Activation-independent platelet aggregation at the interface of immobilized and soluble VWF. (A) Blood prepared as described in the legend to Figure 1, but with 10 μM PG E₁ added to inhibit platelet activation and 5 mM EDTA to prevent ligand binding to integrins, was perfused over immobilized VWF (20 μg/mL coating concentration). The white line delimits VWF-coated (to the left) from uncoated glass. Single platelets adhere when the shear rate is 3000 s^–1 (top); rolling aggregates (some identified by arrowheads) form at 20 000 s^–1 (bottom). Images from Video S2, part 1. (B) Perfusion over immobilized VWF of washed blood cells suspended in Hepes/Tyrode buffer, pH 7.4. In the absence of soluble VWF, single platelets adhere when the shear rate is 3000 s^–1 (top left), and fewer single platelets adhere at 24 000 s^–1 (top right). After adding soluble VWF (20 μg/mL), single platelets adhere at 3000 s^–1 (bottom left; an arrow points to a single platelet shown for reference), but aggregates form at 24 000 s^–1 (bottom right; arrows point to a rolling aggregate and an inset highlights a stretched aggregate during stationary adhesion). Images from Video S2, part 2. (C) Perfusion over immobilized VWF of washed blood cells with added soluble VWF and anti-VWF A1 domain monoclonal antibody (NMC-4, 20 μg/mL).¹⁷  No platelet adhesion is detected.