Elevated VWF Levels Drive Thrombus Instability

Introduction::
After thrombotic event, patients are prescribed dual antiplatelet therapy (DAPT); however, the rate of recurrent ischemic events is still unacceptably high. In many cases, patients that are resistant to this type of intervention have a high von Willebrand Factor (VWF) concentration in relation to ADAMTS13 activity. Because high VWF:ADAMTS13 ratios may limit the effectiveness of antiplatelet therapy, a better understanding of VWF-related thrombosis and platelet activation is needed to enable new therapeutic strategies. Herein, we present a mechanical approach for addressing this problem because the link between biological factors and thrombus biomechanics is not well understood. A microfluidic platform is ideal for addressing these questions because thrombus formation requires a high shear gradient for platelets to adhere, aggregate, and activate. Additionally, this platform gives us a precise way to independently manipulate levels of VWF and ADAMTS13 and to add inhibitors in an effective manner. This goal of this work is to enable a deeper understanding of VWF:ADAMTS13 ratio, its contribution to thrombosis and embolization, and its effect on antiplatelet treatments.




Materials and Methods::
Here, we investigate how high VWF levels in combination with antiplatelet treatments impact thrombus formation and embolization using a novel microfluidic device, with embedded force sensors, that forms platelet-rich thrombi under shear flow (Fig. 1). We assessed aggregation by projected area and platelet activation by contractile force, calcium imaging, and P-selectin expression. We also analyzed detachment events and individual platelet behavior to evaluate plug stability.




Results, Conclusions, and Discussions::
In samples doped with high molecular weight multimers, we observed distinct architectural regions of activated platelets that are strongly adhered, interspersed with weakly activated, loosely packed platelets. The activated portions of the thrombi were prone to microembolization and detached as a unit. We used our grid device to evaluate the stability of shear-induced thrombi for a whole blood sample (Fig. 2A) that was doped with acetylsalicylic acid (ASA) (Fig. 2B), VWF (Fig. 2C), or both VWF and ASA (Fig. 2D). Whole blood, ASA, and VWF + ASA generated similarly sized thrombi (Fig. 2E), but the thrombus in the VWF condition was larger and had an embolization and regrowth event at 120-180 sec. We observed detachment events for the other conditions, but they were smaller in size. This prompted us to evaluate the change in thrombus width as a measure of embolization (Fig. 2F-I). We saw sudden changes in thrombus width that were negative (detachment) or positive (growth). As such, we analyzed the frequency of detachment events that were greater than 10 μm (Fig. 2J). Surprisingly, we found that the highest instances of detachment were for VWF and VWF + ASA conditions, which supports our overall hypothesis that high VWF levels can lead to thrombus instability for patients. Interestingly, these thrombi instabilities persist despite antiplatelet treatments, which may explain why repeat ischemia occurs in patients on DAPT with a high VWF:ADAMTS13 ratio. We consider the sizes of these detachment events to be akin to microemboli that occlude capillaries ( < 10 μm in diameter) or arterioles (10-50 μm in diameter). These findings indicate that high VWF levels can lead to thrombus instabilities and detachment events that can block the flow of blood, starving regions of tissue from oxygen and nutrients.




Acknowledgements (Optional): : Research reported in this publication was supported by the National Heart, Lung, And Blood Institute of the National Institutes of Health under Award Number F31HL156697. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.


References (Optional): : [1] Miles, J. et al. Storage temperature determines platelet GPVI levels and function in mice and humans. Blood Adv. 5, 3839–3849 (2021).