels. My quantitative approaches and advanced instrumentation will bring insights from polymer physics, and advanced single-molecule and fluorescence methods, into the field of hematology. This study will also give me firsthand experience in biochemistry, molecular biology and hematology, enabling me to better apply my quantitative and physical training to more topics in biology research in the future.
Von Willebrand factor (VWF) adheres to platelets at sites of injury to form plugs that stop bleeding. Nature has programmed VWF to sense changes in blood flow near injury sites and to respond by activating its adhesion to platelets. This project will answer how changes in chemical environment and blood flow pattern affect the function of VWF and contribute to vascular diseases such as excessive bleeding, heart attack and stroke.
