Turbulent Flow Promotes Cleavage of VWF (von Willebrand Factor) by ADAMTS13 (A Disintegrin and Metalloproteinase With a Thrombospondin Type-1 Motif, Member 13)

Abstract

Objective- Acquired von Willebrand syndrome is defined by excessive cleavage of the VWF (von Willebrand Factor) and is associated with impaired primary hemostasis and severe bleeding. It often develops when blood is exposed to nonphysiological flow such as in aortic stenosis or mechanical circulatory support. We evaluated the role of laminar, transitional, and turbulent flow on VWF cleavage and the effects on VWF function. Approach and Results- We used a vane rheometer to generate laminar, transitional, and turbulent flow and evaluate the effect of each on VWF cleavage in the presence of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type-1 motif, member 13). We performed functional assays to evaluate the effect of these flows on VWF structure and function. Computational fluid dynamics was used to estimate the flow fields and forces within the vane rheometer under each flow condition. Turbulent flow is required for excessive cleavage of VWF in an ADAMTS13-dependent manner. The assay was repeated with whole blood, and the turbulent flow had the same effect. Our computational fluid dynamics results show that under turbulent conditions, the Kolmogorov scale approaches the size of VWF. Finally, cleavage of VWF in this study has functional consequences under flow as the resulting VWF has decreased ability to bind platelets and collagen. Conclusions- Turbulent flow mediates VWF cleavage in the presence of ADAMTS13, decreasing the ability of VWF to sustain platelet adhesion. These findings impact the design of mechanical circulatory support devices and are relevant to pathological environments where turbulence is added to circulation.

Keywords: aortic valve stenosis; collagen; hemostasis; von Willebrand Factor.

Figure 1.. Vane and cup rheological setup.

A, Top view of the vane setup, which consists of a 4-blade vane rotor that is lowered into a stainless steel cup (internal radius of 13.6 mm) until a 1 mm axial gap between the vane and the bottom of the cup is achieved. B, Side view of the vane rotor, which consists of 4 blades with a 1 mm thickness. C, Measured torque as a function of input rotational rate showing the determination of laminar, transitional, and turbulent flow regimes.

Figure 2.. Western blot and multimer analysis.

Results comparing cleavage under laminar, transitional, and turbulent flows. A, Representative image of western blot from samples tested in the vane rheometer for 10 min. All samples contained VWF (von Willebrand Factor) and ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type-1 motif, member 13). Samples in the figure are representative of the samples subjected to (1) laminar conditions; (2) transitional condition, and (3) turbulent conditions for 10 min. B, Western Blot from VWF and ADAMT13 samples after flow assay under varying flow regimes of samples exposed for 30 min. (1) Laminar flow, (2) transitional flow, (3) turbulent conditions, (4) vortex method (+control), (5) turbulent flow conditions in absence of ADAMTS13. C, Multimer analysis of samples exposed to control CTRL: un-sheared sample, laminar sample, transitional sample, and turbulent conditions for 30 min. D, Multimer analysis of blood samples tested. (1) Laminar flow, (2) transitional flow, and (3) turbulent conditions. Ag indicates antigen; and CB, collagen binding.

Figure 3.. VWF (von Willebrand Factor) function post-exposure to laminar, transitional, and turbulent flows.

A, Control VWF: example of adherent platelets to control VWF. B, Laminar flow: example of adherent platelets to VWF previously subjected to laminar flow conditions. C, Transitional flow: example of adherent platelets to VWF previously subjected to transitional flow conditions. D, Turbulent flow: example of adherent platelets to VWF previously subjected to turbulent flow conditions. E, Number of adherent platelets over 300 seconds (P<0.001). F, Number of adherent platelets at 100 s (*P<0.05, **P<0.01). G, VWF activity to VWF antigen (Ag) levels (*P<0.05, **P<0.01), for the turbulent sample, the VWF activity was ≤10, the detection limit of the assay. H, Collagen-binding (CB) ratio (*P<0.05, **P<0.01).

Figure 4.. Computational fluid dynamics results showing 3-dimensional particle pathlines and velocity magnitude contours on the middle plane for the different rotating speeds.

A, 10 rad/s, (B) 100 rad/s, (C) 450 rad/s. D, Normalized velocity magnitude and pathlines on plane 1 (left) and plane 2 (right) for rotating speed 10 rad/s. E, Normalized velocity magnitude and pathlines on plane 1 (left) and plane 2 (right) for rotating speed 100 rad/s. F, Normalized velocity magnitude and pathlines on plane 1 (left) and plane 2 (right) for rotating speed 450 rad/s. G and H, Parameters used to characterize fluctuations and to predict blood damage from turbulence. G, Reynolds Shear Stress (RSS) normalized. H, Kolmogorov length scale for rotating speed of 450 rad/s.

Figure 5.. Western blots of VWF (von Willebrand Factor).

A, VWF cleavage representative of the results obtained with cone-and-plate. All samples contained VWF and ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type-1 motif, member 13). Samples in the figure are representative of the samples subjected to constant shear rate for 10 min. B, VWF multimer results from cone-and-plate experiments. All samples contained VWF and ADAMTS13. Samples in the figure are representative of the samples subjected to constant shear rate for 10 min. CTRL indicates control.