Streptavidin from streptomyces avidinii

Fibrinogen Alexa Fluor 546 and 647 were from Thermo Fisher Scientific. Corn trypsin inhibitor (CTI) was from Haemotologic Technologies. LC polyP (>1000 phosphate units) and biotinylated polyP (200–1300 phosphate units) were prepared as described previously.^{6 ,27 (link)} Anti-FXI antibodies 1A6, 10C9, and 14E11 were created as described previously.^{5 (link),13 (link)} Hirudin was obtained from Hypen Biomed, lipidated recombinant tissue factor (TF; Dade^®Innovin reagent) was purchased from Siemens, and ellagic acid (EA; aPTT reagent) came from Pacific Hemostasis. HEPES buffered saline (HBS, composed of 25 mM HEPES and 150 mM NaCl, pH 7.4) and polyP-binding buffer (composed of 50 mM Tris HCl, pH 7.4, 1% bovine serum albumin (BSA), 0.05% Tween-20 and 5 mM EDTA) were made in-house. Streptavidin from Streptomyces avidinii was obtained from Sigma Aldrich. Platelet-poor plasma (PPP) was prepared as previously described.^{23 (link)}

Human α-Thrombin (Haematologic Technologies) was diluted in 1X FB to a concentration of 270 nM. Streptavidin from Streptomyces avidinii (Sigma-Aldrich) was diluted in 1X FB to a concentration of 900 nM. For the incubation of 2 x 2 arrays with Streptavidin, 2 x 2 arrays at a concentration of 5.5 nM were mixed with 72 nM Streptavidin in 1X FB and incubated for 30 min to 1 hour at 37°C. For the incubation of α-Thrombin, 2 x 2 arrays at a concentration of 5 nM were mixed with 67 nM α-Thrombin in 1XFB and incubated for 30 min to 1 hour at 37°C. For the incubation of 2D crystals with Streptavidin, DNA origami crystals (25 nM) were mixed with Streptavidin at a concentration of 330 nM in 1X FB and incubated at 37°C for 30 min to one hour. For the incubation of 2D crystals with α-Thrombin, DNA origami crystals (13 nM) were mixed with α-Thrombin at a concentration of 180 nM in 1X FB and incubated at 37°C for 30 min to one hour. After incubation and before imaging, buffer solution was added to adjust the final concentration of DNA origami and proteins to 3 nM and 40 nM, respectively. For the 2D crystals, the final buffer solution has 120 mM to 150 mM NaCl in addition which facilitates the AFM imaging of crystals on mica substrate.