An In Vitro Investigation of the Effect of Shear on von Willebrand Factor Proteolysis

Open Access
Author:
Burke, Shannon Leigh
Area of Honors:
Bioengineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Keefe B Manning, Thesis Supervisor
  • Keefe B Manning, Honors Advisor
  • William O Hancock, Faculty Reader
  • Margaret June Slattery, Faculty Reader
Keywords:
  • von Willebrand factor
  • vWF
  • ventricular assist device
  • VAD
  • proteolysis
  • heart failure
  • cardiovascular disease
Abstract:
Patients suffering from heart failure are frequently put on the support of a ventricular assist device (VAD), due to the lack of viable donor hearts for transplantation. Individuals with VADs are commonly diagnosed with a condition that is referred to as Acquired von Willebrand Disease (AvWD), due to its similarity to congenital von Willebrand disease (vWD). Patients suffering from AvWD exhibit symptoms characteristic of the genetic disorder, prolonged and uncontrollable periods of bleeding, which cannot be explained by the patient’s anticoagulation regimen. Research has shown that these patients experience a loss of the high molecular weight (HMW) multimers of the plasma protein von Willebrand factor (vWF), which are critical to normal coagulation and hemostasis. It is believed that the shear stress experienced by blood circulating through the body alters the conformation of vWF, increasing its susceptibility to proteolysis. Abnormally high shear stresses in VADs cause extensive proteolysis of vWF, producing ineffective proteolytic fragments. This investigation was based on the hypothesis that higher shear rates and longer duration of shear result in increased proteolysis of vWF, and a consequent decrease in HMW multimers. Platelet-poor plasma (PPP) was perfused through an in vitro flow loop via a syringe pump to simulate the high shear conditions experienced by plasma flowing through a VAD. Western blotting was used to analyze the extent of vWF proteolysis as a function of magnitude and duration of shear rate. Sequential centrifugation and size exclusion column chromatography allowed for the isolation and purification of vWF for use as a positive control in the experiment. It was determined that there is a minimal duration of shear exposure below which vWF proteolysis does not occur. At constant shear intensity, proteolysis increases with increasing shear rate. Because high shear rates exist in VADs for very short durations, but VAD patients almost always suffer from AvWD, it is believed that vWF proteolysis is a cumulative effect that builds on itself as the blood travels multiple cycles through a VAD.