INTELLECTUAL MERIT: This research will focus on single-molecule manipulation techniques to study multimeric materials that are activated by shear stress. The structure-function relationships will be determined for the macromolecules and biopolymers in their active form. In particular, the research will focus on the activation of adhesion in von Willebrand factor (VWF), the largest multimeric adhesion ligand circulating in blood, which is activated by shear stress to function as a molecular glue to bind platelets. The objectives of the research are to: (1) Characterize mechanical resistance to domain unfolding of VWF. The PI will determine whether disufide bonds are responsible for the lateral association, hence the fiber formation, of ultra large VWF and sheared VWF. The focus here will be on the mechanical resistance of VWF unfolding to external stretching force. (2) Quantify platelet binding kinetics. Through a single-molecule AFM pulling experiment, the PI will determine the kinetic rate and free energy of plasma VWF binding with glycoprotein (GP) Ib-alpha before and after shear exposure and under flow conditions. These data will quantify activation of the molecular glue by shear. (3) Investigate the viscoelastic and free energies of mesoscopic VWF fibers. We will use thermal fluctuations and dynamically imposed oscillations in the pulling schedule to measure the viscoelastic properties and free energies of VWF, sheared VWF, and ultra large VWF.
BROADER IMPACTS: The proposed research will advance knowledge on the mechanism of activation of VWF, a shear-activated molecular glue important in blood clotting. The lessons learned from nature may help to derive strategies for synthesis of functional smart biopolymers. For the broad society, understanding the blood clotting mechanism, which plays an important role in the blood interactions with biomaterials surfaces, may help people with synthetic devices such as heart valve implants, people with bleeding disorders, and thrombic wound treatment. Experimental findings will be incorporated into the PI?s new undergraduate level course, Introduction to Biological Physics, and a graduate level course, Topics in Biological Physics. Students and postdoctoral associates will have the opportunity to use the state-of-the art instrument for biological physics research. A computer program developed in this project will be used to advance the usefulness of AFM instrumentation. Outreach activities include serving as mentor for the Rice Research Experience for Undergraduates (REU) program through the Rice Quantum Institute (RQI), for the Keck Undergraduate Research Training Program (URTP) through the W. M. Keck Center for Computational and Structural Biology, and for participants in the summer undergraduate research program in HHMI Bionanotechnology at Rice University. Students are recruited from physics, chemistry, mathematics and engineering disciplines that have interests in moving into biologically-oriented research. In addition the PI will serve as mentor for high school students from the Harmony Science Academy, through a collaboration between the Rice Institute of Biosciences and Bioengineering, and the predominantly Hispanic Science Academy of South Texas as well as inner-city schools in Houston.
The research has revealed how stresses in the small blood vessels of the heart and brain could cause a common protein to change shape and form dangerous blood clots. Surprisingly, the project revealed that the proteins could remain in the dangerous, clot-initiating shape for up to five hours before settling back into their normal, healthy shape. The project focused on a protein called von Willebrand factor, or VWF, a key player in clot formation. In the first study of its kind, the Kiang group and her collaborators found that shear forces like those found in small arteries of patients with atherosclerosis cause snippets of non-clotting VWF to change into a clot-forming shape for hours at a time. Prior to this evidence, no one expected to find that this condition would persist for hours. This has profound clinical implications. The finding appeared in Physical Review Letters. Kiang uses atomic force microscopes (AFM) to shed light on the fundamental physical processes involved in protein folding. The AFM has a tiny needle with a tip measuring just a few atoms across. The needle is suspended from a tiny arm that bobs up and down over a surface. Kiang's team uses the bobbing needle to grab and pull apart individual protein molecules. By stretching these like rubber bands, her team has shown it can measure the precise physical forces that hold them in their folded shape. In this project founded by NSF, Kiang group did more than just measure the forces, they used those measurements to see what state the molecule was in. In this way, they were able to study the dynamics of the molecule, to see how it changed over a period of time. VWF is synthesized in the cells that line the walls of blood vessels, and it's stored there until the cells get signals that the vessels are in danger of injury somewhere nearby. In response to those stimuli, the cell secretes VWF. It's a long protein, and one end remains anchored to the cell while the rest unfurls from the wall like a streamer. The act of unfurling makes VWF sticky for platelets, and that begins the process of hemostasis, which is what allows people to avoid bleeding to death when blood vessels are damaged by cuts and wounds. The body recognizes when clotting must stop - when there are too many strings, too much sticking, too many platelet clumps - and it uses an enzyme to clip the long VWF strings. First, it makes large, soluble versions of the strings that remain somewhat sticky, and then these large soluble portions of VWF are reduced into smaller subunits of VWF that circulate in the plasma (PVWF). Under normal conditions, PVWF forms fold into compact shapes and cease to be sticky to platelets. However, previous research had shown that a type of physical stress called shear - which can arise in partially occluded arterial blood vessels with high flow rates - could cause PVWF to become sticky to platelets. However, no one knows how the conformation of the PVWF protein changed. Kiang's research has potential to make a big impact, because knowing more makes it more likely that therapeutic interventions can be more rationally designed. Through a combination of experiments and deductive reasoning, her team determined exactly which portion of PVWF changed its conformation during shear stress. They also determined how long the protein remained partially unfurled before relaxing into its natural shape. The next step will be to design new experiments that allow one to study the different multimeric forms of the protein and its binding to platelets that initiates clot formation. That will tell us even more about the physical properties of the proteins and provide more clues about potential therapies.
