An Experimental Setup to Test the Effects of Shear Stress on von Willebrand Factor

Open Access
Author:
Corsetti, Monica Claire
Area of Honors:
Bioengineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Keefe B Manning, Thesis Supervisor
  • Peter J Butler, Faculty Reader
  • William O Hancock, Faculty Reader
  • Keefe B Manning, Honors Advisor
Keywords:
  • von Willebrand Factor
  • vWF
  • Optical Trap
  • Optical Tweezers
  • Shear Stress
  • Acquired von Willebrand Syndrome
  • ADAMTS-13
Abstract:
This project seeks to develop an experimental setup that will test the effect of applied shear stresses on von Willebrand Factor (vWF) and indicate whether cleavage of that molecule would be seen in-vivo. The design goals are as follows: 1) Quickly calibrate an optical trap to measure its strength before each use. 2) Extract vWF from human plasma, verify the concentration, and attach it to polystyrene beads. 3) Analyze capability to deliver high shear stress to bead in trap. 4) Measure phase shift and amplitude difference between bead motion and fluid velocity. 5) Determine relationship between shear and change in effective radius of the bead. A protocol is developed to perform these tasks, specifically by tracking motion of an untreated polystyrene bead within a flow chamber of DPBS solution by movement of a piezoelectric stage. Extraction of vWF from human plasma requires a repeatable procedure as outlined in Tsai et al. (1), involving centrifugation steps, column chromatography, and verification of vWF extraction through Western blot. Concentrations of vWF are varied through photospectrometry. The vWF is attached to beads through a microcentrifugation procedure as outlined in Arya et al. (2). The maximal shear stress that can be delivered to be beads’ surface is then tested and found to be approximately 180.7 s^-1. Phase shift between fluid velocity and bead motion and the produced amplitude of bead motion between four bead groups is then measured. The bead groups are: 1) Non-vWF coated beads, 2) beads that are vWF-coated but not sheared, 3) vWF-coated beads with 3 cycles of low shear at a shear rate of 157 s^-1 (or a shear stress value of 0.159 N/m^2) for a total of 30 seconds, and 4) vWF-coated beads with 5 cycles of low shear at a shear rate of 157 s^-1 (or a shear stress value of 0.159 N/m^2) for a total of 50 seconds. No difference in phase shift is observed between the four groups, but a difference in amplitude of motion is observed, presumed to be caused by a change in functional radius of the bead due to vWF unraveling. Using Hooke’s Law and Stokes’ Law, a relationship between amplitude of displacement and functional radius of each bead group is obtained.