Fig. 2. Effect of the rate of FXIIIA proteolytic activation on fibrin crosslinking.
A – 1 μM recombinant FXIIIA AP variants were incubated with 30 nM recombinant human thrombin at 37 °C. Aliquots were withdrawn at denoted time points and thrombin was inhibited with 760 nM PPACK. The samples were then subjected to SDS-PAGE on 8% gels. FXIIIA AP cleavage resulting in a 4 kDa molecular weight loss could then be followed by monitoring the appearance of a 79 kDa band. (N=3)
B – Fibrin crosslinking by FXIIIA AP variants. 1 mg/ml human FXIII-free fibrinogen was combined with a recombinant FXIIIA AP-variant (50 nM) at 37 °C, and crosslinking was initiated by addition of 12 nM recombinant human thrombin and 2.5 mM CaCl2. At each denoted time point, the reaction was stopped by addition of reducing sample loading buffer and boiling. The samples were resolved via SDS-PAGE (8% gel). Fibrinogen chains Aα, Bβ, γ, HMW (high molecular weight crosslinks), and γ–γ crosslinks are annotated. * symbol denotes the earliest detection of HMW species during the course of the crosslinking reaction. (N = 3)