Cross-linking of plasminogen activator inhibitor 2 and alpha(2)-antiplasmin to fibrin(ogen)

In this study, we identified lysine residues in the fibrinogen A alpha chai n that serve as substrates during transglutaminase (TG)-mediated cross-link ing of plasminogen activator inhibitor 2 (PAI-2), Comparisons were made wit h alpha(2)-antiplasmin (alpha(2)-AP), which is known to cross-link to lysin e 303 of the Aa chain. A 30-residue peptide containing Lys-303 specifically competed with fibrinogen for cross-linking to alpha(2)-AP but not for cros slinking to PAI-2, Further evidence that PAI-2 did not cross-link via Lys-3 03 was the cross-linking of PAI-2 to I-9 and des-alpha C fibrinogens, which lack 100 and 390 amino acids from the C terminus of the Aa chain, respecti vely. PAI-(2) or alpha(2)-AP was cross-linked to fibrinogen and digested wi th trypsin or endopeptidase Glu-C, and the resulting peptides analyzed by m ass spectrometry, Peptides detected were consistent with tissue TG (tTG)-me diated cross-linking of PAI-2 to lysines 148, 176, 183, 457 and factor XIII a-mediated cross-linking of PAI-2 to lysines 148, 230, and 413 in the A alp ha chain. alpha(2)-AP was crosslinked only to lysine 303, Cross-linking of PAI-S to fibrinogen did not compete with alpha(2)-AP, and the two proteins utilized different lysines in the A alpha chain. Therefore, PAI-2 and alpha (2)-AP can cross-link simultaneously to the alpha polymers of a fibrin clot and promote resistance to lysis.