TG2 cross-links the fibrinogen β-chain to form HMW complexes. (A) Purified human fibrinogen (FIB1) and FXIII-free fibrinogen (peak 1) were incubated with thrombin (ie, to initiate fibrin polymer formation and FXIII-mediated cross-linking) or recombinant human TG2, as described previously.14 Reduced samples were resolved using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and total protein was visualized with Coomassie Blue. (B) FIB1 (2 mg/mL) was cross-linked by TG2 (25 μg/mL), and reduced samples were resolved using SDS-PAGE and visualized with SimplyBlue. A representative gel of at least 3 independent experiments is shown. (C) Fibrinogen β-chain was quantified using densitometry. (D) Representative western blot for fibrinogen β-chain shows HMW cross-linked β-chain in fibrinogen cross-linked by TG2. (E) Thrombin (1 U/mL) or TG2 (25 μg/mL) was added to FIB1 (2 mg/mL). Representative western blots show fibrinogen β-chain detected using western blotting with the polyclonal anti-fibrinogen β-chain antibody (top) or monoclonal anti-fibrin β-chain (59D8) antibody (bottom). (F) Representative western blots show HMW fibrinogen β-chain in fibrinogen cross-linked by TG2 detected using western blotting with the anti-fibrin β-chain (58D8) antibody. For panels D-F, membranes were cut and probed with distinct primary antibody concentrations to resolve β-chain and HMW cross-linked β-chain.
