FVIII-containing, rounded WPBs release UL-VWF strings covered with FVIII. (A) FVIII-GFP–transduced BOECs were stimulated with 100μM histamine at 2.5 dyne/cm2. Shown are fluorescent images taken at indicated time points (in minutes) after the onset of flow. Green represents FVIII fluorescence. Arrowheads indicate released patches and strings. Scale bar represents 20 μm. (B) Nontransduced BOECs were stimulated with 100μM histamine at 2.5 dyne/cm2 for 10 minutes to stimulate exocytosis of WPBs and subsequently incubated with 5 U/mL purified FVIII-YFP for 5 minutes. Images were taken at the indicated time points (in minutes) after the onset of FVIII-YFP perfusion. (C) Nontransduced BOECs were stimulated with 100μM histamine at 2.5 dyne/cm2 for 10 minutes and subsequently incubated with washed platelets for 5 minutes before addition of 5 U/mL purified FVIII-YFP for 5 minutes. (D) Nontransduced BOECs were stimulated with 100μM histamine at 2.5 dyne/cm2 for 10 minutes, incubated with 5 U/mL purified FVIII-YFP for 5 minutes, followed by perfusion of washed human platelets for 5 minutes. Live-cell imaging was performed at 37°C on a confocal laser scanning microscopy using a Zeiss LSM510 equipped with Plan NeoFluar 40×/1.3 oil objective (Carl Zeiss) at 37°C.
