The balance of von Willebrand factor and platelet activation in causing bleeding in an aortic stenosis

Introduction: : Aortic stenoses (AS) occur when the aortic valve does not fully open, creating a stenosis or restriction of blood flow. Severe cases lead to bleeding, known as Heyde’s syndrome. It has been suggested that this disorder may be attributed to the loss of high molecular weight multimers (HMWMs) of the large mechanosensitive protein, von Willebrand factor (VWF). This HMWM loss is known as acquired von Willebrand syndrome (aVWS). VWF is found in varying sizes in circulation due to its natural cleavage by the metalloprotease, ADAMTS13. VWF plays a critical role in blood clotting, with increasing function in hemostasis with increasing size. Platelets also play a crucial role in hemostasis as they adhere to the sub-endothelial matrix via binding of VWF. This adhesion leads to intracellular signaling and activation of the platelet.
Though the current dogma suggests that high shear stress results in the cleavage of VWF, and thus bleeding, high shear alone may not be sufficient to explain the degree of VWF loss seen in patients. Recently, our group discovered unprecedented VWF cleavage when exposing it to turbulent flow.1 Our current study investigates the effects of VWF and shear-induced platelet activation (SIPA), combining in vivo and ex vivo work to better understand bleeding in AS. We determined the shear rates and turbulent kinetic energy through computation fluid dynamics (CFD) so that they can be matched in ex vivo experiments.

Materials and

Methods: : We introduced a transverse aortic constriction (TAC) on the mouse aorta to simulate characteristics of AS in humans, nothing that the scale is much smaller. The consistency of the constriction was maintained by tying a suture around various needle diameters alongside the aorta and then removing the needle. We also used VWF-/- and ADAMTS13-/- genetic lines. We quantified flow through CFD with computed tomography images, matched with Doppler ultrasound.
We recreated flow characteristics of the TAC model using a benchtop vortexer, combined with a 96-well plate, with flow characterized through both CFD and particle imaging velocimetry (PIV). Platelet poor plasma and platelet rich plasma were obtained from centrifugation steps of citrated human peripheral whole blood.
Multimer cleavage analysis was performed through multimer gels. We also quantified platelet activation through flow cytometry and through microfluidic collagen-binding assays. Lastly, we evaluated overall bleeding in mice through a tail bleed assay, where the tail was cut and immersed in saline solution.

Results, Conclusions, and Discussions:: Results
The mice tail bleeding time increased with increased stenosis severity, 7 days post-surgery. VWF-/- mice exhibited extended bleeding times (Figure1). Comparatively, ADAMTS13-/- mice revealed no cleavage of VWF. Cleavage of HMWMs were increased with increased stenosis severity (Figure 2a). HMWMs were a normal level in ADAMTS13-/- mice. Platelet activation, based on integrin activation and granule release, increased with increasing stenosis severity at 24 hours post-surgery, but were significantly reduced after 7 days (Figure 2b). VWF-/- mice exhibited no platelet activation, whereas ADAMTS13-/- continued to display high platelet activation levels after 7 days.
As a systematic way to investigate the observations we saw in our mouse model, four different speeds (34, 112, 150, and 230 rad/s) were used on a vortexer. Flow became transitional at 150 rad/s and turbulent at 230 rad/s. Thrombus growth evaluated in a collagen-coated microfluidic device showed decreases with increasing rotational speeds. HMWMs decreased with increasing rotational speed. Platelet activation also increased with increased rotational speed. The role of turbulence was investigated by increasing the viscosity of our sample, thereby lowering the Reynolds number, while simultaneously increasing the shear stress. Both the amount of VWF cleavage and level of platelet activation were significantly reduced when the viscosity was increased.
Discussion
We demonstrate that ADAMTS13-dependent cleavage of VWF cleavage found in aVWS becomes more severe with increasing AS severity. In addition, we also show that this phenomena is exacerbated if the AS leads to turbulence, as supported by our prior work. We report, for the first time, that SIPA in our system, can occur when HMWM VWF is present, but greatly reduces as VWF size reduces with cleavage. These results demonstrate a delicate balance between VWF cleavage and platelet activation.
On the basis of our results, we propose the role of turbulence to explain the unprecedented degree of VWF cleavage and platelet activation, characteristic of bleeding disorders such as aVWS prevalent in AS and ventricular assist devices. However, these patients are also at risk for thrombosis. We believe this occurs as a result of high levels of SIPA where VWF size potentially remains sufficient to induce SIPA.

Acknowledgements and/or References (Optional)::