Cross-linking of fibrin variants by FXIIIa. Lane 1 of each panel: recombinant normal fibrin; lane 2: γQ398N/Q399N/K406R; lane 3: γK406R. Roman numerals in the 60-minute time point denote origin of cross-link formations as identified by MALDI-MS and Western blotting with chain-specific antibodies; I: γ-dimers, II: α-γ hybrid formation, III: α-α cross-links. After 5 minutes, nearly all γ-chains and a large proportion of the α-chain were cross-linked in normal fibrinogen. In γK406R, FXIIIa preferentially cross-linked the α-chain; after 180 minutes, a substantial amount of γ-chain remained. No γ-chain cross-linking occurred in γQ398N/Q399N/K406R. In all fibrinogens, early, relatively lower molecular weight, cross-linking bands disappeared over time, as these transient oligomers become incorporated into larger-molecular-weight molecular complexes that do not enter the gel.