Characterization of the interaction between rFXIII-A and αC region 233-425 by SPR. (A) Comparison of activated wild-type rFXIII-A (red), with nonactivated wild-type (green), and rFXIII-A double thrombin cleavage mutant R37A/K513A (blue) binding to captured GST α fragment 1. Both wild-type rFXIII-A and R37A/K513A variant were treated with thrombin (5 U/mL) and calcium (1.5mM) for 2 hours at 37°C. Activated wild-type rFXIII-A, R37A/K513A variant and wild-type nonactivated rFXIII-A (1μM) were injected for 60 seconds at a flow rate of 30 μL/min over captured GSTα fragment 1. (B) The effect of calcium during rFXIII-A activation on binding to captured GSTα fragment 1. rFXIII-A (1μM), activated with 1.5mM calcium and 5 U/mL thrombin (red), and rFXIII-A (1μM) activated with 5 U/mL thrombin in the presence of 5mM EDTA (blue) were injected for 60 seconds at a flow rate of 30 μL/min over captured GSTα fragment 1 for comparison. (C) The effect of 50mM iodoacetamide on rFXIII-A binding to captured GSTα fragment 1. Wild-type activated rFXIII-A (0.5μM) was preincubated with (blue) or without (red) 50mM iodoacetamide for 15 minutes at 37°C and injected for 60 seconds at a flow rate of 30 μL/min over captured GSTα fragment 1 for comparison. Sensorgrams shown in panels A, B, and C are representative of one experiment from triplicate runs (n = 3). The binding response was observed with reference subtracted data. Response units are plotted against time in seconds.