VWF project summary. (A) Schematic of how the multimeric VWF mediates platelet adhesion to a damaged vascular wall. (B) VWF captures platelets on a collagen-coated surface under only high shear flow. (C) Experimental (left) and Brownian dynamics simulation (right) studies of VWF conformational change under shear flow. (D) Steered molecular dynamics simulation of A2 domain unfolding under tensile force. (E) A2 domain unfolding/refolding studies by single-molecule force spectroscopy using optical tweezers. (F) Basic elements of our proposed single-molecule–based flow-switchable bioactive materials.

Over the past fifteen years, Dr. Zhang and his group members and collaborators have worked on how the giant blood clotting molecule von Willebrand Factor (VWF) mediates platelet adhesion in the early stage of coagulation. The large multimeric plasma protein VWF plays an essential role in capturing platelets onto the damaged vascular wall, allowing the initiation of blood clotting (Fig. A). The VWF effectively senses blood flow, changing conformation in rapid or elongated flow from an inactive, compact globule to a more elongated shape that allows the VWF to interact with both platelets and collagen on damaged vascular walls.

At Lehigh, we have formed a research thrust among five university faculties. We worked closely together and developed combined single-molecule force spectroscopy, microfluidic imaging, and all-atom and coarse-grained molecular modeling approaches to understand how VWF’s biomechanical aspects regulate its biological functions under blood flow (Fig. B-E). This thrust was funded through an NSF/NIGMS grant. We are also conducting NSF-funded research on designing and characterizing novel single-molecule–based materials with switchable structures and functions responsive to shear flow (Fig. F).

One of our major discoveries was that the VWF’s A2 domain unfolds in response to tensile force and exposes its Tyr1605-Met1606 scissile bond for cleavage by ADAMTS13, a metalloprotease in the circulating blood. This process converts the highly thrombogenic, ultra-large VWF multimers in blood shear to smaller multimeric forms; thus, it prevents thrombus formation. The discovery has enhanced our understanding of VWF-mediated bleeding disorders. The work has been widely cited by many papers and textbooks.

During the formation of platelet plugs, adhesion between VWF and platelets is mediated by the interaction between the A1 (i.e., the domain adjacent to A2) of VWF and the Ibα chain of the platelet receptor glycoprotein (GP) Ib-IX complex. More recently, I collaborated with Dr. Renhao. Li at Emory Medical School and identified an autoinhibitory module (AIM) consisting of N- and C-terminal flanking regions on A1 that can be unfolded by a tensile pulling force of 8–20 pN. AIM unfolding leads to A1 activation and platelet binding. We also discovered the critical mechanisms of how von Willebrand disease can arise due to the weakened AIM mechanical property in A1 mutations and how to treat thrombotic disorders via strengthening AIM’s unfolding forces.

Representative publications:

1. Cao, W., Cao, W., Zhang, W., Zheng, X.L.*, and Zhang, X.F.* (2020): Factor VIII binding affects the mechanical unraveling of the A2 domain of von Willebrand factor. Journal of Thrombosis & Haemostasis, in press.
2. O’Brien, H.E., Zhang, X.F., Sanz-Hernandez, M., Chion, A., Shapiro, S., Mobayen, G., Xu, Y.,  De Simone, A.  Laffan, M.A., McKinnon, T.A.J. (2020): Blocking von Willebrand factor free thiols inhibits binding to collagen under high and pathological shear stress, Journal of Thrombosis & Haemostasis, in press.
3. Pisapati, A.V., Wang, Y., Blauh, M.E., Wittenberg, N.J., Cheng, X., and Zhang, X.F.* (2020): Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence. The Journal of Visualized Experiments, (155), e60784.
4. Dong, C., Kania, S., Morabito, M., Zhang, X.F., Im, W., Oztekin, A., Cheng, X., and Webb, E.B. (2019): A mechano-reactive coarse-grained model of the blood-clotting agent von Willebrand factor. The Journal of Chemical Physics, 151:124905.
5. Wang, Y., Morabito, M., Zhang, X.F.*, Oztekin, A., Webb, E.B., and Cheng, X.*, (2019): Shear-Induced Extensional Response Behaviors of Tethered von Willebrand Factor. Biophysical Journal, 116(11):2092-2102.
6. Morabito, M., Usta, M., Zhang, X.F., Cheng, X., Oztekin, A., and Webb, E.B. (2019): Prediction of Sub-Monomer A2 Domain Dynamics of the von Willebrand Factor by Machine Learning Algorithm and Coarse-Grained Molecular Dynamics Simulation. Scientific Reports, 9:9037.
7. Morabito, M., Dong, C., Wei, W., Cheng, X., Zhang, X.F., Oztekin, A., and Webb, E.B. (2018): Internal Tensile Force and A2 Domain Unfolding of von Willebrand Factor Multimers in Shear Flow. Biophysical Journal, 115(10):1860-1871.
8. Dong, C., Lee, J., Kim, S., Lai, W., Webb, E.B., Oztekin, A., Zhang, X.F., and Im, W. (2018): Long-ranged protein-glycan interactions stabilize von Willebrand Factor A2 domain from mechanical unfolding. Scientific Reports, 8(1):16017.
9. Wei, W., Dong, C., Morabito, M., Cheng, X., Zhang, X.F., Oztekin, A., and Webb, E.B. (2018): Characteristics of von Willebrand Factor adhesion on collagen surface under flow. Biophysical Journal, 114(8):1816-1829.
10. Ouyang, Y., Wei, W., Cheng X., Zhang, X.F., Webb, E.B. III, and Oztekin, A. (2015): Flow-induced conformational change of von Willebrand Factor multimer, Journal of Non-Newtonian Fluid Mechanics, 217:58-67.
11. Zhang, X., Halvorsen, K., Zhang, C.Z., Wong, W.P. and Springer, T.A (2009): Mechanoenzymatic cleavage of the ultra-large vascular protein, von Willebrand Factor. Science, 324:1330-1334.
12. Zhang, Q., Zhou, Y., Zhang, C.Z., Zhang, X., Lu, C., and Springer, T.A (2009): Structural specializations of A2, a force-sensing domain in the ultralarge vascular protein von Willebrand factor. PNAS, 106(23):9226-31.