Fig. 2. FXIIIA undergoes conformational rearrangements upon activation.

(A) fluorescence emission spectra of FXIIIA (500 nM) in the presence of 100 mM CaCl₂ (green), 100 mM MgCl₂ (dark blue), and thrombin-activated FXIIIA in the presence of 4 mM CaCl₂ (purple). Control zymogen FXIIIA is shown in red; (B) nPAGE of FXIIIA: lane 1 – zymogen FXIIIA; lane 2 – FXIIIA activated by thrombin in the presence of 4 mM CaCl₂; lane 3 – FXIIIA treated with 100 mM CaCl₂; lane 4 – FXIII treated with 100 mM MgCl₂; (C) Elution profiles of FXIIIA under different conditions (left): 100 μL of 2 μM FXIIIA was applied to a SEC column and elution profiles were recorded for the zymogen form (red), FXIIIA in the presence of 100 mM CaCl₂ (green), and FXIIIA in the presence of 100 mM MgCl₂ (dark blue). Arrows indicate peak elution volumes (V_e) of the calibration standards (i – thyroglobulin, 670 kDa, V_e = 7.80 ml; ii – bovine γ-globulin, 158 kDa, V_e = 8.40 ml; iii – chicken ovalbumin, 43 kDa, V_e = 9.87 ml; iv – equine myoglobin, 17 kDa, V_e = 11.89 ml); calculation of Stokes radii (R_S) for FXIIIA forms based on their partition coefficients (σ): inverse error function of 1-σ (erf ⁻¹ (1-σ)) was plotted versus Stokes radii (R_S) for the calibration standards (triangles, i – thyroglobulin, R_S = 8.6 nm; ii – bovine γ-globulin, R_S = 5.1 nm; iii – chicken ovalbumin, R_S = 2.8 nm; iv – equine myoglobin, R_S = 1.9 nm). Using the calibration line, the R_S-values were calculated for FXIIIA samples (circles): zymogen (red, R_S = 5.0 nm), FXIIIA treated with 100 mM MgCl₂ (dark blue, R_S = 4.8 nm), and FXIIIA in the presence of 100 mM CaCl₂ (green, R_S = 3.5 nm).