This study was approved by the Medical Research Ethics Committee of Nara Medical University, and blood samples from a patient with type 1 VWD were collected after informed consent following the University ethical guidelines.

Reagents—Standard rFVIII (Advate®), peg-EHL-rFVIII (Adynovate®), and rVWF (Vonvendi®) were obtained from Shire Japan Limited (Tokyo, Japan), and plasma-derived VWF/FVIII concentrate (Confact®; VWF:FVIII ratio; 1.6:1.0; available back then) was purchased from KAKETSUKEN (Kumamoto, Japan). Collagen I/III (MP Biomedicals, Santa Ana, CA), recombinant hirudin (Enzo Life Science, Farmingdale, NY), anti-VWF antibody (DAKO, Santa Clara, CA), Cytoperm/Cytofix (BD, Franklin Lakes, NJ), phalloidin-Alexa 488, Alexa 568 and 647 labeling kit (Molecular Probes, Eugene, OR), were purchased from the indicated vendors. An anti-FVIII C2 polyclonal antibody (polyAb) was obtained from a severe hemophilia A patient with inhibitor was used for immunostaining as previously described [11, 12].

Perfusion chamber experiments–Perfusion experiments were performed as previously described [13,14,15,16]. Briefly, µ-slides VI 0.1 (ibidi, Martinsried, Germany) were coated with collagen I/III at 300 µg/mL in sodium carbonate/bicarbonate buffer at room temperature (RT) overnight, washed three times with phosphate buffer saline (PBS), blocked with 5% bovine serum albumin for 1 h at RT, and washed with PBS prior to the experiments. Blood samples were obtained using hirudin as an anticoagulant to maintain the platelet function as long as possible to form physiological platelet thrombus. Preliminary experiments determined that 25 µg/mL hirudin was the minimum concentration suitable for the experimental procedures (data not shown). Study subjects had not taken any medication that may have affected platelet function or blood coagulation in the two-week period prior to blood sampling. Standard or pegylated rFVIII (1 IU/mL), rVWF (1.6 IU/mL), pd-VWF/FVIII (calculated as 1.6 IU/mL/1 IU/mL) were added to the whole blood and was perfused in the prepared chamber at the indicated shear rates controlled by a syringe pump NE-1600™ (New Era Pump Systems, Farmingdale, NY). Reactions on the collagen-coated surface under these conditions represented thrombus formation at high shear flow.

Immunostaining—After perfusion, thrombi were fixed with Cytoperm/Cytofix® prior to immunofluorescent staining [14,15,16]. The fixed thrombi were permeabilized with PBS containing 1% Triton X-100 for 10 min at RT and blocked with serum free protein block (DAKO) for 20 min, followed by incubation overnight at 4 °C with phalloidin-Alexa488 (6 µg/mL), anti-VWF antibody (10 µg/mL) labeled with Alexa568, and anti-FVIII antibody (1 µg/mL) labeled with Alexa647. The stained thrombi were washed 3 times with PBS and mounted in DAKO-fluorescence mounting medium prior to imaging. Preliminary experiments were performed to confirm sufficient infiltration of the fluorescent antibodies into thrombi. Platelets, VWF, and FVIII were visualized using confocal laser scanning microscopy (FV-1000™, Olympus, Tokyo, Japan). Digital images were obtained at 1 μm intervals to a height of 60 μm from the surface. Surface coverage (SC) was measured by calculating the percentage of the area covered by adhering platelets (labeled with phallodin-Alexa488) based on sliced images at 2 μm from the bottom of the thrombus using ImagePro 6.0™ (Media Cybernetics, Rockville, MD). Thrombus height (TH) was calculated by dividing the total volume of the thrombus by SC. SC and TH were measured as markers of initial thrombus formation and thrombus development, respectively [14,15,16].

Total-thrombus formation analysis system (T-TAS)—A microchip flow chamber system (T-TAS®; Fujimori Kogyo, Yokohama, Japan) was utilized to analyze thrombus formation under flow conditions as previously described [17, 18]. Briefly, PL (platelet) chips were pre-coated with type I collagen. Hirudin-treated whole blood (300 µL) was perfused into the chamber at flow rates of 12 µL min-1, corresponding to initial wall shear rates of 1,000 s-1 (high shear). Platelet thrombus formation was visually monitored using a video-microscope located under the microchip, and pressure inside the chamber was monitored and recorded for 10 min or until occlusion. Times to reach 10 kPa (T10) and 60 kPa (T60) were measured to determine initial platelet thrombus formation. Areas under the curve (AUC) of the obtained pressure readings were calculated as index of thrombus development.

Data analyses—All data and statistical analysis were performed using Graphpad prism 7.0 (Graphpad software Inc., San Diego, CA). The results are shown as means and SD. The statistical differences between groups were evaluated using one-way ANOVA and Tukey’s multiple comparison test. P-values < 0.05 were considered as statistically significant.