OC 59.1 - Presence of red blood cells promotes tension-dependent cleavage of von Willebrand factor in whole blood under high shear

Background: Acquired von Willebrand syndrome (aVWS) involves a substantial loss of high molecular weight multimer (HMWM). Conversely, recent studies reported modest cleavage of von Willebrand factor (VWF) via ADAMTS13 from high shear applied to plasma or recombinant VWF solution, suggesting that an additional unknown mechanism necessarily complements high shear and ADAMTS13 causing excessive cleavage of HMWM in aVWS.

Aims: Test the hypothesis that the presence of red blood cells (RBCs) can promote ADAMTS13-mediated VWF cleavage in whole blood under high shear. Understand the biophysical mechanisms of the RBC-mediated cleavage effect.

Methods: Citrated human blood was centrifuged to collect platelet-poor plasma (PPP), which was re-suspended with washed RBCs to specific hematocrit (Hct). Samples were sheared using a cone-and-plate rheometer. Western blotting was performed to show VWF length distribution. Computational modeling of HMWMs suspended in a concentrated RBC suspension under high shear was employed to visualize and quantify the change of VWF conformation and local tension distribution in real-time under the same Hct and flow conditions.

Results: Coexistence of high shear (5000 1/s) and physiological Hct (40%) significantly enhances VWF cleavage compared to shear alone (Figure 1). Increasing viscosity (4 cP) by Dextran does not enhance cleavage in the absence of RBCs. With physiological Hct (40%), VWF exhibits significant elongation and elevation in VWF tensile forces beyond 20 pN required for A2 unfolding and subsequent cleavage (Figure 2). In contrast, VWF elongation is modest, and tension remains well below the critical tension threshold for A2 unfolding in the absence of RBCs, even with higher viscosity (4 cP).

Conclusion(s): The presence of RBCs in whole blood promotes the stretching of VWF and the tension-dependent unfolding of A2 domain under high shear causing excessive cleavage of VWF and potentially the loss of HMWMs seen in aVWS patients.