Human VWF p.S1494C-p.S1534C is resilient to proteolysis under flow. pCMV6-XL5-VWF-transfected HA-HRH1 HEK293T cells are grown inside poly-l-lysine-coated flow chambers and exposed to mepacrine-labeled whole blood at flow rates of 1500 and 2500 s⁻¹. (A) Corresponding movies can be found in the supplemental Methods. VWF/WT, VWF/p.S1494C, or VWF/p.S1534C strings are systematically severed when exposed to whole blood at shear rates of 1500 s⁻¹ or above. VWF strings containing the p.S1494C-p.S1534C double mutation are, however, utterly resistant to proteolysis, even at higher shear rates (2500 s⁻¹). As a result, VWF/p.S1494C-p.S1534C strings are able to support the formation of massive thrombi by capturing circulating blood platelets (n refers to the number of individual experiments performed for each construct). (B) VWF/p.S1494C-p.S1534C strings vary in length (from ∼100 µm to >1 mm). Reconstruction of a 420-µm string captured over multiple photographic fields is presented as an example. (C) The table summarizes the total number of VWF strings analyzed (n) for each construct. VWF/WT, VWF/p.S1494C, and VWF/p.S1534C strings were found to be systematically cleaved on exposure to plasma proteases. None of the 93 VWF/p.S1494C-p.S1534C strings analyzed at 1500 s⁻¹ were cleaved after up to 5-minute exposure to whole blood. Similarly, no cleavage of VWF/p.S1494C-p.S1534C strings was observed at 2500 s⁻¹; however, of the 44 strings observed, 3 could not be included in the analysis because of the detachment of the cells they were tethered to (*). Original magnification, ×63 (aperture 1.4). All videos were captured on a LSM 510 Meta confocal microscope at 37°C and processed with the LSM 510 software.