Localization of the rFXIII-A–binding region on the αC of fibrinogen with the use of SPR. With the use of a CM5 sensor chip anti-GST antibody capture approach, a serial dilution of activated rFXIII-A (7.8-1000nM) was injected for 60 seconds over captured GSTα fragments 1-9 at a flow rate of 30 μL/min. Sensorgrams shown are representative of triplicate data and display the double subtracted binding data for GSTα fragments 1 (A) and 9 (B). Response units are plotted against time (n = 3). (C) Competitive inhibition of activated rFXIII-A binding to captured GSTα fragment 1. Thrombin-activated rFXIII-A (125nM) was preincubated for 5 minutes at 25°C with increasing molar concentrations (12.5nM, 125nM, 1250nM, 6250nM) of GST-cleaved α fragments 1-7. The mix of rFXIII-A/α fragment was injected for 60 seconds at a flow rate of 30 μL/min over captured GSTα fragment 1. The same experiment was performed with α fragments 1, 8, and 9 (D). The binding response (RU) for each sample was converted to the percentage of binding of activated rFXIII-A to the captured GSTα fragment 1 (y-axis) and plotted against the molar concentration of each αC fragment 1-7 (nM) added to the activated rFXIII-A (C) and α fragments 1, 8, and 9 (D; n = 3). Error bars show ± 1 SD.