P nitrophenyl phosphate liquid substrate

Human soluble collagen type I, III, IV, V, or rat tail type I (25 µg/mL in PBS; pH 7.4) were immobilized overnight at 4°C. Wells were washed with PBS and blocked with BSA (2% v/v, in PBS) for two hours. Simplagrin (0–3 µM) diluted in PBS-T (PBS, 1% BSA, 0.05% Tween, pH 7.4) was added in quadruplicates. After two hours, wells were washed in PBS-T and incubated with anti-Simplagrin (1 µg/mL) in the same buffer. After one hour, wells were washed 3× and incubated with alkaline phosphatase-coupled anti-rabbit IgG (1∶5000, in PBS-T). After one hour incubation at 37°C, the wells were washed four times with PBS-T and stabilized p-nitrophenyl phosphate liquid substrate (Sigma) was added. Colorimetric analysis was performed by measuring absorbance values at 405 nm.

Polystyrene plates were coated with 100 µL of collagen type III (0.3 µg/mL) or a 2% (w/v) solution of BSA diluted in PBS for two hours at 37°C. After washing twice with PBS to remove unbound protein, residual binding sites were blocked by adding 5 mg/mL denatured BSA overnight at 4°C. After washing 3× with 50 mM Tris HCl, 150 mM NaCl, and 0.05% (v/v), TBS-T, pH 7.4, increasing concentrations of recombinant Simplagrin (ranging from 0.0015 to 1.5 µM) were added to the wells and incubated at 37°C for one hour. Wells were washed again and incubated with 3 nM of vWF factor VIII free (Haematologic Technologies Inc.) in TBS-T supplemented with 2% (w/v) BSA. After one hour at 37°C, wells were washed 3× with TBS-T, and a polyclonal rabbit anti-human vWF (DakoCytomation) was added (1∶500 in TBS-T) and incubated for one hour at 37°C. After three washes with TBS-T, an alkaline phosphatase conjugate anti-rabbit IgG (whole molecule; Sigma) was added (1∶10000) and incubated at 37°C for 45 minutes. Before adding the stabilized p-nitrophenyl phosphate liquid substrate (Sigma), wells were washed 6× with TBS-T. After 30 minutes of substrate conversion, the reaction was stopped with 3 N NaOH and absorbance read at 405 nm using a Thermomax microplate reader (Molecular Devices). Wells coated only with BSA were used as assay blanks. All experiments were performed in triplicate.