SDS-PAGE analysis displaying the effect of Pep1 on rFXIII-A₂*–mediated cross-linking of fibrin γ-γ, α-α, and α-γ. (A) An FXIII-A₂B₂/Pep1 mix (4.4 µg/mL and 735 µM Pep1, respectively) was added to 1.47 µM recombinant fibrinogen, followed by an activation mix containing human α-thrombin (0.05 U/mL) and CaCl₂ (1.5 mM). The samples were incubated at 37°C for set time points (0-180 minutes), and the reaction stopped using NuPAGE 10× SDS-PAGE reducing buffer with NuPAGE loading dye and boiled for 15 minutes. The SDS-PAGE image shown is representative of 3 independent experiments (n = 3). (B) SDS-PAGE gel densitometry displaying reduction in the fibrin γC and αC over time in the presence (+) and absence (-) of Pep1. (*γ-chain reduction ^+/− Pep1, P < .03; **αC reduction ^+/− Pep1, P < .01). (C) SDS-PAGE gel densitometry displaying the formation of γ dimers (left axis) and γ-α hybrids (right axis) over time in the presence (+) and absence (-) of Pep1 (P = .01). Net intensities were converted to fold increase over time point 0. γ-Dimer formation in the presence and absence of Pep1 gave a P value of .01. γ-α–Hybrid formation in the presence and absence of Pep1 gave a P value of .004. (All experiments were a total of n = 3; the results are expressed as means [SEM].)