In this study, we identified lysine residues in the fibrinogen A␣ chain that serve as substrates during transglutaminase (TG)-mediated cross-linking of plasminogen activator inhibitor 2 (PAI-2). Comparisons were made with ␣ 2 -antiplasmin (␣ 2 -AP), which is known to cross-link to lysine 303 of the A␣ chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to ␣ 2 -AP but not for crosslinking to PAI-2. Further evidence that PAI-2 did not cross-link via Lys-303 was the cross-linking of PAI-2 to I-9 and des-␣C fibrinogens, which lack 100 and 390 amino acids from the C terminus of the A␣ chain, respectively. PAI-2 or ␣ 2 -AP was cross-linked to fibrinogen and digested with trypsin or endopeptidase Glu-C, and the resulting peptides analyzed by mass spectrometry. Peptides detected were consistent with tissue TG (tTG)-mediated cross-linking of PAI-2 to lysines 148, 176, 183, 457 and factor XIIIa-mediated cross-linking of PAI-2 to lysines 148, 230, and 413 in the A␣ chain. ␣ 2 -AP was crosslinked only to lysine 303. Cross-linking of PAI-2 to fibrinogen did not compete with ␣ 2 -AP, and the two proteins utilized different lysines in the A␣ chain. Therefore, PAI-2 and ␣ 2 -AP can cross-link simultaneously to the ␣ polymers of a fibrin clot and promote resistance to lysis.

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