(G-268) Aptamer Inhibitor of Von Willebrand Factor Improves Vascular Patency in a Microfluidic Model of Occlusion and Prolonged Retraction

Introduction:: Von Willebrand factor (VWF) is mechanistically critical in arterial thrombosis. Previous studies have shown that aptamer BB-031, a VWF-A1 inhibitor, induces thrombolysis in vivo in ischemic stroke models. As the search persists for effective acute ischemic stroke treatment, especially after prolonged periods after stroke onset, BB-031 could be a safe and more efficacious therapeutic alternative to current standards of care. This study aims to utilize a microfluidic model of arterial occlusion and prolonged retraction to evaluate BB-031 thrombolysis.


Materials and Methods:: A channel comprising an upstream wye and a downstream region of stenosis was fabricated and used to manufacture microfluidic chambers made of polydimethylsiloxane (PDMS) bonded to a glass slide. Stenosis regions were coated with collagen overnight prior to use. Heparinized whole blood from healthy volunteers (n=10/group; repeat donations to ensure pairing across treatment groups) was incubated with fluorescent anti-CD41 and perfused via gravity through the microfluidic channel to simulate arterial thrombosis. Thrombosis was imaged in real time. Initial wall shear rate in the microfluidic system was 10,000/s at the throat of the stenosis and outflow mass was collected on a scale to determine complete occlusion of the microfluidic channel via a plateau of the mass curve. All experiments reached complete arterial occlusion within 20 min. Thrombi were left to retract in situ for up to 6 hours. A subset of thrombi were formed with exogenous fluorescent fibrinogen, anti-CD41, and anti-VWF and imaged during retraction via z-stacks on confocal microscopy. After 0, 3, or 6 hours post-occlusion, the wye system allowed for reperfusion with treatment conditions immediately upstream with matched donor blood. Reperfusion occurred via gravity and at a pressure head mimicking the middle cerebral artery for 120 minutes. The reperfusion blood was stained with distinct anti-CD41 from original thrombus and containing either 1692nM (~1mg/kg) BB-031, 0.7nM alteplase (ALT), 0.7nM tenecteplase (TEN), or vehicle. Thrombolysis and patency during reperfusion were monitored via outflow mass measurement and z-stacked immunofluorescent imaging. Results are reported as mean +/- standard deviation.


Results, Conclusions, and Discussions:: After 6 hours of retraction, thrombi in the microfluidic system substantially remodeled (Figure 1A). Intensity-derived profiles of occlusive clots demonstrated even distribution post-occlusion shifting to a central peak after 6 hours. Time lapsed fluorescent confocal microscopy images further show fibrinogen, platelets, and VWF incorporated in the thrombi and dynamically remodeled over time (Figure 1B). Upon treatment of thrombi 0 hours post-occlusion, BB-031 did not substantially increase outflow compared to vehicle, ALT, or TEN. Upon treatment 3 or 6 hours post-occlusion, BB-031 outflow was substantially increased compared to vehicle, ALT, and TEN (Figure 1C). Altogether, BB-031 maximum outflow mass was 0.85 +/- 1.2 g and was significantly higher compared to control outflow (0.17 +/- 0.33 g), ALT outflow (0.35 +/- 0.56 g), and TEN outflow (0.22 +/- 0.30 g), inclusive of all retraction times (Figure 1D). Z-stacked binary threshold renderings pre- and post-reperfusion from a subset of experiments illustrate an average volumetric decrease in total thrombus size, including original thrombi platelets (PLTthrom) and new platelet deposition during the reperfusion treatment period (PLTrep), with BB-031 when treated after 3 or 6 hours of clot retraction (Figure 1E,F). After 6 hours of clot retraction, vehicle showed a minimal change in the average total clot volume, whereas ALT and TEN showed increased average total clot volumes. While all treatment conditions showed an average decrease in the originally formed occlusive thrombus volume (Figure 1G), BB-031 showed minimal deposition of new platelets from the reperfusion sample, especially after a 6-hour retraction period (Figure 1H).

This study establishes an in vitro high-throughput microfluidic platform to study arterial occlusion, clot retraction, drug delivery, and recanalization. BB-031 significantly improves patency in this model compared to controls and current standards of care, which is most pronounced 3 hours and 6 hours post-occlusion, showing promise for BB-031 treatment of stroke when tPA is no longer effective. Volumetric changes in clot volume before and after the treatment period may illustrate improvement in vascular patency due to inhibition of re-occlusive platelet deposition as opposed to thrombolysis of original arterial thrombi.


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