Cross-linking of Wild-type and Mutant α2-Antiplasmins to Fibrin by Activated Factor XIII and by a Tissue Transglutaminase

• Published in: The Journal of biological chemistry 2000-12, Vol.275 (48), p.37382-37389
• Main Authors: Lee, Kyung N., Lee, Chung S., Tae, Weon-Chan, Jackson, Kenneth W., Christiansen, Victoria J., McKee, Patrick A.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M003375200

Human α2-antiplasmin (α2AP), the main inhibitor of plasmin-mediated fibrinolysis, is a substrate for plasma transglutaminase, also termed activated factor XIII (FXIIIa). Of 452 amino acids in α2AP, only Gln2 is believed to be a fibrin-cross-linking (or FXIIIa-reactive) site. Kinetic efficiencies (kcat/Km(app)) of FXIIIa and the guinea pig liver tissue transglutaminase (tTG) and reactivities of Gln substrate sites were compared for recombinant wild-type α2AP (WT-α2AP) and Q2A mutant α2AP (Q2A-α2AP). [14C]Methylamine incorporation showed thekcat/Km(app) of FXIIIa to be 3-fold greater than that of tTG for WT-α2AP. With FXIIIa or tTG catalysis, [14C]methylamine was incorporated into Q2A-α2AP, indicating that WT-α2AP has more than one Gln cross-linking site. To identify transglutaminase-reactive sites in WT-α2AP or Q2A-α2AP, each was labeled with 5-(biotinamido)pentylamine by FXIIIa or tTG catalysis. After each labeled α2AP was digested by trypsin, sequence and mass analyses of each labeled peptide showed that 4 of 35 Gln residues were labeled with the following reactivities: Gln2 > Gln21 > Gln419 > Gln447. Q2A-α2AP was also labeled at Gln21 > Gln419 > Gln447, but became cross-linked to fibrin by FXIIIa or tTG at approximately one-tenth the rate for WT-α2AP. These results show that α2AP is a better substrate for FXIIIa than for this particular tTG, but that either enzyme involves the same Gln substrate sites in α2AP and yields the same order of reactivities.