Characterization of crosslinking sites in fibrinogen for plasminogen activator inhibitor 2 (PAI-2).

PAI-2 is a serpin that can be crosslinked to fibrin(ogen) via the Gln-Gln-Ile-Gln sequence (residues 83-86). We have characterized the lysine residues in fibrinogen to which PAI-2 is crosslinked by tissue transglutaminase and factor XIIIa. There was no competition with the crosslinking of alpha 2-antiplasmin, another inhibitor of fibrinolysis, which was specific for Lys 303 in the A alpha chain. PAI-2 was crosslinked to several lysine residues, all in the A alpha chain, 148, 176, 183, 230, 413, and 457, but not to Lys 303. The contrast with alpha 2-antiplasmin was clear from studies with truncated fibrinogens and competition by peptides. This was confirmed and extended by mass spectrometry of peptides after protease digestion of crosslinked products, which identified the lysine residues to which the inhibitors were crosslinked. PAI-2 remained active after cross-linking and inhibited fibrin breakdown, even by two-chain t-PA. Thus, a second inhibitor of fibrinolysis, in addition to alpha 2-antiplasmin, is crosslinked to fibrin and protects it from lysis.