Cross-linking of wild-type and mutant alpha(2)-antiplasmins to fibrin by activated factor XIII and by a tissue transglutaminase

Human alpha (2)-antiplasmin (alpha (2)AP), the main inhibitor of plasmin-me diated fibrinolysis, is a substrate for plasma transglutaminase, also terme d activated factor XIII (FXIIIa). Of 452 amino acids in alpha (2)AP, only G ln(2) is believed to be a fibrin-cross-linking (or FXIIIa-reactive) site. K inetic efficiencies (k(cat)/K-m(app)) of FXIIIa and the guinea pig liver ti ssue transglutaminase (tTG;) and reactivities of Gin substrate sites were c ompared for recombinant wild-type alpha (2)AP (WT-alpha (2)AP) and Q2A muta nt alpha (2)AP (Q2A-alpha (2)AP), [C-14]Methylamine incorporation showed th e k(cat)/K-m(app) of FXIIIa to be 3-fold greater than that of tTG for WT-al pha (2)AP, With FXIIIa or tTG catalysis, [C-14]methylamine was incorporated into Q2A-alpha (2)AP, indicating that WT-alpha (2)AP has more than one Gin cross-linking site. To identify transglutaminase-reactive sites in WT-alph a (2)AP or Q2A-alpha (2)AP, each was labeled with 5-(biotinamido)pentylamin e by FXIIIa or tTG catalysis, After each labeled alpha (2)AP was digested b y trypsin, sequence and mass analyses of each labeled peptide showed that 4 of 35 Gin residues were labeled with the following reactivities: Gln(2) > Gln(21) > Gln(419) > Gln(447). Q(2)A-alpha (2)AP was also labeled at Gln(21 ) > Gln(419) > Gln(447), but became cross-linked to fibrin;by FXIIIa or tTG at approximately one-tenth the rate for WT-alpha (2)AP, These results show that alpha (2)AP is a better substrate for FXIIIa than for this particular tTG, but that either enzyme involves the same Gin substrate sites in alpha (2)AP and yields the same order of reactivities.