K13 - Red Blood Cell Promotes Shear-induced Cleavage of von Willebrand factor

Introduction: : The multimeric plasma protein von Willebrand factor (VWF) plays a crucial role in hemostasis through a mechanosensitive process, where it extends and opens key binding domains, thereby tethering platelets to the injured vasculature. Additionally, as globular VWF is subjected to fluid forces, a separate domain can reveal the cryptic cleavage site for the metalloprotease ADAMTS13. Flow-dependent cleavage of VWF by ADAMTS13 is essential for maintaining a healthy balance of VWF function in the blood.
Excessive cleavage leading to a reduced number of high molecular weight multimers (HMWMs) leads to the bleeding disorder acquired von Willebrand syndrome (aVWS). It is most commonly found in lymphoproliferative disorders, cardiovascular disorders, or myeloproliferative neoplasms. Despite relatively well-understood mechanisms contributing to the first two, there is no apparent cause for aVWS in myeloproliferative neoplasms, such as polycythemia vera (PV). PV is a cancer where the bone marrow overproduces blood cells, leading to elevated hematocrit and increased platelet counts. Patients with PV, primarily characterized by elevated blood cells, exhibit a heightened risk of bleeding.3 Here, we investigate the biomechanical effect of elevated hematocrit relative to platelet counts on VWF cleavage.

Materials and

Methods: : Anticoagulated blood was collected from healthy individuals and PV patients. Components were separated through centrifugation steps and reconstituted at controlled cell counts.
Samples were exposed to flow through either straight tubing that mimics the geometries of arterioles or through a cone-and-plate viscometer that creates a uniform shear environment. Particle tracking velocimetry (PTV) was performed with ghost RBCs to identify transport within the flow at varied hematocrit levels.
Multiscale cellular blood flow simulations of red blood cell (RBC) suspensions under shear were conducted to quantify the intercellular fluid mechanics in comparison to the PTV experiments to provide insights on observed biological phenomena.
Gels were performed to identify cleavage, and densitometry was used to quantify the distribution of multimer sizes, a method for detecting reduced high-molecular-weight VWF multimers.

Results, Conclusions, and Discussions:: Results
PV samples exhibit a wide range of hematocrit and platelet counts due to variability in disease and therapy. For hematocrit below 40%, there is no marked change in multimer size. However, a strong negative correlation (r = -0.8585, P < 0.0001) is found between VWF size and hematocrit when hematocrit levels exceed a physiological level of 40% (Figure 1). Platelet counts did not have any identifiable relationship with changes in VWF size.
To further investigate the role of platelets and hematocrit, we adjusted cell counts for blood coming from healthy donors. To amplify any effects of cell count on VWF cleavage, we applied a uniform pathophysiological shear rate of 5000 s-1. There is a slight reduction in VWF multimer size when platelets are present. Still, strikingly, there is a significantly greater reduction in size with increases in hematocrit (Figure 2a). To more directly relate to elevated hematocrit for physiological shear, experiments were repeated for arteriole-sized conduits at physiological shear rates. For an elevated hematocrit of 55%, there is a significant decline in high-molecular-weight VWF multimers (Figure 2b).
To better quantify micro-rheology, we employed experiments and computations to understand the flow environment associated with the elevated hematocrit. Experimental PTV reveals increased chaotic particle paths for combinations of increasing shear and hematocrit. Multiscale computational simulations were used to quantify intercellular flow while accounting for RBC deformation and rotation. These simulations demonstrate similar fluctuating fluid velocity comparable to the fluctuations found in the PTV experiments. Together, the data supports that the fluctuating intercellular flows caused by rotating and deforming RBCs can substantially enhance VWF cleavage.
Discussion
We provide evidence, for the first time, that the presence of RBCs leads to enhanced enzymatic cleavage of VWF. Increased VWF proteolysis as a combined function of shear stress and hematocrit helps to close the gap between aVWS found in non-physiological shear stress in cardiovascular disease and aVWS seen in PV. We found that the increased chaotic flow paths we observe mirror the chaotic flow environment of unstable flow (especially turbulence) that we’ve shown can cause cleavage in cardiovascular disorders.

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