Development of recombinant proteins. (A) Schematic representation of recombinant GSTα fragments 1-9. FXIII-A cross-linking glutamines Q237, Q328, Q366, and the α2 anti–plasmin lysine K303 are shown. The tandem repeat region (αC region 270-373) is represented by the shaded area. (B) Expression of recombinant GSTα fragments 1-9. Four percent to 20% Bis-Tris (tris(hydroxymethyl)aminomethane)–reducing SDS-PAGE. GSTα fragments 1-9 are shown left to right, respectively. (C) Sixteen percent% Tris-tricine SDS-PAGE of α fragments 1-9 (in the absence of the GST tag) are shown left to right, respectively. (D) Four percent to 12% Bis-Tris SDS-PAGE of representative samples used in this investigation. Lane 1, reduced nonactivated rFXIII-A; lane 2, reduced thrombin-activated rFXIII-A in the presence of calcium; lane 3, reduced nonactivated rFXIII-A R37A/K513A double thrombin cleavage variant; lane 4, reduced thrombin-treated R37A/K513A variant in the presence of calcium; lane 5, nonreduced purified FXIII-A₂B₂ displaying FXIII-A and -B subunits; lane 6, reduced purified fibrinogen displaying αβγ chains. Dashed gray vertical lines have been inserted to indicate a repositioned gel lane. (E) Biotin-labeled pentylamine incorporation FXIII-A activity assay that compares the activity of FXIII-A₂B₂ (▴) with wild-type rFXIII-A (■) and double thrombin cleavage mutant rFXIII-A R37A/K513A (●; n = 3). Error bars show ± 1 SD.