R29 - RBC-enhanced Tension-Dependent Cleavage of von Willebrand Factor under Shear

Introduction: : von Willebrand factor (VWF) is the largest blood protein crucial for thrombosis and hemostasis. Once secreted from the endothelium, VWF remains ultra-large and must be cleaved by ADAMTS13 to maintain healthy hemostasis. This process requires unfolding of the A2 subdomain of VWF via locally stretching the subdomain beyond a critical tension threshold (~10pN). Recent flow-based experimental studies demonstrated that high shear alone does not lead to significant VWF cleavage, contrasting with physiological conditions where cleavage is essential for maintaining hemostatic balance. This highlights a critical gap in understanding the flow-based mechanisms beyond simple shear that contribute to the cleavage of freely suspended VWF.
Through rheometer tests on red blood cell (RBC) suspensions and examining VWF cleavage under various hematocrit levels using Western blotting, we observed that RBCs at high concentrations can promote VWF cleavage under shear.
To understand the mechanism behind this finding, we hypothesize that microscale intercellular rheology determined by blood a as concentrated suspension can lead to RBC-enhanced Tension-dependent Cleavage (RT-cleavage) of VWF. To test our hypothesis, we developed and employed a multiscale computational model to directly visualize and quantify the molecular conformation and tensile mechanics of freely suspended VWF in a concentrated RBC suspension.

Materials and

Methods: : A multiscale computational method for concurrently simulating cellular and molecular blood flow has been developed to visualize and quantify VWF multimer conformational dynamics in concentrated RBC suspensions. The technique has been validated to capture realistic hemorheology, cell mechanics, and VWF multimer biophysics. A shear rate at the bulk scale, covering physiological to pathological levels, is imposed to drive the flow of the RBC-VWF suspension. In the rheometric experiment, RBC suspensions were reconstituted to different hematocrits and sheared at various shear rates for comparison. Sodium dodecyl sulphate-agarose discontinuous gel electrophoresis was performed on platelet-poor plasma (PPP) separated from the sheared RBC suspensions to examine the VWF molecular weight distribution.

Results, Conclusions, and Discussions:: RESULTS
RBCs undergoing collective tank-treading motion under shear significantly facilitate with the VWF folding-unfolding cycle (Fig. 1) compared to zero-hematocrit cases (Fig. 2a and Fig. 2b). RBC-enhanced VWF conformational change stretches VWF significantly leading to elevated tension surpassing the tension threshold needed for VWF-A2 unfolding (Fig. 2c). Hct > 20% can elevate VWF tension by 1-2 orders of magnitude to 10~100 pN, surpassing the A2 unfolding threshold (10 pN), making VWF susceptible to ADAMTS13 enzymatic cleavage. Zero Hct leads to a tension level of 1pN or less. As a negative control, elevating the bulk viscosity by 4x without RBCs shows only 4x tension, insufficient for A2 unfolding and VWF cleavage (Fig. 2c). This suggests microscale intercellular rheology created by concentrated RBCs, rather than the bulk viscosity increase, is the driver for elevated VWF tension and cleavage. To validate this RT-Cleavage mechanism, we compare VWF tension (Fig. 2d) against the VWF cleavage percentage of high molecular weight (HMWM) (Fig. 2e) among different hematocrits. Strong correlation between the two further confirms the RT-cleavage. To further prove this mechanism is biophysical, we replace RBCs with microbeads at the same concentrated volume fraction (20~40%) and observe a similar suspension-enhanced cleavage effect on VWF. Further microrheological analysis of RBC suspensions suggests that VWF high tension occurs when squeezing between RBCs (Fig. 3ab), directly highlighting that the RT-cleavage of VWF is driven by intercellular microrheology instead of bulk rheology.
CONCLUSION AND DISCUSSION
Our study reveals that whole blood, as a concentrated RBC suspension, induces instantaneous elevations in VWF tensile forces, which significantly enhance the enzymatic cleavage of VWF under shear. This finding provides a potential resolution to the controversy that pathological high shear alone is insufficient to cause excessive VWF cleavage. Our work shifts the paradigm by proposing a new RT-cleavage mechanism, highlighting how microscale intercellular rheology can lead to increased VWF tension and consequently enhanced VWF cleavage.

Acknowledgements and/or References (Optional)::