The effect of Pep1 on the rFXIII-A2*–mediated incorporation of biotinamido-pentylamine and α2-antiplasmin to fibrin. (A) Biotinamido-pentylamine incorporation assays: rFXIII-A2*/Pep1 mixtures (final concentrations per well, 0.5 µg/mL and 6.95-55.2 µM, respectively) were added to fibrinogen-coated wells. The reactions were activated by adding DTT (0.1 mM), biotinamido-pentylamine (0.3 mM), CaCl2 (10 mM), and human α-thrombin (1 U/mL). Reactions were stopped at 5-minute intervals by addition of 200 mM EDTA. Incorporated biotinamido-pentylamine was detected at an absorbance of 405 nm (n = 3). Error bars show ± 1 SD. (B) α2-Antiplasmin incorporation assays: An rFXIII-A2*/Pep1 mix (final concentrations per well, 1.1 µg/mL and 6.95-55.2 µM, respectively) was added to fibrin-coated wells. An activation mix containing 0.1 mM DTT, 5 mM CaCl2, 1 U/mL human α-thrombin, and 10 µg/mL α2-antiplasmin was added, and the reaction stopped at 20-minute intervals using 200 mM EDTA. Cross-linked α2-antiplasmin was detected using an α2-antiplasmin HRP-conjugated antibody and OPD substrate at an absorbance of 490 nm. Blank wells were absent of rFXIII-A2*. Samples were performed in triplicate (n = 3). Error bars show ± 1 SD.
