Bc thrombin

Films of the argatroban-bound E2As polymer was formed in 96-well plates by adding 100 μl of the modified polymer solution to each well and allowing the THF solvent to evaporate slowly at RT by partially covering plate with its lid. After 24 h, plates were stored at 4°C until assay was run. In order to determine argatroban levels in the modified E2As polymer, the antithrombin activity of the films were compared to the antithrombin activity of a free argatroban standard curve. The assay utilizes a chromogenic substrate which mimics fibrinogen, the natural substrate for the serine protease, thrombin [16 (link)]. Briefly, the chromogenic substrate (i.e., N-p-tosyl-Gly-Pro-Arg-p-nitroanilide acetate) (Sigma-Aldrich, St. Louis, MO) is cleaved by thrombin (BC thrombin, Siemens, Newark, DE) into a residual peptide (i.e., Tosyl-Gly-Pro-Arg-OH) and free p-nitroanilide, which is measured at 405 nm in a Biotek Cytation 3 microplate reader (Winooski, VT). The absorbance difference is used for the determination of thrombin inhibition in the assay.

The AG polymeric solution was evaluated for activity using an assay previously described^{18 (link)}. Briefly, films of the AG-bound CarboSil® polymer were formed in 96-well plates by adding 100 μl of the modified polymer solution. To determine AG levels in the modified CarboSil® polymer, the antithrombin activity of the films were compared to the antithrombin activity of a free AG standard curve. The chromogenic substrate (i.e., N-p-tosyl-Gly-Pro-Arg-p-nitroanilide acetate) (Sigma–Aldrich, St. Louis, MO) is cleaved by thrombin (BC thrombin, Siemens, Newark, DE) into a residual peptide (i.e., Tosyl-Gly-Pro-Arg-OH) and free p-nitroanilide, which is measured at 405 nm in a Biotek Cytation 3 microplate reader (Winooski, VT).

Materials for synthesis of the argatroban polymer solution were obtained from the following: CarboSil^® polymer from DSM Biomedical (Berkeley, CA); anhydrous hexane from Fisher Scientific (Fair Lawn, NJ); anhydrous tetrahydrofuran (THF), anhydrous hexamethylene diisocyanate (HMDI), argatroban monohydrate ((2F,4R)-1-[(2S)-5-(diaminomethylideneamino)-2-[[(3R)-3-methyl-1,2,3,4-tetrahydroquinolin-8-yl]sulfonylamino]pentanoyl]-4-methyl-piperidine-2-carboxylic acid) all from Sigma-Aldrich (St. Louis, MO). Hanks Balanced Salt Solution (HBSS) with calcium and magnesium was purchased from Gibco^® (Grand Island, NY). Materials for the chromogenic antithrombin assay were obtained from the following: chromogenic substrate (N-p-tosyl-Gly-Pro-Arg-p-nitroanilide acetate) from Sigma-Aldrich (St. Louis, MO); BC thrombin from Siemens (Newark, DE). ECC circuits were constructed from the following: Tygon^® poly(vinyl chloride) (PVC) tubing from Fisher Healthcare (Houston, TX); polyurethane angiocatheters, 16-gauge and 14-gauge, from Kendall Monoject Tyco Healthcare (Mansfield, MA).