Native HNPs bind to GST-VWF73-H, soluble VWF, and ultra-largeVWF on endothelial cells under flow. Purified native HNPs (0-6 µM) were flown on a CM5 surface covalently immobilized with purified recombinant GST-VWF73-H peptide (A) and multimeric VWF (B) under the flow rate of 20 µL per minute for 10 minutes. The bound HNPs are demonstrated by the increase in response units (RU) as a function of time. The representative curves of 3 independent experiments are shown. The dissociation constants K_D (s) were determined by fitting the sensorgrams using the 1:1 Langmuir interaction model. (C) Purified native HNPs (0.2, 0.4, 0.8, 1.0, and 2.0 µg per lane) were incubated with purified plasma VWF (10 µg per lane) for 30 minutes. HNPs bound to VWF multimers were detected by western blotting with mouse anti-HNP1-3 IgG (green, on the left), followed by Alexa Fluor 488–conjugated anti-mouse IgG after electrophoresis with 1% agarose gel. VWF multimers were detected on the same membrane by incubation with rabbit anti-human VWF IgG (red, in the middle), followed by Alexa Fluor 594–conjugated anti-rabbit IgG (red). The merged image (HNP1/VWF) is shown on the right. (D) Alexa Fluor 488–conjugated native HNPs (green, on the left) (1 µg/mL) were incubated with cultured HUVECs on a BioFlux microfluidic channel after stimulation with histamine (100 µM) for 2 minutes under flow (5 dyne/cm²). The fluorescent images were obtained after fixation of HUVECs with 4% paraformaldehyde in PBS and stained with Alexa Fluor 594–conjugated rabbit anti-VWF IgG (1:1000) (red, in the middle). The merged image (HNP1/ULVWF) is shown on the right.