Nanopost array fabrication and VWF coating

Nanopost arrays were replicated in polydimethylsiloxane (PDMS) from a silicon master, which was manufactured by electron-beam lithography and inductively coupled plasma etching (Fig. S1). Details of the fabrication are provided in Supporting Methods in the Supporting Material. To coat the tips of the nanoposts, we placed a 300 μL droplet of VWF solution (10 μg/mL in phosphate buffered saline (PBS)) on the tips of nanopost arrays and let the protein adsorb for 2 h. VWF was purified from cryoprecipitate as previously described (17). We observed that VWF from the droplets formed fiber-like strands between the tips of the nanoposts (Fig. S2).

Nanoposts were then treated with Alexa Fluor 594-conjugated bovine serum albumin (BSA) (20 μg/mL in PBS; Sigma, Bloomington, MN) for 1 h to facilitate the fluorescent visualization of the nanoposts and block the adsorption of other proteins. For additional blocking, we submerged the nanoposts in a solution of 0.2% Pluronic F-127 (BASF Ludwigshafen, Germany) in PBS for 1 h. The quality of the blocking layer was evaluated by incubating platelets with nanoposts treated with Alexa Fluor 594-conjugated BSA and Pluronic F-127, but with no VWF coating. Platelets did not attach to these coatings.