Von Willebrand factor (VWF) is critical in arterial thrombosis. Previous studies have shown that aptamer BB-031, a VWF inhibitor, induces thrombolysis in vivo in ischemic stroke models, making it a promising stroke therapeutic. We hypothesized that BB-031 will induce thrombolysis in an ex vivo ischemic stroke model and that advanced imaging techniques will provide mechanistic information. A microfluidic model was utilized to simulate ischemic stroke and drug delivery without disturbing the occluding thrombus. Heparinized whole blood was obtained from healthy volunteers (N=10). A gravity-based constant pressure microfluidic system mimicking arterial perfusion induced occlusive thrombosis at a collagen-coated region of stenosis. After resting for 0, 3, or 6 hours to allow retraction in situ thrombi were reperfused with blood containing 1692nM (~1mg/kg) BB-031, 0.7nM alteplase (ALT), 0.7nM tenecteplase (TEN), or vehicle 500um upstream of the thrombus using a separate inlet. Platelets were labelled prior to thrombus formation and reperfusion using fluorescent anti-CD41. Thrombolysis and patency were then monitored for 2hrs via outlet mass recording and z-stacked immunofluorescent imaging. Upon immediate reperfusion, 1692nM (~1mg/kg) BB-031 increased mean mass output by 0.250±0.629g compared to 0.031±0.065g with vehicle, 0.074±0.061g with ALT, and 0.101±0.150g with 0.7nM TEN. After a 3-hour retraction period, BB-031 reperfusion mean mass output was 0.951±1.279g compared to 0.301±0.380g with vehicle, 0.412±0.681g with ALT, and 0.569±0.714g with TEN. After 6-hours resting, reperfusion mass output increased by 1.354±1.433g with BB-031, 0.368±0.538g with vehicle, 0.158±0.195g with ALT, and 0.520±0.539g with TEN. Representative z-stacked renderings of the thrombus pre and post reperfusion from a 0-hour resting period show a volumetric decrease in thrombus size of 39% with BB-031 compared to 25% and 18% reduction with vehicle and ALT respectively in a donor-matched dataset. BB-031 improves patency in a microfluidic model of biofidelic thrombolysis compared to current standards of care, which is enhanced at 3 and 6 hours post-occlusion. This may be due to VWF-dependent lysis resulting in reduced thrombus volume.